Cheese Making Technology

8/23/2017 Cheese Making Technology eBook
Cheese Making Technology eBook

Cheese making has been an important Canadian domestic and export industry for the past 400 years-almost as
long as the fur trade. The Canadian cheese industry is in continuous growth with respect to both quantity and
variety of cheese.
This Cheese Making Technology book is one book in our Dairy Education Series. The author of this book is Dr.
Arthur R. Hill. Please email comments and questions about this site.
I hope you find this site useful as a reference, or for teaching or training purposes. If you do use information
from this site, please cite your source as Professor Arthur Hill, Cheese Technology. Dairy Science and
Technology Education Series, University of Guelph, Canada.
Reproduction of this site, or any portions thereof, in either print or electronic form without permission of the
author is strictly prohibited.
I hope you enjoy this site and that you'll consider taking our five day hands-on cheese course. For more
information go to www.foodscience.ca
We would also like to draw your attention to a dairy science and technology website - www.dairyscience.info -
developed by Dr. Michael Mullan in Ireland, which is focused heavily on cheese technology.
Section A: Getting Started

Introduction to Cheese Making
The Basic Process
Cheese making can be described as the process of removing water, lactose and some minerals from milk to
produce a concentrate of milk fat and protein. The essential ingredients of cheese are milk, coagulating enzyme
(rennet), bacterial cultures and salt. Rennet causes the milk proteins to aggregate and ultimately transform fluid
milk to a semi-firm gel. When this gel is cut into small pieces (curds), the whey (mostly water and lactose)
begins to separate from the curds. Acid production by bacterial cultures is essential to aid expulsion of whey
from the curd and largely determines the final cheese moisture, flavour and texture. A flow chart showing the
general operations of cheese making is in Figure 1.1.
Cheese Families
The objectives of cheese making are: (1) To obtain the optimum cheese composition with respect to moisture,
acidity (pH), fat, protein and minerals (especially calcium); (2) Establish the correct structure of the cheese at the
microscopic level; and (3) Ripen to perfection. Objectives (1) and (2) are achieved by varying initial make
procedures and it is then possible to achieve objective (3). Most of these variations in initial make procedures are
different means to control the rate and extent of acid development, and the rate and extent of moisture release.
Grouped according to texture and basic manufacturing procedures, seven cheese families are described below
and summarized in Table 1.1. Table 1.2 contains composition data for some common cheese varieties.
https://www.uoguelph.ca/foodscience/book/export/html/2011 1/70
Family 1. Acid-coagulated Fresh Cheese

In North America, 'fresh cheese' normally refers to cheese produced by acid coagulation at 30 - 32C with little
or no added rennet. Acid is normally produced via fermentation by lactic cultures but some fresh cheese may
also be produced by direct acidification with glucono-delta-lactone. Cheese made for fresh consumption is also
made via rennet coagulation (Family 2) and a procedure known as heat-acid precipitation (Family 3).
Varieties: Cottage, Quark and Cream
Coagulation: The distinguishing characteristic of these varieties is that coagulation is achieved by acidification
to pH 4.6 - 4.8, with little or no coagulating enzyme. Acidification is normally by lactic acid producing cultures.
Most other American and European cheese varieties also use lactic acid producing cultures, but gelation is
induced by a coagulating enzyme at pH 6.5 - 6.7, before much acid development has taken place.
pH Control: After cutting at pH 4.6 - 4.8, the curd is cooked to 52C which is sufficient to inactivate the culture
and prevent further acid development. Acidity is also reduced by washing the curd before salting.
Moisture Control: Curd moisture is reduced by syneresis during cooking but remains high, 60 - 70%, in the
finished cheese.
Curing: Fresh cheese as the name implies is consumed fresh and has a shelf life of only 2 - 3 weeks.
Family 2. Rennet-coagulated Fresh Cheese

In Latin American, Middle Eastern and some European countries, fresh rennet cheese is produced with little or
no culture. Without acid production by lactic acid bacteria, cheese pH remains high and the resulting cheese does
not melt when used in a stir fry or other cooked recipes. For reasons of safety and quality, these varieties must be
handled with extra attention to sanitation and refrigeration.
Varieties: Queso Blanco, Queso Fresco, Italian fresh cheese, Halloumi
Coagulation: The distinguishing characteristic of rennet coagulated fresh cheese is that little or no culture is
used. Coagulation is, therefore, entirely by rennet at the natural pH of milk.
pH Control: The pH is determined by the amount of culture. If no culture is used, the pH remains in the range of
6.5-6.7. In some Queso Blanco varieties a small amount of culture is used to reduce the pH to about 5.8 which
reduces the growth of both spoilage (increases shelf life) and pathogenic (increases food safety) microorganisms.
Further acidification is inhibited by cooling and salting. Too much acidification below pH<5.8 will produce a
meltable cheese which is unsuitable for frying.
Moisture Control: Curd moisture may be reduced by syneresis during cooking and limited acidification, but is
still 50 - 70% in the finished cheese. Some varieties exhibit syneresis after packaging.
Curing: Consumed fresh and has a shelf life of only 2 - 4 weeks.
Family 3. Heat-Acid Precipitated Cheese

All cheese making involves a coagulum of milk proteins which is normally formed in one of three ways.
1. Enzymatic coagulation of the primary milk protein, casein, where the enzyme, rennet, is the primary
coagulating agent. Acid production by lactic cultures encourages coagulation and has important effects on
the final cheese texture, but the primary coagulating agent is rennet. This is true for Cheese Families 2,
and 4 - 7.
2. The second type of coagulation is acid induced coagulation of casein, as in Cheese Family 1, where the
acid is produced by natural fermentation or sometimes by the slow release acidulating agent, glucono-
delta-lactone. All the cheese in Family 1, are acid coagulated in the temperature range of 20 - 35C. In this
temperature range, a pH of less than 4.9 is required to form the coagulum, although some fresh cheese is
fermented to pH as low as 4.4.
3. The third type of coagulation, like the second, is primarily acid induced, but no fermentation is involved
and the acid is added to hot milk at temperatures in the range of 75 - 100C. This process has the unique
properties that: (i) The heat treatment denatures the whey proteins which can then be coagulated along
with the casein and recovered in the cheese, hence, a huge yield advantage; (ii) The recovered whey
proteins have a great capacity to bind water so that a high moisture but firm cheese can be produced,
hence, another huge yield advantage; (iii) Acid coagulation at high temperatures requires less
acidification, so the final cheese is much less acid with pH in the range of 5.2 to 6.0 rather than the range
4.4 - 4.8 required for the Family 1 varieties.
4. Finally, the inclusion of whey proteins prevents cheese melting so this process can be used to produce
frying/cooking cheese such as ricotta and Paneer.
Varieties: Ricotta (Italy), Channa and Paneer (India), some varieties of Latin American white cheese.
Coagulation: Coagulation is accomplished by direct acidification of heated milk. High heat treatment of milk
(temperatures greater than 75C) causes denaturation of the whey proteins. Subsequent acidification of the hot
milk coagulates both casein and whey proteins, so that most of the milk protein is recovered in the cheese.
pH Control: The final acidity (pH) is determined by the amount of acid added. Final pH is normally in the range
of 5.3 - 5.8. Any organic acid can be used, but lactic and citric acids are most common.
Moisture control: Moisture can be reduced by holding the curd in the hot curd-whey mixture after coagulation,
and by draining and pressing procedures. Moisture is generally high (55 - 80%) due to the high water holding
capacity of whey proteins.
Curing: Heat-acid precipitated varieties are normally consumed fresh. An exception is Mizithra, a type of ricotta
cheese which is cured, dried, and consumed as a grating cheese. It is also possible in some cases to hot pack
heat-acid varieties to obtain extended shelf life. High concentrations of whey proteins decrease cheese
meltability and account for the excellent cooking properties of heat-acid precipitated cheese.
Family 4. Soft-Ripened Cheese

Varieties: Feta, Camembert, Brie, Blue
Coagulation: Coagulation is primarily rennet (enzymatic) with three important differences relative to cooked and
pressed varieties (Families 5-7).
1. The amount of lactic acid bacteria inoculum is large and the ripening period before renneting is extended.
The result is that acidification has considerable influence on the development of curd structure during
setting and demineralization of the curd is decreased.
2. Cutting is delayed (i.e., setting time increased) to further encourage acidification and demineralization
before cutting.
3. Cutting is accomplished with large knives or just broken up with paddles to minimize moisture and fines
losses before filling the forms.
pH Control: The distinguishing feature of these cheese is that the curd is placed in the forms while still sweet
and let stand in a warm room for several hours. Acidification (i.e. conversion of lactose to lactic acid) continues
until the accumulation of lactic acid inhibits culture growth. Acid development is also influenced by the time
and amount of salting. The pH is normally about 4.4 - 4.6 on the day following manufacture and in the case of
Feta remains low during curing, The pH of mould ripened varieties increases during curing (i.e., acidity
decreases), especially Camembert and traditional Brie.
Note that most current versions of Brie use mild acid producing culture system to produce a sweeter Brie (lowest
pH during early ripening is 5.0 - 5.2). This product ripens more slowly than conventional Brie and has a much
greater shelf.
Moisture Control: Syneresis is induced by acid development after forming and by brine salting. Moisture content
is typically 45 - 60%.
Curing Time: 2 - 8 weeks.
Family 5. Semi-hard Washed Cheese

Varieties: This is the largest and most diverse group of cheese including Gouda, Edam, Colby, Brick, Montasio,
Oka, Muenster and many others.
pH Control: The distinguishing feature of these cheese is the practice of washing to remove lactose. Part or all of
the whey is removed and replaced with water to leach lactose from the curd. The objective is to limit the amount
of lactose to a level which permits sufficient lactic acid development to produce a minimum pH of 5.0 - 5.2, but
not enough to ferment and produce cheese pH less than 5.0.
Moisture Control: The amount of syneresis is controlled mainly by the temperature and time of cooking and by
the temperature of the wash water. Higher temperatures during cooking or washing cause the curd to contract
and expel moisture. Also, important are the rate of acid development and salting treatments. Washed curd cheese
typically have moisture contents of 40 - 50%. With some exceptions, washing treatments are used to make
cheese with a moisture content of 40% or greater and pH greater than 5.1.
Curing: 2 weeks - 9 months.
Family 6. Hard Cheese: Low temperature

Hard cheese (Families 6 and 7) are characterized by lower moisture (some pasta filata types excepted) than other
families. Lower moisture permits removal of sufficient lactose by syneresis to avoid the necessity of washing.
Low moisture is achieved by high temperature cooking (Family 7) or by controlled fermentation and curd
handling (Family 6).
Varieties: Cheddar types and Pasta Filata. types. Cheddar and Pasta Filata manufacture are similar in the early
stages. Pasta filata varieties are distinct in that they are worked and stretched in hot water and brine salted.
Cheddar types are salted before hooping and pressing.
pH Control: The distinguishing feature of these cheese is that acid development is mainly controlled by the
amount of syneresis. As with semi-hard cheese, the objective is to obtain a minimum pH of 5.0 - 5.2 within 1 - 3
days after manufacture. Lactose content is substantially reduced by fermentation with associated moisture loss
during cheddaring and vat salting.
Moisture Control: Moisture is controlled by cooking temperature and time, stirring out after draining,
cheddaring, amount of culture, and salting treatments. Typical moisture content is 35 - 39% for Cheddar types
and up to 52% for Pasta Filata types.
Curing: 1 - 36 months.
Family 7. Hard Cheese: High Temperature

Varieties: Romano, Parmesan, Swiss
pH Control: Type of culture, time-temperature profile during pressing until cooling, lactose removed by
syneresis. Little acid development before draining.
Moisture Control: Rapid syneresis induced by high renneting temperature and high cooking temperature.
Curing: 1 - 36 months.
Other Technological Criteria

The cheese families described above provide a useful 'coat rack' to help organize cheese according to the initial
manufacturing procedures which determine cheese composition and its primary micro-structure. The following
is a more comprehensive summary of technological parameters which determine cheese characteristics.
Species: cow, goat, sheep, buffalo, yak, other

Milk standardization
Fat and protein contents
Whey and milk blends
Coagulation
rennet gel
acid gel
heat-acid precipitate
Moisture control
Cooking temperature and time
Mesophilic versus thermophilic cultures
Amount and acidifying properties of the culture
Heat treatment of the milk
Type of pH control
Direct acidification vs fermentation
Amount and type of culture
Lactose removal:
Washing (American, Dutch)
High temperature syneresis (Swiss, Hard Italian)
High acid syneresis (Feta, Cheshire)
Cheddaring (Cheddar, Pasta Filata)
Extent of acid development
Low acid (minimum pH > 5.8), Latin American fresh cheese
Medium acid (minimum pH 4.9 - 5.5), most European varieties
High acid (minimum pH < 4.9), Fresh cheese, soft ripened cheese
Salting procedures
Salt before forming
Surface salt after forming
Immersion in salt brine
Type and duration of ripening
Fresh versus ripened
Interior, including blue veined cheese
Interior and surface ripened
Bacterial/yeast smears
White surface mould
Type of rind
Rindless-waxed, film wrapped, painted
Dry rind (cured at 85% relative humidity)
Surface ripened (cured at 90-95% relative humidity)
Texture
Openings: mechanical, small holes, large holes
Firmness
Melting properties
No melt: softening without flow (frying cheese)
Stretching: Low melt and strongly elastic (Mozzarella)
Fondue: Medium melt, medium elasticity (Raclette)
High melt: flows readily with no stretch (aged Cheddar)
Recommended references
See also References in the Dairy Science and Technology Education website.
Alfa-Laval. Dairy Handbook. Alfa-Laval, Food Engineering AB. P.O. Box 65, S-221 00 Lund, Sweden. [Well
illustrated text. Excellent introduction to dairy technology].
American Public Health Association, Standard Methods for the examination of dairy products. 1015 Eighteenth
St. NW, Washington, D.C.
Battistotti, B., Bottazzi, V., Piccinardi, A. and Volpato, G. 1983. Cheese: A guide to the world of cheese and
Cheese making. Facts on File Publications, New York, NY.
Berger, W., Klostermeyer, H., Merkenich, K. and Uhlmann, G. 1989. Processed Cheese Manufacture, A JOHA
guide. BK Ladenburg, Ladenburg.
Carroll, R. and Carroll, R. 1982. Cheese making made easy. Storey Communications Inc., Ponnal, Vermont.
[Well illustrated manual for small and home cheese making operations]
Chandan, R. 1997. Dairy Based Ingredients. Amer. Assoc. Cereal Chemists, St. Paul, Minnesota.
Davis, J.G. 1965. Cheese. American Elsevier Publ. Co., New York.
Eck, A. and Gillis, J.-C., 2000. Cheesemaking from Science to Quality Assurance, Lavoisier Publishing, Paris..
Emmons, D.B., Ernstrom, C.A., Lacroix, C. and Verret, P. 1990. Predictive formulas for yield of cheese from
composition of milk: a review. J. Dairy Sci. 73: 1365-1394.
Fox, P.F., Guinee, T.P., Cogan, T.M., McSweeney, P.L.H. 2000. Fundamentals of Cheese Science. Aspen
Publishers, Inc. Gaithersburg, Maryland.
Hill, A.R. 1995. Chemical species in cheese and their origin in milk components. In Chemistry of Structure
Function Relationships in Cheese, E.L. Malin and M.H. Tunick, Editors. Plenum Press, NY.
International Dairy Federation Special Issue No 9301. Factors Affecting Yield of Cheese.
Irvine, D.M. 1982. Cheddar Cheese Manufacture. A bulletin produced by the Ontario Ministry of Agriculture
and Food. [Out of print]
Irvine, D.M. and Hill, A.R. 1985. "Cheese". In Comprehensive Biotechnology. M. Moo-Young, Editor.
Kosikowski, F.V. and Mistry, V.V. 1997. Cheese and Fermented Milk Foods, 3rd Edition, F.V. Kosikowski and
Associates, Brooktondale, NY.
La Fondation de Technologie Laitiere et Department de Science et Technologie des Aliments Universite Laval.
1985. Dairy Science and Technology: Principles and Applications. Les Presses de l'Universite Laval, Quebec.
Law, B. 1999. Technology of cheese making Sheffield Academic Press, Sheffield, UK.
Lawrence, R.C., Heap, H.A. and Gilles, J. 1984. A controlled approach to cheese technology. J. Dairy Sci. 67:
1632-1645.
Lelivre, J., Freese, O.J. and Gilles, J. 1983. Prediction of Cheddar cheese yield. N.Z.J. Dairy Sci. Technol. 18:
169-172.
Masui, K.. and Yamada, T. 1966. French Cheeses: The Visual Guide to More than 350 Cheeses From Every
Region of France. DK Publishing, New York.
Official Methods of Analysis of the Association of Official Agricultural Chemists, P.O. Box 540, Benjamin
Franklin Station, Washington, D.C.
Pfizer Cheese Monographs. C. Pfizer and Co., New York.
1. Italian Cheese Varieties

2. American Cheese Varieties
3. Cottage Cheese and Other Cultured Milk Products
4. Ripened Semi-soft Cheeses

5. Swiss Cheese Varieties
6. Lactic Starter Culture Technology
Price, W.V. and Bush, M.G. 1974. The process cheese industry in the United States: A review. I. Industrial
growth and problems. J. Milk and Food Technology 37: 135-152. II. Research and Development. Ibid 37: 179-
198.
Robinson, R.K., Editor. 1990. Dairy Microbiology, Volumes 1 and 2. Elsevier Applied Science, NY.
Scott, R., Robinson, R.K. and Wilbey, R.A. 1998. Cheese making Practice. 3rd Edition. Applied Science. Publ.
Ltd., London.
Troller, J.A. 1993. Sanitation in Food Processing. 2nd Edition. Academic Press. New York.
Walstra, P., Geurts, T.J., Noomen, A., Jellema, A. and van Boekel, M.A.. 1998. Dairy Technology. Marcel
Dekker Inc. New York, NY.
Wong, N.P., Jenness, R., Keeney, M. and Marth, E.H. 1988. Fundamentals of Dairy Chemistry. Van Nostrand
Reinhold Company, New York, NY.
Websites
Dairy Science and Technology Education website at the University of Guelph
Centre For Dairy Research, Madison, WI. http://www.cdr.wisc.edu/
Agriculture Canada, http://res2.agr.ca/
Others:
http://www.cheese.com/
Section B: Analytical
Process and quality control procedures
Chemical and microbiological analyses of cheese milk, finished cheese and cheese whey are required to
maintain efficient operations and to ensure food safety and quality. This chapter describes some analytical
procedures relevant to cheese making operations, but it is not intended to be a comprehensive process and
quality control manual. The following general comments are intended to orient the reader to the general types of
analyses required in cheese operations. Subsequent chapters will identify process and quality control
requirements in the context of each step in the cheese making process.
Milk Analysis
Milk composition analyses should include both fat and protein, determined by infrared milk analysers. Note that
casein content rather than total protein content is the critical parameter with respect to cheese yield. Cheese
makers are, therefore, advised to regularly monitor the relative amounts of casein, whey proteins and non-
protein nitrogen in their milk. Monthly or bimonthly analysis of protein distribution by Rowland fractionation is
sufficient to monitor seasonal trends. Alternatively, an indication of casein and whey protein distribution can be
obtained by comparing protein concentration in cheese whey to the protein concentration in the initial milk. This
has the advantage that infra red milk analysers can be calibrated to measure protein in cheese whey.
See Chapters 6 and 12 for details on standardization of milk composition and the importance of casein to cheese
yield.
Quality measurements of cheese milk should include total counts (and/or psychrophilic counts), tests for
inhibitors and somatic cell counts. Depending on the types of controls in place at the producer level, cheese
makers may need to monitor bacteria counts, inhibitors, and somatic cell counts of individual producer milks.
Cheese Analysis
Cheese composition analyses should include fat (by Babcock, Mojonnier, or near infra red procedures),
moisture, salt and pH. Cheese pH should be measured at the time of manufacture, 3 - 4 days after manufacture
and periodically during curing. Other composition parameters should be determined several days after
manufacture to permit time for equilibration of soluble components. Salt in particular, requires time to become
evenly distributed throughout the cheese and in the case of brine or surface ripened cheese, uniform salt
distribution may never be achieved. For Cheddar cheese and other vat salted cheese, representative samples for
accurate determination of salt content can be usually be obtained as early as seven days after manufacture.
With respect to process and quality control, the 'pH profile' during manufacture and curing is vital. 'pH profile is
a term I use to describe the set of pH values at critical process control points in the cheese making process. Other
critical process control parameters are the ratio of salt to moisture (S/M), the moisture in the nonfat substance
(MNFS), and the fat in the dry matter (FDM). These ratios are normally reported as percentages and calculated
as in Equations 1a, 1b, 1c, below. Note that percent total solids is 100 minus percent cheese moisture.
Routine cheese microbial analyses should include yeasts and moulds, total coliforms and staphylococci. For raw
milk cheese, all vats must be tested for the presence of Salmonella, Staphylococci, Listeria and
enteropathogenic E. coli. Cheese made from heat treated but not pasteurized milk must also be considered higher
risk and should be monitored on a regular basis for the presence of common pathogens. Microbial analyses
should be performed at the time of manufacture and after curing. Cheese whey should be monitored for the
presence of bacteriophage specific for the culture currently in use.
Analytical Quality Control
A simple but vital truism, is that inaccurate analytical results are of less value than no analytical results. The
most important causes of poor quality, poor yield efficiency and poor process control are insufficient and
inaccurate chemical and microbial analyses. Effective control of quality and plant efficiency requires effective
quality control of analytical procedures. Smaller cheese manufacturers generally find it's more economical and
reliable to have most analyses performed by an outside laboratory. But, whether the analyses are performed in
house or by an outside laboratory, be certain that your laboratory services are accurate and reliable. In Canada,
dairy laboratory reliability can be assured by certification with the Canadian Laboratory Accreditation
Programme (LAP), Ottawa, (613) 247-1395. The LAP is able to provide ongoing certification for both milk
analysis (composition and quality) and cheese composition analysis. I strongly recommend that cheese
makers use LAP or a similar certified testing, whether lab services are provided from inside or outside the
company.
Some analytical procedures are detailed in subsequent sections. The reader is also referred to:
1. Standard Methods for the examination of dairy products. American Public Health Association, 1015
Eighteenth St. NW, Washington, D.C.
2. Official Methods of Analysis of the Association of Official Agricultural Chemists, P.O. Box 540,
Benjamin Franklin Station, Washington, D.C.
Cheese Sampling
Chemical Analysis
Depending on the size and shape, firm to hard cheese should be sampled using a cheese trier (at least 100 g
sample) or by taking a sector sample. Soft cheese can be blended for sampling or sector sampled depending on
its texture. Cheese samples are stored in opaque air tight containers and fragmented using a grater or other
device before analysis. It is important to grind and mix the sample well before subsampling for analysis.
If the analytical procedure requires less than a 1 gm sample it is desirable to prepare a liquid cheese homogenate
and a subsample from the homogenate. An homogenate suitable for most purposes can be prepared as follows.
Weigh 40 g cheese into a blender container

Add about 100 g of 7% sodium citrate solution
Blend until homogenous (high speed blender such as Polytron is most suitable).
Rinse blender shaft into container and make up to final weight of about 200g.
Note that cheese is notorious for inhomogeneous composition. Brine salted cheese have pronounced salt and
moisture gradients, namely, higher salt and lower moisture near the surface. Large blocks or wheels of pressed
cheese, will have moisture and pH gradients, namely, increasing moisture and decreasing pH towards the
interior. In addition to moisture and salt gradients, surface ripened cheese also has pH gradients, namely, pH
increases at the surface during curing. These difficulties greatly complicate the matter of obtaining accurate
composition and mass balance (yield) data. A useful approach to improve yield control of large blocks is to set
aside small blocks (eg., 20 kg blocks of Cheddar) for early composition and quality testing, and subsequently,
conduct representative sampling of the large blocks (eg., 240 kg blocks of Cheddar) during the cut/wrap process.
Microbial Analysis
Obtain samples as described above for chemical analysis. Triers or knives used for sampling must be flame
sterilized. Samples should be stored in sterile bags such as Whirl Pack bags, stored at 0-4C and analysed within
24 hours.
Equipment
1. Balance, 1,000 g capacity

2. Blender
3. Blender container autoclaved or sanitized with 200 ppm chlorine solution for 5 min.
Procedure
1. Break the cheese into small pieces while still in the bag. Use a pestle or similar device if necessary.
2. Heat dilution blanks of sterile aqueous 2% sodium citrate to 40C. Transfer 30 g of cheese to sterile blender
container, add 270 ml diluent and mix for 2 min. at speed sufficient to emulsify the cheese properly. If
temperature exceeds 40C during blending, use a shorter mixing time or decrease initial temperature of
citrate solution. This 1:10 dilution should be plated or further diluted immediately.
3. Further dilutions can be prepared as required. Pipette 11 ml of the 10-1 dilution of the homogenate,
avoiding foam, into 99 ml dilution blank (0.1% peptone) or 10 ml into 90 ml dilution blank. Shake this
and all subsequent dilutions vigorously 25 times in a one foot arc. Prepare 10-1, 10-2, and 10-3 dilutions.
Total Solids
Oven Method
1. Pre-dry aluminum dishes (105C, 1 h) and weigh to the nearest 0.1 mg on an analytical balance.
2. Weigh quickly 3-5 g of fragmented cheese into the aluminum dish. The weight of sample is the total
weight minus the weight of the dish from Step 1.
3. Dry to constant weight (about 16 h) at 105C. To check for constant weight: weigh at least two samples,
return both samples to the oven for an additional 20 minutes, and re-weigh. The difference between the
weights before and after the additional drying period should be less than 1 mg.
4. Cool in desiccator and determine total dry weight. Sample dry weight is the total dry weight less the
weight of the dish determined in Step 1.
5. Report total solids and moisture contents on weight percent basis as follows:
Note: Several rapid moisture tests based on infrared or microwave drying are available. Check with your
laboratory equipment supplier.
Application
Accurate cheese moisture analysis is critical to composition and yield control. Rapid moisture tests (e.g.,
microwave moisture oven) can be used to obtain early feed back (e.g., cheese moisture immediately after
pressing) information to help with process control.
Titratable Acidity
Principle
See discussion of pH and acidity in Section 3.5.
Apparatus and Reagents
1. An acidimeter equipped with a burette graduated in tenths of a ml up to 10 ml, and some means of filling
the same without undue exposure of the solution to the carbon dioxide of the atmosphere.
2. N/10 sodium hydroxide solution.
3. A dropping bottle containing a 1% alcoholic phenolphthalein solution.
4. White cup, glass stirring rod, 17.6 ml pipette (or 8.8 or 9.0 ml pipette)
5. For cream, Torsion balance and 9 g weight.
Method
1. Mix sample thoroughly by pouring it from one container to another. The temperature of the sample should
be near 20C.
2. Pipette 17.6 ml of milk or cream into a white cup. Note: 8.8 ml pipettes may also be used but are no longer
as readily available as 17.6 ml pipettes. Readily available 9 ml pipettes are also used but require
application of a correction factor to the final result.
3. Add six drops of phenolphthalein indicator solution to milk, 10 drops if the product is cream.
4. Titrate the sample with the N/10 sodium hydroxide solution (0.1 Normal NaOH) while stirring the sample
with the glass rod. Look for the appearance of a faint pink colour which signals the endpoint. Add another
drop or half a drop of NaOH if the pink colour does not persist for 30 s.
5. Record the number of ml of NaOH used to reach the endpoint. This value is called the 'titre'. Titratable
acidity reported as percent lactic acid is dependent on the volume of sample.
For the 8.8 ml pipette, % Lactic acid = titre
For the 17.6 ml pipette, % Lactic acid = 0.5 x titre
For the 9.0 ml pipette, % Lactic acid = 0.98 x titre.
Note that there is practically no lactic acid in fresh milk, but it is a North American convention to report TA in
terms of % lactic acid.
Application
As described in the next section, both titratable acidity (TA) and pH are measures of acidity. However, for most
process control purposes, pH is a more useful measurement. Many cheese makers, however, still use TA to
monitor initial acid development (that is to check for culture activity) during the first hour after adding the
culture. For this purpose, TA is a more reliable indicator because relative to pH measurement, it is more
sensitive to small changes in milk acidity.
When using TA to monitor initial culture activity note that:
1. You are looking for a measurable increase in TA to confirm that the culture is active. For example, if the
initial TA taken immediately after the culture was added is 0.183% lactic acid, and the TA after one hour
of ripening is 0.194 % lactic acid, the change in TA is 0.194 - 0.183 which is 0.011%.
2. Different people will interpret the coloured endpoint differently, so it is important that the same person
takes both the initial and final TA measurements.
3. Carefully performed, it is possible to reliably measure a change in TA of 0.05% lactic acid, so if the TA
increase is greater than 0.05% you can conclude that the culture is active. In most cases TA increases in
the range of 0.05% to 0.10% are obtained after about 30 minutes of ripening (that is, 30 minutes after
adding the culture).
4. It is critical to take the initial TA reading after the culture is added, because the culture (especially the bulk
culture) is acidic.
pH
Concepts of Acidity and pH
All aqueous systems (including the water in you and in cheese) obey the following relationship (Equation 3)
between the concentration of hydrogen ions (H+) and hydroxyl ions (OH-). Note, the square brackets indicate
concentration in moles per litre. A mole is 6 x 1023 molecules, that is, the numeral six with 23 zeros after it.
[H+] x [OH-] = 10-14
Because the actual concentrations in moles per litre are small, it is customary to express the values as exponents.
For example, if we know that the concentration of hydrogen ions [H+] in a sample of milk is 0.000001 moles/l
which is equivalent to 10-6 moles/l, we can calculate the concentration of hydroxyl ions as 10-14/10-6 = 10-
8 moles/l which is the same as 0.00000001 moles/l.
If [H+] = [OH-] the solution is neutral with respect to acidity.

If [H+] > [OH-] the solution is acidic.
If [H+] < [OH-] the solution is basic or alkaline.
Chemicals which contribute H+ or absorb OH- are acids, while bases contribute OH- or absorb H+.
The concept of pH evolved as a short hand method to express acidity. We have already seen that a hydrogen ion
concentration of 0.000001 moles/l can be expressed as [10-6], an expression which defines both the unit of
measurement and the numerical value. The concept of pH is a further abbreviation which expresses the
concentration of hydrogen ions as the negative log of the hydrogen ion concentration in units of moles/l. This
sounds complex but is quite easy to apply. For example, the log10 of hydrogen ion concentration of [10-6] is
equal to -6. The final step is to take the negative of the log, that is -1 x -6 which is 6. So, 0.0000001 moles/l =
[10-6] = pH 6. From the relationship expressed in Equation 3, if the concentration of one of OH- and H+ is
known, it is always possible to calculate the concentration of the other. So, if the pH of a solution is 6, the pOH
is 14 - 6 = 8. Because this relationship is understood, the convention is to only report pH. Note, that because
the negative sign was dropped by convention, decreasing pH values mean increasing acidity, that is,
increasing concentration of H+ ions. So, although both TA and pH are measures of acidity, pH decreases with
increasing acidity.
All of this can be summarized by a description of the pH scale. The pH scale for most practical purposes is from
1 to 14, although a pH of less than one is theoretically and practically possible.
pH 7.0 is neutral acidity [H+] = [OH-]
pH < 7.0 = acid condition [H+] > [OH-]
pH > 7.0 = alkaline condition [H+] < [OH-]
pH Versus Titratable Acidity
TA and pH are both measures of acidity but, for most purposes, pH is a better process control tool, because the
pH probe measures only those H+ which are free in solution and undissociated with salts or proteins. This is
important because it is free H+ which modifies protein functionality and contributes sour taste. It is also the pH
rather than titratable acidity which is the best indicator of the preservation and safety effects of acidity. It must
be emphasized, that the most important factor available to the cheese maker to control spoilage and
pathogenic organisms is pH control. The pH history during and after cheese manufacture is the most important
trouble shooting information. Cheese moisture, mineral content, texture and flavour are all influenced directly by
the activity of free hydrogen ions (i.e. pH).
Titratable acidity (TA) measures all titratable H+ ions up to the phenolphthalein end point (pH 8.5) and,
therefore, varies with changes in milk composition and properties. During cheese manufacture, the pH gives a
true indication of acid development during the entire process so that the optimum pH at each step is independent
of other variables such as milk protein content. However, the optimum TA at each step in cheese making will
vary with initial milk composition and the type of standardization procedure used.
A good practical illustration of the difference between TA and pH is the effect of cutting. Up to the time of
cutting, TA of the milk increases with the development of acidity by the culture. After cutting the TA of the
whey is much lower. This does not mean that acid development stopped. It simply means that titratable H+ ions
associated with the milk proteins are no longer present in the whey. This leads to the concept of buffer capacity,
which is an important principle in cheese making. The effect of protein removal on the TA of whey, is related to
the ability of protein to 'buffer' the milk against changes in pH. That same buffer property is the reason it helps
to take acidic medication, like aspirin, with milk.
Buffer capacity can be described as the ability of an aqueous system, such as milk, to resist changes in pH with
addition of acids (added H+) or bases (added OH-). Specifically, buffer capacity is the amount of acid or base
required to induce a unit change in pH. For example, a small addition of acid to distilled water will cause a large
reduction in pH. The same amount of acid would have a small effect on the pH of milk because milk proteins
and salts neutralize the acidity.
The two most important buffer components of milk are caseins (buffer maximum near pH 4.6) and phosphate
(buffer maxima near pH 7.0). The buffer maximum near pH 5.0 is extremely important to cheese manufacture
because the optimum pH for most cheese is in the range of 5.0 - 5.2. As the pH of cheese is reduced towards pH
5.0 by lactic acid fermentation, the buffer capacity is increasing (i.e., each incremental decrease in pH requires
more lactic acid). The effect is to give the cheese maker considerable room for variation in the rate and amount
of acid production. Without milk's built in buffers it would be impossible to produce cheese in the optimum pH
range.
Another way to illustrate the difference between TA and pH is to consider typical ranges of pH and TA for
normal milk. TA is a measure of the total buffer capacity of milk for the pH range between the pH of milk and
the phenolphthalein end point (about pH 8.3). The pH of milk at 25C, normally varies within a relatively narrow
range of 6.5 to 6.7. The normal range for titratable acidity of herd milks is 0.12 to 0.18% lactic acid In other
words, pH is a good indicator of initial milk quality, while the traditional measurement of TA to indicate
bacterial growth in milk is less precise.
pH Measurement
The pH of cheese milk, whey and soft cheese can be measured directly. Firm and hard cheese must be
fragmented before analysis. Always measure cheese pH in duplicate and use extreme care in handling the
electrode. Place the fragmented cheese in a 30 ml vial or small beaker and gently push the electrode into the
cheese ... too much haste is likely to break the electrode on the bottom of the beaker. To ensure good contact,
press the cheese around the electrode with your fingers. There is no need to rinse the electrode between cheese
samples. However, if the electrode is stored in buffer it should be rinsed with distilled water before measuring
cheese pH. Always store the electrode in pH 4 buffer or as directed by the manufacturer. Do not rub the
electrode. The electrode should be washed with detergent and rinsed with acetone occasionally to remove fat and
protein deposits.
Babcock Methods for Milk Fat

Apparatus and Materials
1. Babcock centrifuge.
2. Water bath at 55C.
3. Torsion balance, 9 and 18 g weights.
4. Babcock shaker.
5. Glassware: 8% milk bottles, 50% cream bottles, 50% Paley bottles, 17.5 ml cylinders, 17.6 ml pipette, .
6. Reagents: - Babcock sulphuric acid (Sp. Gr. 1.82-1.83)
N-butyl alcohol
Glymol.
Milk
1. Temper sample to 20C and mix by pouring gently from original container to a beaker of similar capacity
4-5 times.
2. Transfer 17.6 ml (18.0g) of milk to 8% bottle with 17.6 ml pipette. Allow pipette to drain then blow out
the remaining drop into the bottle.
3. Add 17.5 ml sulphuric acid (Sp. Gr. 1.82-1.83) in at least three increments using special cylinder. Rotate
bottle between thumb and fingers while adding acid to wash milk from neck. Mix thoroughly 2 min. after
each addition of acid by moving the bulb of the bottle in rapid circular motion. Final colour of mixture
should be chocolate brown.
4. Centrifuge 5 min.
5. Add distilled water at 60C to bring contents to within one-quarter inch of base of neck. Do not mix.
7. Add water at 60C to float fat into neck of bottle. Top meniscus should be about even with the top of the
graduated portion. Do not mix.
9. Temper bottles in water bath at 55C for 5 min.
10. Measure length of fat column with dividers from top of upper meniscus to bottom of lower meniscus.
Place one divider point at zero mark and read percentage fat by weight directly where other point touches
the scale.
Cream and Cheese

1. Temper cream sample to 20C and mix. Grind cheese to small particles.
2. Weigh 9 g of cream into 50% cream bottle and add 9 ml of distilled water at 200C. Weigh 9 g of cheese
into a 50% Paley bottle and add 10 ml of distilled water at 60C.
3. Add 17.5 ml sulphuric acid in at least three increments. Mix until colour is uniform chocolate brown and
all cheese particles are dissolved.
5. Add distilled water at 60C to bring contents to within one-quarter inch of base of neck. Do not mix.
7. Add water at 60C to float fat into neck of bottle. Do not mix.
9. Temper bottles in water bat at 55C, for 5 min.
10. Place 4-5 drops glymol on the fat column letting these run down the side of the neck. Measure the length
of the fat column from the demarcation between fat and glymol to the bottom of the lower meniscus.
11. Report fat in percent by weight.
Skim milk, Buttermilk, Whey
1. Temper sample to 20C and mix gently.

2. Transfer 2 ml N-butyl alcohol and then a 9 ml sample to an 18 g double neck bottle. Mix thoroughly with
a circular motion.
3. Add 9 ml of Babcock sulphuric acid for skim milk or buttermilk, 7 ml for whey.
4. Centrifuge 6 min. Place bottles in the centrifuge cup with the small neck facing the outside.
5. Add water at 60C to bring contents 1 cm from the base of the neck. Do not mix. Centrifuge 2 min.
6. Temper bottles in water bath at 55C for 5 min.
7. Place a finger over the large neck and press down until the lower meniscus of fat in the small neck
corresponds to a major division.
Cheese Salt
Cheese salt determination using the Volhard procedure is described below. Other methods which have proven to
give accurate results are:
1. Automatic Chloride Titraters operate on the principle of coulometric silver ion generation to titrate
chloride ions in the sample. When all chloride ions are titrated free silver ions cause a conductivity change
which signals the end of titration.
2. Quantab Chloride Titrater depends on the reaction of chloride ions with silver dichromate, which is
brown, to form silver chloride chromate ion and silver chloride which is white. The reaction takes place on
a calibrated strip which permits direct estimation of chloride content.
Volhard procedure for salt determination
Apparatus and Materials
1. A torsion moisture balance

2. 250 ml erlenmeyer flask and a 500 ml beaker
3. Two graduated cylinders, one 50 ml and the other 100 ml
4. A 10 ml pipette and a 5 ml graduated pipette
5. Burette graduated in ml and 1/10 ml, and burette stand

6. An electric or gas hot plate
7. Chemically pure concentrated nitric acid
8. Saturated potassium permanganate solution
9. 0.1711 N potassium thiocyanate solution (contains 16.63 g per litre) in a brown glass bottle
10. 0.1711 N silver nitrate solution (contains 29.07 g per litre) in a brown glass bottle
11. A saturated solution of ferric ammonium sulphate
12. Sucrose
13. Boiling chips such as carborundum granules or glass beads.
14. Fume hood
Method
1. Prepare cheese sample as for cheese moisture test.

2. Weigh about 3 g cheese into a clean dry 250 ml erlenmeyer flask.
3. Add 10 ml of 0.1711 N silver nitrate solution as accurately as possible to the flask. If cheese contains
more than 3% salt, add more silver nitrate.
4. Add 15 ml of the chemically pure nitric acid.
5. Add 50 ml of distilled water.
6. Add a few boiling stones.
7. Place flask on hot plate in fume hood and boil.
8. When contents of flask are boiling uniformly, carefully add 5 ml of saturated potassium permanganate.
Continue boiling until purple colour disappears, then add a second charge of 5 ml of potassium
permanganate. When purple colour again disappears, add another 5 ml of potassium permanganate.
Continue boiling until all cheese particles are digested. To ascertain when digestion is complete, remove
flask from hot plate and allow to stand quietly for a few moments. Undigested cheese particles will float
upon the surface, while the white precipitate of silver chloride will sink to the bottom of the clear liquid.
When no more white particles are seen upon the surface, digestion is complete.
9. Add sufficient distilled water to bring the volume up to approximately 100 ml. Allow precipitate to settle
and very carefully pour off the liquid into a beaker. Be careful not to pour off any of the white precipitate
of silver chloride.
10. Add 100 ml of distilled water to flask and swirl contents to wash precipitate.
11. Add 3 ml of saturated ferric ammonium sulphate as an indicator and titrate the excess silver nitrate with
0.1711 N potassium thiocyanate. A reddish colour denotes the end point.
12. The number of ml of 0.1711 N silver nitrate originally added minus the titration value found in step 11,
divided by the weight of the cheese in the sample equals the percentage of salt in the cheese.
EXAMPLE
3.00 g of cheese to which 10.00 ml of 0.1711 N silver nitrate had been added gave a reading of 4.00 ml in Step
11.
4.00 ml 0.1711 N potassium thiocyanate required to combine with excess silver nitrate.
6.00 ml 0.1711 N silver nitrate combined with salt in cheese.
Therefore per cent salt by weight = 6.00/3.00 = 2.00
Because the salt in the cheese is measured by its chloride content, it is necessary to test the reagents used for
chloride, or related substances content. This is done by carrying out a test using sucrose instead of cheese. The
titration value subtracted from the original amount of silver nitrate added is subtracted from the value found in
Step 12 before dividing by weight to find the percentage salt in the cheese.
To check the strength of the 0.1711 N silver nitrate solution, dissolve 10 g chemically pure dry sodium chloride
in sufficient water to make up one litre of solution. Each ml of this solution is equivalent to one ml of 0.1711 N
silver nitrate. When the silver nitrate has been standardized, each ml of silver nitrate is equivalent to one ml
0.1711 N potassium thiocyanate.
Culture Activity Test

Purpose
This simple test is useful to ensure that cheese cultures have adequate activity before inoculating the cheese vat.
For most cheese a general rule of thumb is that the activity and amount of inoculum should be sufficient to
produce a titratable acidity of about .34% lactic acid, in 10% reconstituted skim milk, after 4 h of incubation at
37C. The test is also useful to compare types of cultures or bulk cultures prepared under different conditions. For
these purposes a pH versus time chart is quite useful (See Figure 3.1). A further application is to check
sensitivity of the culture to bacteriophage in the plant (See Section 3.9 and Figure 3.1).
Procedure
1. Mix 10 g of low-heat, antibiotic-free skim milk powder in 90 ml of distilled water in a 100 ml Erlenmeyer
flask.
2. Sterilize at 15 lb pressure (1.05 kPa.) for 10 min.
3. Cool to 37C.
4. Inoculate with 3.0 ml starter or other amount as appropriate. Rinse pipette twice by drawing the sterile
milk into it.
5. Incubate at 37C for at least 4 h. Longer if desired for pH versus time profile.
6. Check pH at 30 min. intervals.
7. Titrate 17.6 ml with N/10 sodium hydroxide (NaOH) using 1 ml phenolphthalein. Divide the required ml
of NaOH by 2 the obtain titratable acidity in units of percent lactic acid.
8. Record starter activity as follows:
Active, over 0.34%
Slow 0.26 to 0.30%
Detection of Bacteriophage
The following tests are based on the principle that bacteriophage specific to the culture in use will be present in
high numbers in the cheese whey. Therefore, by monitoring whey for the presence of phage "a dead vat" on
subsequent days can be avoided.
Culture Activity Test

The culture activity test described above can be used to detect the presence of phage in cheese whey. Prepare
300 ml of reconstituted skim milk and place 99 ml in each of three beakers. Add 1 ml of whey to Beaker 1 (100
x dilution), then transfer 1 ml from Beaker 1 to Beaker 2 (10,000 x dilution) and finally, transfer 1 ml from
Beaker 3 to Beaker 4 to make a 1 million times dilution. Add culture and monitor pH as described in section 3.8.
Bromocresol Purple (BCP) Phage Inhibition Test
This test is quite simple to perform, and produces more accurate results than the culture activity test.
1. Prepare Materials
BCP stock solution (1 g/100 ml water)

Test tubes containing 9.9 ml sterile BCP-milk (5 ml BCP stock solution/litre milk)
30-32C water bath or heating block
1 ml graduated pipettes
Membrane filter (0.45 u) -- optional
Disposable syringe -- optional
Clinical centrifuge -- optional
Whey sample for phage testing
Freshly grown culture, frozen syringe, or frozen can of each strain
2. Add Whey to BCP Milk and Make Dilutions
Transfer 0.1 ml of fresh (or filter-sterilized) whey to the first dilution tube (10-2) and mix well. Transfer 0.1 ml
from the first to the second dilution tube and mix well. Repeat process for the third dilution tube. (If unfiltered
whey is used, a control tube containing BCP milk and whey only, must be prepared. This control tube tests for
the presence of active culture in the whey that could mask phage inhibition of a strain.) Whey samples should be
refrigerated immediately after collection and held cold until tested for phage.
3. Add Culture to Control and Whey Dilution Tubes
Cheese culture (0.2 ml) is added to whey dilution tubes and to a control tube for each strain. If you are using
direct-to-the-vat culture, dilute 1 ml of culture in 9 ml of milk and then add 0.2 ml of the mixture to the dilution
tubes. The control tube contains only BCP milk and culture---NO whey. The control tube serves to show starter
strain inhibition by colour comparison with the other tubes.
Incubate Tubes and Interpret Results. Incubate both control and dilution tubes for 6 hours at 30-32C. Compare
the colour of the whey dilution tubes to that of the control tube. Ignore coagulation. An uninhibited culture will
produce sufficient acid to turn the BCP dye from blue to yellow. Strains should be removed from the culture
blend when full inhibition persists at the 10-6 dilution level. The following system should be used to record
phage inhibition:
0 = No inhibition at any dilution
1 = Partial inhibition at 10-2 dilution
2 = Full inhibition at 10-2 dilution
Inhibitory Substances(1)
This section is adapted from two reports prepared by: Mark Mitchell (1995), Ontario Ministry of Agriculture,
Food and Rural Affairs, Guelph, Ontario
Regulations
Most jurisdictions have regulations concerning the testing methods and limits of certain antibiotics in raw
milk. The Milk Act of Ontario, Regulation 761, Section 52, Subsection, states:
"The milk of every producer shall be tested at least once a month for the presence of an inhibitor by an official
method."
An official method is described in a separate inhibitor policy document which states:
The minimum sensitivity of an official method to test for the presence of an inhibitor under section 52 of
Regulation 761 shall be:
1. 0.01 international units of penicillin per millilitre of milk by the Standard Disc Assay (Bacillus
stearothermophilus) procedure.
2. 10 parts per billion sulfamethazine by the High Performance Liquid Chromatography (modified Smedley
and Weber) procedure.
A concentration of .01 international units of penicillin per millilitre of milk is equivalent to 6 parts per billion
(ppb). Note: 1 ppb is equivalent to a single penny in $10 million or one second in 32 years.
Detection Methods
It is beyond the scope of this manual to discuss any specific methods in detail. What follows is are brief
descriptions of five types of inhibitor tests which are currently used in the dairy industry. For each category one
or more brand name tests are listed to indicate possible choices. For cheese manufactures seeking assistance with
inhibitor testing, there are many private labs which provide suitable services. In Ontario, a wide range of
expertise and methodologies are available from Laboratory Services Division, University of Guelph.
Growth Inhibition Assays
Examples: Delvotest P, Delvotest SP, BR test, BR-AS test, Charm Farm, and the Disk Assay
This test format involves a standard culture of a test organism in an agar growth media, usually Bacillus
stearothermophilus, that is inoculated with a milk sample and incubated for periods of up to several hours. If the
milk contains sufficient concentrations of inhibitory substances the growth of the organism will be reduced or
eliminated. The presence of an inhibitory substance is indicated by zones of inhibition or a change in colour of
the media (pH and redox indicators).
The major disadvantages of these tests are that they are not very specific for identification purposes, have
limited sensitivities to many antibiotics and take a long time before results are available. Growth inhibition tests
are only able to classify residues into either the -lactam (penicillin like antibiotics) or other than -lactam
antibiotic families. A further concern is that growth inhibition tests are subject to the effects of natural inhibitors
(eg. lysozyme, lactoferrin, complement and defensins) which can be found in high levels in mastitic milk and
may give false positive test results, particularly when used at the cow level. These effects can be minimized by
heating individual cow samples at 82C for 2-3 minutes in a microwave oven or water bath before testing to
destroy natural inhibitors and allow antibiotics which are more heat stable to remain.
The advantages of these tests are that they are cheap, easy to perform and have a very broad detection range.
Enzymatic Colorimetric Assays

Example: Penzyme Test for -lactams
The penzyme test is based on the inactivation of an enzyme by -lactam antibiotics. The enzyme (DD-
carboxypeptidase or penicillin binding protein) is present in all bacteria and is involved in the synthesis of the
bacterial cell wall. -lactam antibiotics will bind specifically with this enzyme and block it's activity, thus
preventing the formation of the bacterial cell wall. This enzyme has been freeze dried and placed in sealed vials
to which the milk sample is added. After addition of 0.2 ml (200 l) of milk sample to the vial the sample is
incubated for 5 minutes at 47C. During this time any -lactams present in the milk bind to the enzyme and
inactivate a certain amount depending on the concentration present.
Reagent tablets specific for the enzyme (D-alanine peptide and D-amino acid oxidase) are then added to the milk
sample and the sample is incubated at 47C for 15 minutes. During incubation any remaining active enzyme will
react with the reagent added. The end product of the substrate and enzyme reaction (pyruvic acid and hydrogen
peroxide) is measured by a redox colour indicator and the final colour is compared to a colour chart provided
with the kit.
An orange colour (reduced) indicates a negative test result.
A yellow colour (oxidized) indicates a positive test result.
Microbial Receptor Assays:

Example: Charm II
This test uses bacterial cells (Bacillus stearothermophilus), which contain natural receptor sites on or within the
cells for antibiotics, and radio labelled (C14 or H3) antibiotics. Milk sample is added to a freeze dried pellet of
bacterial cells (binding reagent) in a test tube and the sample is mixed and incubated. During incubation any
antibiotic present in the milk will bind to it's specific receptor site. Radio labelled antibiotic (tracer reagent) is
then added and the sample is mixed and incubated. Unbound receptor sites on the bacterial cell will be bound by
the radio labelled antibiotic. The sample is then centrifuged to collect the bacterial cells in the bottom of the test
tube and the supernatant and butterfat is discarded. The bacterial cells are then resuspended and mixed in
scintillation fluid. Binding is measured with a scintillation counter and compared to a positive and negative
control. The more antibiotic present in the sample the lower the scintillation counts determined by the
equipment.
Charm currently has test kits in this format for -lactams, macrolides, aminoglycosides and sulfonamides.
Immunoassays
Unlike other residue testing methods immunoassays are fast, sensitive, inexpensive, reproducible, reliable and
simple to perform. The technique depends upon the measurement of the highly specific binding between
antibodies (Ab) and antigens (Ag). Antigens are substances which are foreign to the body (eg. bacteria, viruses,
toxins, pollens, drugs, hormones and pesticides) and that when introduced into the body give rise to the
production of antibodies. Antibodies are proteins produced in the body by white blood cells (lymphocytes) as a
result of exposure to antigens (destroy invading pathogens). The extreme sensitivity of the immunoassay is due
to the development of certain labelling techniques for molecules (conjugates), enabling the measurement of very
small masses (picogram or parts per trillion) of substances.
Immunoassays are classified according to the label which is attached to either the antigen (the anolyte being
measured) or the antibody. The label may be a radioactive atom as in radio immunoassays (RIA), or an enzyme
as in enzyme immunoassays (EIA or ELISA (Enzyme- linked immunosorbant assay)) or a fluorescent substance
as in fluorescence immunoassays (FIA).
There are 3 major types of immunoassays used commonly for the detection of antibiotics in milk:
1. Enzyme-Linked Immunoassay (eg. LacTek tests, SNAP for Tetracyclines, Single Step Block for SMZ)
2. Enzyme-Linked Receptor Binding Assay (eg. SNAP for -lactams, Delvo-X-Press)
3. Radio immunoassay (CHARM II for tetracyclines and chloramphenicol)
Rennet Activity
Coagulation Time versus Setting Time
Rennet is generally described in the industry as single, double or triple strength. Single strength is considered to
be that concentration where 200 ml is sufficient to set 1,000 kg of milk in 30 - 40 min. at 30 - 32C. Setting time
is the point where the curd will break cleanly and exude clear whey. Coagulation time is the point where flecks
of curd first appear on a spatula or slide dipped into the milk. Coagulation time is about half of setting time, so
typically, coagulation using single strength rennet requires 15-20 minutes followed by setting at 30-40 minutes.
The following simple test can be used to check coagulation time which can be measured much more accurately
than setting time. The test uses skim milk because the presence of fat globules makes it difficult to see the first
sign of coagulation.
Measurement of Coagulation Time
1. Prepare 200 ml samples of 10% reconstituted low heat skim milk powder in 250 ml beakers. Add 0.02%
calcium chloride dihydrate (40 mg per 200 ml).
2. Temper to 32C in a water bath.
3. Add 1.0 ml of 5% rennet solution to each sample.
4. Determine the clotting time by dipping a clean spatula or glass slide into the milk. When coagulation has
occurred flecks of curd will appear in the milk film on the slide.
Relative Milk-Clotting Activity Test
A more rigorous test of coagulant activity is the "Relative Milk-Clotting Activity Test" (RMCAT) which
measures the activity of rennet and other coagulants in "International Milk-Clotting Units" (IMCU). The method
is described in International Dairy Federation standard 157:1992.
Yeasts and Moulds

Selective media for yeasts and moulds include acidified media and antibiotic media. The method described
below uses acidified potato dextrose agar.
Equipment and Material
Potato dextrose agar
Equipment for plating
Tartaric acid solution (10 aqueous)
Incubator set at 22-25C
Procedure
1. Prepare cheese homogenates and serial dilutions as described in Section A.

2. Predetermine the quantity of sterile 10% tartaric acid solution necessary to obtain a pH of 3.5 + 0.1. Put a
portion of the medium in a small beaker and titrate to pH 3.5 at 45C. Check the accuracy of the titration by
allowing the agar to cool to incubation temperature, place electrodes directly into the solidified medium,
and read the pH. It should be 3.5 + 0.1. Calculate the amount of sterile 10% tartaric acid solution
necessary for the volume of tempered agar to be used for pouring plates.
3. Place 5 ml of the 0.1 dilution and 1 ml of additional dilutions as required into each of duplicate petri
dishes.
4. Add the tartaric acid solution to the tempered agar immediately before pouring 15 - 20 ml into each of the
plates containing the sample dilutions.
5. Mix well and let solidify before inverting the plates. Incubate at 22 - 25C.
6. Count the plates at 3 and 5 days of incubation. Yeast cells will appear as cream coloured shiny colonies.
Presumptive Coliforms
Prepare cheese homogenates and serial dilutions as described in Section A.
Place 5 ml of the 0.1 dilution (= 0.5 g of original sample or Omega dilution) and 1 ml of additional
dilutions as required into each of duplicate petri dishes and add molten VRB agar. (Note: Do not sterilize
VRB agar.) When solidified, pour over layer (5 ml VRB).
Incubate at 35C + 10C for 18 - 24 hrs.
Count the dark red colonies, at least 0.5 mm in diameter, and record results as coliforms per g of sample.
Samples of cottage cheese and other acid milk products should be plated within 24 hrs. after manufacture
because coliform counts decline under acid conditions. Coliforms also decrease in number during aging of
ripened cheese varieties.
It must be emphasized that this method provides a presumptive count only. If presumptive counts are
consistently high, colonies should be confirmed (see Standard Methods). The Canadian Food And Drug Act
and Regulations permit 500 coliforms/g of cheese made from pasteurized milk and 5,000 coliforms/g of
cheese made from unpasteurized milk. Permitted counts of Eshericia coli are 100 and 500/g respectively.
Staphylococci
Procedure A
The method described here enumerates total Staphylococci by surface plating on Baird-Parker media. A
coagulase test can be used to determine if individual colonies are S. aureus. Canadian Food And Drug Act and
Regulations permit up to 100 coagulase positive S. aureus in pasteurized milk cheese and up to 1,000/g in
cheese made from unpasteurized milk.
Equipment and Materials
Plating equipment
Glass spreaders (hockey stick-shaped glass rods)
Incubator set at 37C
Baird-Parker Agar
Procedure
1. Pour plates of B.P. agar (15 ml/plate) and dry surfaces (using sterile laminar airflow cabinet -- 2 hrs).
2. Pipette 0.1 ml of homogenate and of subsequent dilutions onto surface of agar and spread evenly with a
sterile bent glass rod until surface appears dry. Prepare duplicate plates. Use 10-1, 1--2, and 1--3dilutions.
3. Incubate at 37C for 48 hrs.
4. Count the number of colonies in each of the following groups:
1. convex, shiny, black, with or without narrow gray-white margin, surrounded by clear zone
extending into opaque medium.
2. convex, shiny, black, with or without narrow gray-white margin, surrounded by clear zone
extending into the opaque medium with an inner opaque zone.
3. convex, shiny, black, with or without narrow gray-white margin, >1 mm in diameter.
Procedure B
Pipette 1 ml or 0.1 ml of homogenate and of subsequent dilutions into petri dish.
Add approximately 10 ml Baird-Parker medium. Mix well. Let stand on bench.
When solidified, invert and put in incubator at 37C (for 48 hours).
Read the same as Procedure A.
Note: Add 5 ml of well mixed Ey Tellurite, enrichment, at 5C to Baird-Parker agar prior to pouring plates.
Figure 3.1. Culture Activity Test

This section is adapted from two reports prepared by: Mark Mitchell (1995), Ontario Ministry of Agriculture,
Food and Rural Affairs, Guelph, Ontario
Figure 3.1. Culture activity test: example.
Conditions
Culture Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. cremoris
Temperature 37 C
Inoculum 2% of mother culture prepared with 10% reconstituted skim milk powder.
Test media 1 10% skim milk powder, low heat, antibiotic free.
Test media 2 Same as one with 1% cheese whey.
Results
Titratable acidity after 4 hours:
Treatment 1 0.34%
Treatment 2 0.25%
pH versus time
Time 0 1 2 3 4 5 6 7 8 9
Skim powder 6.62 6.59 6.5 6.4 6.15 5.74 5.39 5.08 4.92 4.87
Skim with whey 6.61 6.57 6.5 6.42 6.35 6.31 6.3 6.3 6.29 6.29
Interpretation
1. Test media 1 shows normal growth. 0.34% acidity after 4 h with a 2% inoculum is adequate for most types
of cheese. pH versus time plot is typical, reaching pH 5.2 between 6 and 7 hours.
2. Test media 2, containing cheese whey, shows inadequate acid development, indicating the probable
presence of bacteriophage in the cheese plant.
Section C: Milk
Raw milk quality
4.1 The Principal Milk Components
See also Dairy Chemistry and Physics in the Dairy Science and Technology Education website.
Cheese can be made from the milk of many mammals including goats, sheep, buffalo, reindeer, camel, llama,
zebra and yak. The milk of ruminants is the best milk for cheese making because it contains high levels of the
milk protein casein which is required to provide an adequate coagulum. Our consideration of milk composition
will include only a summary of the proximate analyses of the most common dairy species and a few, relevant
with respect to cheese making, comments about each component.
Proximate Analysis
Gross composition of food (also referred to as proximate analysis) means distribution of the total amounts of
fats, proteins, carbohydrates, ash (mainly minerals such as calcium) and moisture or total solids. Typical
proximate analysis profiles for cows', sheeps' and goats' milk are listed in Table 4.1. Further discussion refers
only to cows' milk unless otherwise stated.
Milk fat
Fat content ranges from 2.0 to 7.0 kg/hl. An approximate average for regions where Holstein Fresian cattle
predominate is about 3.9 kg/hl. With respect to cheese manufacture and quality the following properties are
important.
Most diverse of all natural fats. We are routinely quantifying over 100 fatty acids ranging from four
carbons to 22 carbons in length. Many more have been identified.
Traditional, but not entirely justified, nutritional concerns are: (1) About 70% of milk fatty acids are
saturated; and (2) It contains cholesterol.
Positive nutritional factors are butyric acid (anticarcinogenic), saturated but mid to short length fatty acids
(antihypertensive), and rumenic acid (anticarcinogenic).
Unique flavour of dairy fat is due to short chain fatty acids, especially butyric acid
The two major spoilage reactions in milk fat are (1) break down of the triglyceride fat structure releasing
fatty acids such as butyric to create a rancid flavor; and (2) Oxidation of unsaturated fats creating an
oxidized flavour (flat, cardboard flavour)
The melting properties of butter fat are significant to cheese texture.
Milk Proteins
Total milk protein ranges from about 2.5 to 5.5 kg/hl. The average for regions in which Holstein Fresians
predominate is about 3.3 kg/hl. There are two major groups of proteins, the caseins (about 2.6 kg/hl) which I
refer to as the 'cheese proteins' and the whey proteins (about 0.7 kg/hl) which as the name suggests are usually
lost in the whey during cheese making. Caseins are not water soluble and so the cow packages them in water
dispersable particles called micelles which along with caseins include most of the milk calcium, magnesium,
phosphate and citrate (more about casein micelles in Coagulation. Unlike whey proteins which are very sensitive
to heat, caseins are little affected by heating except that they react with heat denatured whey proteins. Table
4.2 lists the principal caseins and some properties which are most relevant to cheese making. Similarly, Table
4.3 lists some properties of the principal whey proteins.
Factors affecting gross milk composition

Species
(see also Table 1)
Cheese making principles are similar for milk of all species with some modifications required to account for
high solids of some species such as buffalo and sheep. Cows' milk and goat's milk have similar cheese making
properties except that:
Goats' milk cheese tends to ripen by lipolysis (fat breakdown) more than cows' milk cheese.
Goats' milk has smaller fat globules which allows higher fat recovery and possibly a smoother texture..
Cheese making quality of goats' milk is also improved due to higher levels of alpha-S1 casein in goats'
milk which permit better coagulation.
Genetics
Through out the modern history of dairying, farmers have selectively bred dairy cattle to increase production or
fat content or both. Recently, genetic selection has focussed on other milk properties such as increasing the
proportion of milk protein to fat. Three genetic effects are most relevant to cheese making.
(1) Relative proportions of fat and protein (P/F ratio)
Fat content and protein content generally increase or decrease in parallel, but fat varies more with feed and
season then protein. The same is true for breed (genetic) effects, such that genetic selection has produced the the
following practical effects in modern dairying.
Higher fat breeds have lower protein/fat ratio. For example, a typical protein/fat ratio in Jersey milk is 0.7
relative to 0.84 in Holstein milk (Table 4.4).
Genetic selection over the last 100 years has produced considerable within breed improvement with
respect to both milk production and increased protein and fat content. The result is greatly increased per
cow production of milk protein and fat. However, once again, because fat responds more to genetic
selection than protein, the result in many areas has been a gradual decrease in the average P/F ratio. For
example in Ontario, the P/F ratio decreased from about 0.88 in 1970 to 0.85 in 1995 (estimated from data
provided by Laboratory Services Division, University of Guelph.
(2) Relative proportions of fat and protein to other solids
With respect to other solids, mineral content (mainly Ca, Mg, and P) generally varies in proportion to protein
content and lactose content is relatively stable. Because lactose is largely a wasted component, increasing
protein and fat by feed or genetic selection has economic advantages in terms of feed conversion, milk
transportation costs, and waste handling.
(3) Stage of lactation
Fat content tends to increase during lactation as milk production decreases. The result is that the relative
proportion of protein to fat (protein/fat ratio or P/F) is highest at the peak of lactation (about 60 days of
lactation) and lowest at the end of lactation. Protein distribution also changes during lactation with resulting
effects on cheese ripening and flavour. In particular, the proportion of alpha-caseins decrease during lactation
while the proportion of beta-casein increases.
Feed
Depending on the relative demand
for butter fat versus milk non-fat
solids, there may be incentive to
change the relative proportions of
milk protein and fat. The only
short term means to do this is by
changing the diet. Generally less
roughage and more high energy
feeds will encourage lower fat
content with little decrease in
protein content to provide a higher
P/F ratio.
Season
Seasonal variation in milk

composition is most important to
cheese yield efficiency and
composition control. Some
important seasonal effects are listed below and illustrated to the right. These observations are based on Ontario
data.
Fat content reaches a minimum in August and a maximum in October.

Protein content changes roughly in parallel with fat content, but the seasonal variations are smaller,
causing high protein fat ratios (P/F) during the summer and low P/F ratios in the winter.
Casein content also varies with season which is most important because cheese making is dependent on
casein NOT on total protein (more on this in Treatment of milk for cheese making, Standardization of
milk for cheese making and Yield efficiency).
Milk as a growth medium

Cheese making depends on the growth of bacteria to produce acidity, flavour compounds, and ripening enzymes.
It is, therefore, important to understand the characteristics of milk as a growth medium.
General Nutrients
Milk is a good source of all principal nutrients, including carbon, nitrogen and macro-minerals. Many
micronutrients such as vitamins and micro-minerals are also available. However, milk is unique with respect to
its sugar.
Milk Sugar
Carbohydrates, especially simple sugars such as sucrose (table sugar), can be utilized as sources of energy more
quickly than fats and proteins. However, the energy currency of the cell is glucose (also called dextrose) so to
use available carbohydrates, microorganisms must be able to convert them to glucose.
The only sugar naturally present is milk is lactose. Most microorganisms lack the enzyme lactase which is
required to break lactose into its two component sugars, namely, glucose and galactose. Lactic acid bacteria
which do have lactase readily break down lactose and use glucose as an energy source. Lactic acid bacteria,
therefore, have a competitive advantage in milk; that is, they are able to out grow other bacteria which are
unable to obtain glucose from lactose. Further, some lactic acid bacteria are able to convert galactose to glucose.
Acidity (pH)
Acidity as measured by pH is one of the most critical parameters with respect to both food safety and both
process and quality control of fermented foods such as cheese. The concepts of acidity and pH are explained in
Sections 3.5. The titratable acidity of milk typically varies from 0.12 to 0.19% lactic acid depending on
composition, especially protein content. The pH of milk is near the physiological pH of 6.8 which, considering
the following points, means that milk is a good growth medium with respect to acidity (pH).
Most organisms grow best at pH near physiological pH of 6.8. As explained in Section 3.5, titratable
acidity (TA) is not a good predictor of acid effects on microbial growth and chemical properties such as
protein functionality.
The major groups of microorganisms important to food preservation are in order of increasing acid
tolerance: bacteria, yeasts and moulds.
Natural fermentation of warm raw milk by lactic acid bacteria reduces milk pH to less than 4.0 which
prevents the growth of pathogenic bacteria and most spoilage bacteria
Moisture
Milk has a high moisture content (typically 87% for cows' milk) and with respect to available moisture, is an
excellent growth medium. But, it must be understood that with respect to microbial growth, the critical
parameter is water activity not moisture content. Water activity (aw) is an index of the availability of water for
microbial growth. It is the availability of water in the food reported as a fraction of the availability of water from
pure water. In other words, the aw of water is 1 and the aw of other substances is reported as decimal fractions of
1. Water activity is reduced by dissolved substances, varying directly with number of dissolved molecules rather
than the weight. For this reason, relative to large molecules such as proteins, small molecules such as sugar and
salt have a large effect on water activity. For example, jams are preserved by their high sugar content.
Microorganisms vary greatly in their ability to survive and/or grow at reduced water activity. However,
acknowledging that exceptions exist, the minimum water activity for the principal groups of microorganisms are
as follows:
Most bacteria: 0.90 - 0.91

Most yeast: 0.87 - 0.94

Most moulds: 0.70 - 0.80
Compare these values with typical aw values for milk, cheese and a few other foods.
Milk, fresh fruits and vegetables, fresh meats 60 - 98% moisture, aw 0.97 - 1.00
Most baked products, some cheese, some cured meats, 20-60% moisture, aw 0.88 - 0.96.
Dehydrated foods such as breakfast cereals. Less than 5% moisture, aw 0.20 - 0.30
Typical aw values for some cheese at the marketing stage are given below (Eck and Gillis, 2000). See also
typical aw values for cheese families in Table 1.1.
Availability of oxygen
Cottage 0.988
Brie 0.980
Munster 0.977
Saint-Paulin 0.968
Edam 0.960
Cheddar 0.950
Parmesan 0.917
Availability of oxygen
With respect to oxygen requirements, microorganisms may be:
Aerobic: must have oxygen to grow

Anaerobic: can only grow in the absence of oxygen
Microaerophilic: require small amounts of oxygen
Able to grow with or with out oxygen.
Moulds require oxygen, so they can be eliminated by vacuum or gas flush packaging. Most yeast are aerobic
(require oxygen) but some can grow anaerobically (in the absence of oxygen). Bacteria may fall into any of
these categories, but lactic acid bacteria are micoaerophilic or anaerobic.
Milk will acquire some dissolved oxygen during milking, storage and handling, but it is used up quickly during
bacterial growth.
Types of microorganisms and their activity in milk

The numbered list below identifies seven types of bacteria according to how they change the properties of milk.
Often these changes are negative (spoilage) but as we will see in later sections, many of these bacteria are
important to the development of cheese flavour. Before proceeding to the list, please note the following
definitions:
Psychrotrophic refers to microorganisms which are able to grow at temperatures less than 7C. Cold milk
storage and transport selects for psychrotrophic bacteria which are often proteolytic and lipolytic.
Common psychrotrophic bacteria in milk are species of Micrococci, Bacilli, Staphyloccoci, Lactobacilli,
Pseudomonas, and coliforms. Pseudomonas species are the most common and typically have the most
impact on quality. At temperatures of 2 - 4C, bacterial growth in milk is mainly due to strains
of Pseudomonas flourescens. Little growth occurs at temperature less than 2C.
Spore forming bacteria are able to exist in a highly stable form called 'spores'. In the spore state, these
bacteria are able to withstand greater extremes of acidity, temperature and desiccation.
Enzymes are biological catalysts that accelerate the rates of biochemical reactions. Bacterial enzymes are
most significant to milk spoilage and cheese ripening but it is important to distinguish between the
enzyme and the bacterial source. For example, many psychrotrophic bacteria produce heat stable enzymes
which remain active in milk and cheese even after the bacteria are killed by pasteurization.
Keeping the above definitions in mind, note the following types of microorganisms, grouped according to their
impact on milk quality.
(1) Lactic acid bacteria which ferment lactose to lactic acid and other end products. Lactic acid bacteria (LAB)
important to cheese making will be described further in Chacultures7. For now note the following:
As noted earlier, LAB are able to readily metabolize lactose so they have some competitive advantage
over other microorganisms.
Notwithstanding, their ability to metabolize lactose, LAB prefer temperatures greater than 30C, so,
depending on initial relative counts, psychrotrophic bacteria including some coliform and pseudomonas
bacteria are able to outgrow LAB at room temperature.
(2) Proteolytic bacteria which degrade protein and cause bitterness and putrefaction. Most important in cheese
milk are species of:
Pseudomonas which are psychrotrophic and produce heat stable lipases.

Bacillus which form heat stable spores and survive pasteurization
(3) Lipolytic bacteria which degrade fats and produce lipolytic rancidity. Again, the most common example in
milk is the genus Pseudomonas. Several psychrotrophic species of Pseudomonas produce heat stable lipases as
well as proteases.
(4) Gas producing microorganisms which cause cheese openness, floating curd in cottage cheese, and gassy
milk.
Yeasts are always present in milk and are common contaminants during the cheese making process. They
may cause 'yeast slits' in cheese and contribute to ripening of surface ripened cheese.
Coliform bacteria are always present in milk but their numbers can be minimized by good sanitation.
Also, coliform bacteria compete poorly with lactic acid bacteria, so their numbers rapidly decrease in the
presence of a rapidly growing lactic acid culture.
Clostridium tyrobutyricum is a thermoduric (survives pasteurization) spore forming organism of legendary
fame among cheese makers. C. tyrobutyricum causes gas formation (carbon dioxide) during the later
stages of ripening of Swiss and Dutch type cheeses. The resulting craters and cracks in the cheese are
called 'late gas defect'. European cheese makers frequently check raw milk for thermoduric and/or spore
forming bacteria to estimate potential for late gas defects. Five hundred spores per litre of milk are
sufficient to cause late gas defect.
Propioni bacterium produces the desirable gas formation in Swiss type cheese.
Some lactic cultures, called heterofermentative, also produce carbon dioxide. See Cultures.
(5) Ropy bacteria cause stringy milk due to excretion of gummy polysaccharides. Usually ropy bacteria such
as Alcaligenes viscolactis are undesirable. However, in some fermented dairy products, ropy lactic acid bacteria
such as certain subspecies of Lactococcus lactis are used to develop texture.
(6) Sweet curdling bacteria produce rennet-like enzymes which may coagulate milk. Common examples are the
psychrotrophic spore formers Bacillus subtilis and Bacillus cereus.
(7) Numerous off flavours have been associated with specific milk contaminates. Some examples are:
Malty: S. lactis var maltigenes

Bitter: see (2) Proteolytic bacteria
Rancid: see (3) Lipolytic bacteria

Unclean: coliform bacteria
Fishy: Pseudomonas
Fruity: Pseudomonas
Pathogenic Bacteria
This short course makes no attempt to provide comprehensive training on food safety with respect to cheese
manufacture. However, some food safety principles will be discussed in the context of other topics, for example,
acid control and food plant sanitation. Here, we mention only some characteristics of a few pathogens which are
particularly significant to cheese making. We begin with definitions to distinguish between food infection and
food intoxication.
Food infections are caused by organisms which grow in the gastro intestinal track. Illness occurs after ingestion
of an infectious dose which depends on many factors including the health status of the person.
Food intoxication results from toxins produced by bacteria. Toxins may be present within the bacteria
(endotoxin) or excreted outside the bacteria (exotoxin). The organism need not be alive or even present to cause
illness. A good example, is Staphylococcus aureus. Like all the other pathogenic bacteria listed
below, Staphylococcus aureus is destroyed by pasteurization but its enterotoxin survives pasteurization.
Pathogens: common before 1940

Corynebacterium diptheriae: causes diphtheria
Brucella abortus: causes brucellosis in cows and undulant fever in people.
Mycobacterium tuberculosis: causes TB in people.
Coxiella burneti: causes Q fever in people.
Pathogens which emerged during 1940 - 1970
Staphylococcus aureus: food intoxication caused by heat stable enterotoxins of which enterotoxin A is the
most common.
Salmonella species: Salmonellosis is an infection caused by many species and strains of species
of Salmonella. Salmonellosis is of great concern to the dairy industry, especially the cheese and milk
powder sectors. Infectious doses can be extremely low, perhaps as low as a single organism.
Enteropathogenic E. Coli produce enterotoxins, some of which are heat stable. Of numerous species and
strains, the most famous is E. Coli 0157 H7, which occurs frequently in raw milk. E. Coli0157 H7 is of
particular concern because it is quite acid tolerant and is able to grow at refrigeration temperatures.
Recent Pathogens
Yersina enterocolitica is a psychrotrophic infectious agent.

Campylobacter jejuni, is an infectious agent which has passed Salmonella as the leading cause of
diarrhoea all over the world.
Listeria monocytogenes is a psychrotrophic infectious agent, which requires special caution because it is
acid tolerant and more heat stable than most pathogens, although it does not survive proper pasteurisation.
Bacillus cereus is mainly important as spoilage agent. However, some strains are mildly pathogenic which
is problematic because Bacillus cereus forms heat stable spores which survive pasteurization and are able
to grow at refrigeration temperatures.
Antibiotics
Lactic cultures are very sensitive to antibiotics. In most jurisdictions increasing penalties have greatly reduced
antibiotic residues in milk. Nevertheless, antibiotic testing of all cheese milk is still recommended. See rapid
screening tests in Section 3.10.
Mastitic Milk
Mastitis is an infection of the udder which negatively impacts milk quality. Pooling milk dilutes the effect of
single infected cows and herds but in most jurisdictions the cumulative effect of mastitis, especially subclinical
mastitis, is significant. Olson as cited in Eck and Gillis (2000) estimates a cheese yield loss of 1% if 10% of the
milk is from cows with subclinical mastitis. Further, as noted below, the quality effects of mastitic milk are
probably of more economic importance than the yield effects.
Causative organisms include human pathogens such as E. coli and Staphylococcus aureus. Nonbacterial
infections such as prototheca infection also cause high SCC. Based on new automated procedures for bacteria
counting, Ontario producer milk data suggests that prototheca is a common mastitic agent and frequently
contributes to high SCC and bacterial counts.
Typical Ranges of Somatic Cells

Somatic cells include any type of 'body' cell in the milk, such as skin cells (epithelial) from the cows' udders and
leucocytes of several types. Leucocytes are white blood cells which are part of the cow's immune response to
infection in the udder, so they are used as an index of mastitis or udder infection. Several observations are
relevant:
The milk of healthy cows should contain less than 100,000 somatic cells per ml of milk. Higher counts
indicate subclinical mastitis (infection in the udder).
Clinical mastitis is associated with counts greater than 1,000,0000/ml
Producer milks in Ontario average about 200,000 cells/ml but counts less than 100,000 can readily be
achieved with good herd management
Critical Ranges With Respect to Milk Quality
There is evidence that counts as low as 100,000 cells/ml affect cheese yield (Barbano et al, 1991, J. Dairy Sci.
74:369) and the quality of other dairy products such as ultra-high temperature milk. SCC in the range of 250,000
- 500,000 are associated with altered milk composition and decreased cheese yield. When counts exceed
1,000,000 cells/ml, altered milk composition and reduced cheese yield, are obvious.
Composition Effects
Gross composition effects of udder infection are not significant for SCC less than about 250,000/ml. Above that
level the following trends are observed:
Little change in fat content

Increased mineral content, particularly more cloride.
Decreased lactose content which balances the osmotic effects of increased mineral content.
Protein effects:
Less casein
More whey proteins, especially immunoglobulins
More nonprotein nitrogen
Increased pH (up to 7.5 whereas 6.7 is normal)
Bacteriological Properties:
High SCC are normally associated with shedding of pathogenic (to humans) bacteria in the milk including E.
coli, S. aureus and others. Basically, whatever organism is causing the udder infection, including the algae,
prototheca will be present in the milk. Further, growth factors present in high SCC milk encourage growth of
both E. coli and S. aureus (Amer. J. Vet. Res. 45:2504). The growth rates of some lactic cultures are also
affected; Streptococcus thermophilis grows faster and Lactobacillus acidophilus is inhibited.
Significance To Cheese Milk
Cheese yield is affected in two ways:
Mastitic milk contains more plasmin, a heat stable milk protease which degrades protein and causes more
protein to be lost in the whey.
Reduced casein directly affects cheese yield.
Poor curd formation (longer flocculation time, slower rate of curd firming, and reduced maximum
firmness) contributes to yield loss as fines.
Perhaps more important than yield are the effects of subclinical mastitis on cheese quality (J. Dairy Res. 53:645).
Modest levels of SCC cause several quality problems:
Decreased curd strength due to high whey proteins, low caseins, high pH and altered calcium-phosphate-
caseinate balance. As noted above, these changes affect cheese yield, but they also impact quality.
Higher moisture cheese due to impaired curd syneresis.
Soft, less elastic, sticky and grainy cheese texture.
Increased flavour intensity, usually with off flavours
Significance to Fluid Milk
Very high counts (>2 million) will cause milk to taste salty and result in many quality problems. Lower counts,
even as low as 300,000 can increase development of bitter flavour due to increased levels of plasmin. This is a
particular problem with ultra-high-temperature processed milk because the enzyme is heat stable and the storage
time is long enough to permit significant protein degradation.
Raw Milk quality tests

The following list is a summary of the most important raw milk quality tests. Procedures for some milk quality
tests are described in Process and quality control procedures.
1. Organoleptic
2. Total plate counts: good < 3,000/ml; maximum raw milk 100,000
3. Coliforms: good < 10/ml; concern > 25; max 100
4. Psychrotrophes (grow at T < 7C): good < 1,000
5. Somatic cell counts: good <100,000; concern >300,000;
6. Rapid test for inhibitors
7. Disk assay (official test for inhibitors)
8. Added water: maximum freezing point -0.505C (-525H)
9. Composition: fat, protein, lactose, total solids, and casein if possible.
Treatment of milk for cheese making

5.1 Clarification
See also Clarification, Separation and Standardization in the Dairy Science and Technology Education website.
Clarification may be as simple as filtering out debris or may include standardization of micro flora by removing
microbial cells and spores. The principal clarification/standardization procedures are as follows.
(1) Cloth filters are common to remove debris at the farm but should not be necessary at the processing plant.
(2) Centrifugal clarifiers, medium speed centrifuges, remove particles which escape filtration. Cream separators
effectively double as centrifugal clarifiers because small particles of debris collect at the periphery of the
separator bowl and are ejected as sludge. The loss of milk solids by this process is minimal.
(3) Bactofugation is a high speed centrifugal process which separates bacterial cells and spores. This process is
particularly important in Europe where problems arise due to spore formers such asClostridium tyrobutyricum.
Bactofugation removes 95%

of the spores of milk which
means the risk of late gas
defect due to germination
and growth of Clostridium
tyrobutyricum is much
reduced but not eliminated.
1-2% of milk solids is
transferred to the
bactofugate which, in
particular contains casein
along with somatic cells and
bacteria. To avoid yield loss
the bactofugate which
contains 12-16% of dry
matter, is sterilized by
ultrahigh temperature
processing and added back
to the milk.
(4) Microfiltration is a membrane

process which has been used in a few European cheese plants since 1985. Think of microfiltration as an ultrafine
sieve. Microfiltration and related membrane processes are illustrated in Figures on the right and below and are
further described in Cheese making from ultra filtered milk. Microfiltration achieves about 99% reduction of
spore forming bacteria relative to 95% by bactofugation. The disadvantage is that microfiltration can be applied
only to skim milk because the milk fat globules are too large to pass through the microfiltration membrane
(See the figure below).
Standardization of cheese milk composition

In addition to standardization of microflora, it is normally necessary to adjust milk fat or protein or both. The
objective of milk composition standardization is to obtain the maximum economic return from the milk
components. In practice, this means that milk composition is adjusted to achieve the most economically
favourable balance of the cost of ingredients and the percent transfer of milk solid components to cheese while
maintaining cheese quality.
Cheese yield is mainly determined by the recoveries of protein and fat in the cheese (that is the percent of fat and
protein transferred from milk to cheese) and by cheese moisture, but other components also contribute
significantly. Cheese yield is discussed in Yield efficiency. Standardization of milk for cheese making is a
detailed practical guide to milk standardization, including the necessary calculations for manual standardization.
Here we summarize general considerations on milk standardization.
Government standardized cheese varieties

Food regulatory agencies in many jurisdictions have mandated standardized foods for which specific criteria
with respect to composition and/or quality must be met. Section 28 Table Part 1, Canada Agricultural Products
Act and Regulations lists maximum moisture and minimum fat levels (percent by weight) for 46 cheese
varieties. No other composition or quality standards are prescribed, so, the identities of cheese varieties are not
protected. For example, American mozzarella is NOT pasta filata cheese like Italian stretch mozzarella, but it is
mozzarella according to Canadian regulations.
Cheese fat on a dry matter basis
Table 6.1 includes data for target fat and moisture content according to the respective minimum and maximum
values as prescribed by the Canada Agricultural Products Act. It also includes a column for fat in the dry matter
(FDM) which is the target cheese fat content reported as a percentage of the target total solids content, where
total solids is calculated as 100 minus the target moisture content. Because the principal nonfat component in
cheese is casein, the target FDM value is useful to estimate the proportions of fat and protein required in the
cheese milk. For example cheese makers generally consider a full fat cheese contains 50% FDM which
corresponds to a protein fat ratio in the cheese milk of 0.94 - 0.96. By this criteria, both Cheddar and Feta are
full fat cheese because they both contain about 50% FDM, although on a wet basis their respective fat contents
are 31 and 22%.
Protein/fat ratios (P/F)

P/F (ratio of protein to fat) is exactly what the name implies. Having no units, it is an index of the relative
proportions of fat and protein in the milk. Please be clear that the P/F value indicates nothing about the absolute
value of fat and protein. P/F ratio is generally lower in low fat milk and higher in high fat milk, so that Jersey
milk, for example, has a less favourable P/F for cheese making than Holstein milk. This is partially offset by a
higher casein number (casein as a percentage of total protein) in Jersey milk.
Standardizing to target protein/fat ratios
Standardization normally means adding skim milk or skim milk solids, or removing cream to increase the ratio
of protein to fat (P/F). Several practical points are relevant.
Multiple component pricing makes it possible to cost milk components as individual ingredients. P/F can
then be optimized according to relative costs of protein and fat, transfer rates of protein and fat from milk
to cheese, and the value of fat in the cheese relative to its value as cream.
Component yield economies must be balanced against cheese quality.
Calculation of P/F to produce cheese with required moisture and fat depends on retention of fat, casein
and serum solids in the cheese, where serum solids refers to recovery of the soluble components of milk,
namely, sugars, whey proteins, nonprotein nitrogen and some minerals. Specifically the important
principles with respect to serum solids are:
Higher serum solids recovery means that a lower P/F is required (that is more fat or less protein) in
the cheese milk to achieve the target FDM in the cheese.
Serum solids recovery is increased in high moisture cheese because the moisture retained includes
dissolved solids.
Serum solids recovery is reduced by curd washing treatments.
Serum protein (whey protein) recovery is increased by milk pasteurization (there is more discussion
on heat treatments in the next section).
Standardizing to casein/fat ratios

Better process and composition control can be achieved by standardizing to fixed casein/fat ratios rather than
protein/fat ratios. This requires accurate casein measurement which is still not feasible for most plants. See
further discussion in Standardization of milk for cheese making and Yield efficiency.
Sources of milk proteins
Standardization usually requires the addition of protein or removal of fat. The former has the advantage that it is
possible to produce cheese quantities beyond what's possible from the available fresh milk. This is significant in
areas where fresh milk is in short supply or as in Canada, where milk purchases are limited by quotas. Several
sources of milk proteins are available for cheese milk standardization.
(1) Skim milk powder is convenient for small or remote cheese plants. It can be used effectively with the
following limitations:
Use only certified LOW HEAT (Whey Protein Index > 6) and antibiotic free powders.
Reconstitute the powder thoroughly and filter to remove undissolved particles before blending with the
cheese milk. Incomplete solubilization may cause over set Swiss cheese and poor stretching of pasta filata
cheese.
Nonfat solids of cheese milk should not be raised above about 11% (normal level is 9%). This can be
avoided by adding more water with the powder.
Protein from skim milk powder is usually more expensive than from other sources.
(2) Skim milk and condensed milk are convenient sources because they can be handled and measured in liquid
form. The only cautions are to limit heat treatment to minimum pasteurization requirements and limit nonfat
milk solids to less than 11 kg/100 kg. Again, nonfat solids can be adjusted by adding water.
(3) Culture media contribute nonfat milk solids which must be accounted for in calculations for milk
standardization. For example, the high heat treatment involved in bulk culture preparation ensures that most milk
proteins (including whey proteins) present in the culture will be transferred to the cheese.
(4) Protein concentrates and isolates available to supplement cheese milk are numerous. A few are listed below.
The feasibility of using one or more of these products, depends on, among other things, the type of cheese. For
example, relative to most other varieties, high levels of whey proteins can be used in Feta cheese without
compromising quality.
Liquid or dried milk concentrates prepared by ultra filtration of skim milk contain caseins and whey
proteins in the normal proportions found in milk.
Specially prepared blends of caseins and whey proteins.
Liquid or dried casein concentrates prepared by microfiltration of skim milk.
Liquid concentrates of denatured whey proteins.
Sources of milk fat

Most jurisdictions prohibit the use of non dairy fat in cheese. That leaves a number of choices:
Milk and cream unaltered other than by pasteurization and gravity or centrifugal creaming.
Recombined cream prepared from skim milk and butter oil. This process requires homogenization which
is generally considered undesirable with some exceptions including Feta, Blue and cream cheese.
According to some recent work quality problems associated with homogenization can be eliminated by
homogenizing the cream rather than the milk. Homogenization of cheese milk is further discussed in
Section 5.4 below.
In cases where nondairy cream is desirable, the limitations are:
Altered flavour, especially the absence of short chain fatty acids such as butyric which are only found in
dairy fat. The flavour problem can be addressed by dairy flavour additives.
Preparation of filled cheese milk (filled means containing fat other than dairy fat) requires
homogenization, which as noted normally creates inferior texture.
The fat should have melting properties similar to butter fat.
Manual standardization
In the absence of online systems equipped with customized algorithms, it is necessary to create spread sheets to
calculate milk formulae and monitor yield parameters. The first step is to determine the optimum P/F, a process
that always involves some experimentation. The estimates given in Table 6.1 can be used for a first
approximation and then adjustments can be made on succeeding days based on the cheese analysis. This
emphasizes the need for consistent and accurate records of milk and cheese composition and manufacturing
parameters.
Detailed procedures, including calculations, for manual standardization are described in Standardization of milk
for cheese making.
Automated standardization
Automated composition control systems separate warm milk into cream and skim and then automatically and
continuously recombine the two streams in the proportion required to obtain the desired P/F ratio. The
standardized milk is tempered to the correct setting temperature and delivered directly to the setting vats. Two
general types of control are possible.
Fully automated using online milk analysers based on near infra red or light scattering technology.
Partially automated control where composition is monitored with an in line density metre which is
calibrated using an off line milk analyser.
Recombined Milk
Considering the limitations described above for protein and fat sources, it is possible to manufacture cheese from
recombined milk.
Failure to achieve optimum standardization for maximum yield efficiency is a major cause of economic loss in
many cheese plants.
Heat treatments
See also Pasteurization in the Dairy Science and Technology Education website.
Many people assume that all dairy products in Canada, including cheese, are made from pasteurized milk. Not
so; several alternatives are possible as outlined below. Note, however, that the Food and Drugs Act and
Regulations, recognizes only two types of cheese with respect to milk heat treatment, namely, fully pasteurized
milk and raw milk. That is, if the milk is not fully pasteurized the resulting cheese is considered raw milk
cheese.
1. No heat treatment results in raw milk cheese which has more flavour. Raw milk cheese by law must be "held
at 20C or more for a period of 60 days or more from the date of the beginning of the manufacturing process, "
Food And Drugs Act And Regulations, Sections B.08.030 and B.08.043. The question of raw milk cheese is an
ongoing concern to consumer groups and to health authorities. Suffice it to say that with respect to regulations
on cheese milk heat treatments, 'one size doesn't fit all'.
2. Thermisation (63-65C, short hold) results in phosphatase positive milk which must be fully pasteurized before
cheese making. The purpose is to prevent raw milk spoilage (eg. over a weekend) due to acid or protease
producing bacteria.
3. Pasteurization (63C, 30 min. or 72C, 16 s) is generally considered the safest alternative, but the full flavour of
traditional ripened cheese can not be achieved. Note that over pasteurization causes denaturation of whey
proteins which subsequently adsorb to the casein particles. The effects are:
Longer flocculation times

Weak or no curd formation
Excessive loss of fines
Poor syneresis (moisture release)
Coarse textured curd with reduced ability to stretch, mat and melt.
4. Heat treat (55 - 65C, 16 s) is trade lingo for subpasteurization treatment which is applied to destroy most
pathogens but allow some bacteria to survive and contribute to cheese ripening. This process permits fuller
flavour of cheese with better control of culture growth (i.e., acid development) than with raw milk. For current
regulatory purposes, heat treat is equivalent to raw. Most aged Canadian Cheddar is safely made from heat
treated milk
Homogenization
See also Homogenization in the Dairy Science and Technology Education website.
The process of homogenization reduces milk fat globule sizes from 1 - 15 micrometer to less than 2 micrometer
(a micometer is 0.000,0001 m). The natural membrane on the fat globule is replaced by milk proteins, mainly
caseins. This results in increased interaction between fat globules and the casein particles in the rennet gel. For
some cheese homogenization is desirable:
Homogenization promotes lipolysis, whitening, and flavour development in cheese made from cows' milk
but which are traditionally made from goats' or sheep's milk, e.g., Blue and Feta.
Homogenization increases fat recovery and creates smoother texture in cream cheese.
With respect to most firm to hard ripened cheese, many workers have observed that cheese made from
homogenized milk are too tough and firm after pressing. However, there is evidence that homogenization for
Cheddar cheese making has some advantages if medium pressure (6.9 MPa) is used and if only cream (35% fat)
is homogenized and subsequently blended with unhomogenized skim milk (Nair et al. 2001, Int. Dairy
Journal 10:647).
Increased rate of gel firming and higher curd firmness at cutting.

Increased cheese yield due to greater moisture retention and improved fat and protein recovery.
Notwithstanding higher moisture content, cheese texture and flavour was not decreased by
homogenization.
Additives to Cheese milk

(1) Calcium Chloride is frequently added at a level of about 0.02% to aid coagulation and reduce amount of
rennet required, especially if milk is set immediately after pasteurization. The role of calcium in milk
coagulation will be discussed in Coagulation.
(2) Nitrates (sodium or potassium nitrate) be added at levels of about 200 ppm to Edam, Gouda, Swiss to inhibit
growth of gas forming Clostridium tyrobutyricum.
(3) Annatto cheese color is added to some cheese to standardize seasonal changes in color or to create orange
cheese such as Cheddar and Cheshire.
The following are some facts about annatto.
Annatto is a carotenoid similar to -carotene and Vitamin A in structure, but it has no Vitamin A activity.
Annatto color is red to yellow pigment but it usually appears as orange. The red constituent is more
apparent with decreasing pH (6-4.8) changing the orange to pink while at pH < 4.8 the pink fades and
becomes nearly white. This explains the phenomenon of 'acid-cut cheese'.
Bleaching and pinking of annatto is also caused by oxidizing agents such as copper, iron, chlorine and
light.
Oxidation of annatto is also encouraged by heat, so annatto is an unsuitable colorant for process cheese
Alternatives to annatto are:
Beta-carotene which is too yellow and makes the cheese taste like carrots.
Apo-8-carotenal which has the advantage that it is not lost in the whey.
Decolorants
Goats' and sheeps' milk are flat white in color because they lack -carotene. Cows' milk may be whitened to
mimic goats' or sheeps' milk. Chlorophyll based products which mask the natural yellow colour can be used as
whitening agents. Titanium dioxide is an effective whitening agent but is no longer a legal additive for cheese.
(5) Ripening Agents
A wide range of products are available to accelerate cheese ripening or to develop a broader flavour profile.
Relative to traditional cheese varieties, several factors suggest the need for ripening supplements:
Nontraditional cheese making methods

Pasteurized or heat treat versus raw milk
Cows' milk substituted for the milk of other species
Traditional rennet pastes containing a wide range of enzymes including lipases and proteases have
been replaced with purified extracts
Cold storage and transport of milk severely alters natural milk micro flora
Economic pressure to reduce ripening time
Marketing pressure to standardize quality attributes
Lipases (lipolytic enzymes) are traditionally added to cows' milk to produce cheese such as Feta, Romano,
Kefalotyri, and Parmesan which are traditionally made from goats' or sheeps' milk. That's because goats' and
sheep's milk, especially goats' milk, have more natural lipase than cows' milk. Commercial lipases are
commonly extracted from kid goats.
Enzyme Cocktails
Mixtures of enzymes from various sources added to the milk to accelerate ripening of aged cheese such as
Cheddar. These cocktails include both lipases and proteases, with a predominance of proteases for Cheddar.
Bacterial enzyme extracts from lactic acid bacteria have also been used. Accelerated ripening is further
discussed in Ripening and packaging.
Standardization of milk for cheese making

Standardization refers to the practice of adjusting the composition of cheese milk to maximize economic return
from the milk components while maintaining both cheese quality and cheese composition specifications.
Composition specifications may be self imposed (eg., low fat cheese) or imposed by government standards of
identity. In Canada, standards of identity are defined for 46 cheese varieties (Table 6.1). These standards only
include limits for maximum moisture and minimum fat so they do little to standardize other cheese
characteristics. For example, American Mozzarella is made by a different process and has different properties
than Italian stretch Mozzarella, but by Canadian regulations American Mozzarella can be called Mozzarella
provided it contains less than 52% moisture and more than 20% fat.
Important parameters of composition

Standardization of cheese milk normally requires increasing the proportion of protein relative to fat, which can
be done by adding protein or taking away fat. The relative amount of protein and fat in milk is called
the protein-fat ratio or P/F. The P/F is the principal factor which determines the amount of fat in the cheese
relative to other milk solids in the cheese. Because it is easy to measure cheese fat and total solids, the
proportion of fat in the cheese is reported as (1) fat on a wet basis; and (2) the ratio of cheese fat to cheese total
solids. This ratio is called 'fat in the dry matter' or F/DM. The F/DM in cheese is determined mainly by P/F of
the milk but the percent moisture is also important. Because cheese whey contains soluble solids, higher cheese
moisture means that more soluble solids (mostly non-fat solids) are also retained in the cheese so that the ratio of
F/DM decreases. The target value of F/DM in the cheese is used to determine the first approximation of the P/F
required in the milk to give the desired fat content of the cheese. See Table 6.1.
There is a third ratio, namely, casein number (CN), which we will use in the standardization procedures given
below, but which is important to understand. Total protein content of cows' milk is about 3.3Kg/hL of which
about 2.6 /Kg/hL is casein. The remainder is whey protein (about .7 Kg/hL) including about .1 Kg/hL of some
nitrogenous compounds which are not true protein and are referred to collectively as non-protein nitrogen
(NPN). Casein is mostly recovered in cheese (i.e., transferred from milk to cheese during cheese manufacture).
Whey proteins remain soluble in whey so that only small amounts are recovered depending on how much whey
is retained in the cheese. Casein content is, therefore, most relevant to cheese yield, so when cheese makers
standardize milk on the basis of protein content, they are using total protein as an index of casein content. Direct
measurement of casein would be better because the proportion of casein in total protein varies with breed,
season, region and other factors. However, wet chemical analysis of casein is not feasible for most plants and
rapid instrumental methods are still under development.
The percentage proportion of casein in total protein is referred to as the casein number (CN).
Methods of Standardizing
There are three methods of standardizing milk, namely:
1. Addition of concentrated non-fat milk solids (i.e., skim milk powder or condensed skim).
2. Addition of skim milk.
3. Removal of cream.
These methods are based on the assumption that the milk has a high fat content relative to the protein content.
This is normally the case, so that cows' milk usually has excess fat over that required to produce a legal cheese.
The exceptions are high fat cheese such as cream cheese or double cream blue cheese.
It is not always economical to standardize milk. The cheese maker must compare the costs of standardizing with
the extra yield of cheese or cream. Many cheese makers simplistically assume that all they have to do is
standardize milk to meet the official composition standards. But the objective of standardization is to maximize
the total return from all milk components while meeting regulations and without compromising quality. If the
value of butter fat is low relative to protein, it is more economical to sell the fat as cheese rather than as cream
provided that the extra fat can be retained in the cheese without compromising quality.
Units
Raw milk composition for payment purposes is reported in units of Kg of component per hL of milk at 4C. This
is referred to as weight over volume (w/v) measurement. Measurement in units of w/v is dependent on milk
density which in turn is affected by both composition and temperature. Weight over weight (w/w) measurement
(eg., Kg component per 100 Kg of milk) results in a significantly smaller value because the density of milk is
more than 1 Kg/L. Measurement by w/w has the advantages that: (1) most wet chemical reference analyses used
to calibrate milk analysers report composition in units of w/w; (2) w/w values are independent of milk
temperature. However, milk composition for payment purposes is reported in units of w/v because the volume of
milk is easily measured with dip sticks or volumetric meters. Weight measurement would require installation of
farm bulk tanks on expensive load cells. Volume rather than weight measurement of milk and other liquids is
also more convenient in the plant.
In any case, the important point with respect to accurate standardization is to ensure that all measurements and
calculations use the correct units. When component estimates are given as percentages, the basis of measurement
must be stated as w/w percent (eg., Kg fat per 100 Kg milk) or w/v percent (eg., Kg fat/hL of milk). In this
manual composition values given in percent always mean w/w. Cheese composition will always be stated in
percent w/w (eg., 30% fat in Cheddar cheese means 300 g fat per Kg cheese). Similarly, 3.3 % fat in milk means
3.3 Kg fat per 100 Kg milk. If weight over volume units are used I will always state the specific units, eg., 3.3
Kg/hL. Because composition of producer milk is reported to processors in units of Kg/hL and because milk
metering systems are volumetric, I will usually report milk composition in units of Kg/hL.
It is important to ensure that milk analysers are calibrated in the appropriate units and the correct units are
subsequently used for milk standardization calculations and calibration of automated standardizing systems. Wet
chemical analysis is normally done by weight, so reference results for milks used to calibrate milk analysers are
normally reported in units of percent by weight and it is convenient to calibrate milk analysers in percent by
weight (eg., Kg/100 Kg). If required, w/v values can be estimated using the following equation.
w/v=w/w x pT where pT is density at temperature T
Note, that the density must be known at the given temperature. For example, if the milk composition was given
in units of w/w and you are metering milk into your cheese vat at 32C you need to know the density of the milk
at 32C. For milk of average composition (4.0 % fat), the density can be estimated according to the following
equation(1).
pT = 1.0366 - .00035T where pT is density at temperature T
Density values for milk of average composition (4% fat) at some temperatures relevant to cheese manufacture
are:
Calculations
The following steps are required to calculate the amount of powder or skim milk to be added, or the amount of
cream to be removed. Suppose a cheese maker wishes to fill a 10,000 l (100 hl) setting vat for the manufacture
of Cheddar cheese.
Step 1. Determine the protein and fat contents of the milk using an automatic milk analyzer. If a milk analyser is
not available the protein content of pooled milk can be crudely estimated from the fat content using the
following formula:
Kg/hL of protein = (0.4518 x Kg/hL of fat) + l.521
For the purpose of this example, assume the available milk contains 3.50 Kg/hL of fat and 3.l0 Kg/hL of protein.
Step 2. Determine the required fat, moisture and F/DM of Cheddar cheese. 'Dairy Products Regulations' of
the Canada Agricultural Products Standards Act require Cheddar cheese to contain a minimum of 31% fat and a
maximum of 39% moisture. Therefore,
F/DM = % fat/% dry matter = 30.0/(100.0 - 39.0) = 49.2%
Step 3. Determine the required P/F of the milk. The P/F required to yield F/DM = 50% as required for Cheddar
cheese is about 0.96 (See Table 6.1).
Step 4. Calculate the amount of: skim milk powder to be added; or fat to be removed; or skim milk to be added.
Standardization by Adding skim milk powder
(i) Calculate the % protein required to give P/F = 0.96
The required level of protein = 0.96 x % fat = 0.96 x 3.50 = 3.36
(ii) The % protein to be added = 3.36 - 3.10 = 0.26 Kg/hL
(iii) Calculate the weight of protein which must be added per 100.00 hL of milk.
The required weight of protein = 0.26 Kg/hL x 100 hL = 26.0 Kg
(iv) Calculate the amount of powder which must be added assuming the skim milk powder (SMP) contains
35.0% protein. If possible the skim powder should be analyzed so the exact protein content is known. The
supplier may be able to provide this information. Protein content can also be estimated using a milk analyzer to
test the reconstituted skim milk.
Required amount of powder = 26.0 Kg/0.35 = 74.3 Kg
(v) Check calculations:
Weight of fat in milk: 3.50 Kg/hL x 100.00 hL = 350.0 Kg.

Weight of protein in milk: 3.10 Kg/hL x 100.00 hL = 310.0 Kg
Weight of protein in SMP: 0.35 Kg/Kg x 74.0 Kg = 26.0 Kg
Total Protein: 310.0 Kg + 26.0 Kg = 336.0 Kg
P/F ratio of standardized milk: 336.0 Kg/350.0 Kg = 0.96
Standardization by Removing fat
(i) Calculate the level of fat required to give P/F = 0.96.
The required level of fat = Kg/hL of protein/.96 = 3.10 Kg/hL/.96 = 3.23 Kg/hL
(ii) Use a Pearson's square to calculate the litres of cream that must be removed, assuming that the separator
removes cream containing 30.00 kg/hl of fat.
Un-standardized Milk 30.00 - 3.23 = 26.77 Parts

3.50 Kg/hL Stanrdardized Milk
Standardized Milk
3.23 Kg/hl
Cream 3.50 - 3.23 = 0.27 Parts
30.00 Kg/hL Cream
Total Parts 26.77 + 0.27 = 27.04
This means that the required proportions of cream and fresh milk are 0.27 and 26.77 parts, respectively, for a
total of 0.27 + 26.77 = 27.04 parts. On a percent basis, the components are:
Standardized Milk 100 x 26.77/27.04 = 99.0% w/v

30% cream 100 x 0.27/27.04 = 1.00% w/v
(iii) Calculate how much cream must be removed from 10,000 Kg of milk to provide standardized milk
containing 3.23% fat.
Cream to be removed = 1.00% of 100 hL = 1.00 hL.
(iv) Check calculations:

Weight of fat in milk 3.50 Kg/hL x 100.00 hL = 350.0 Kg

Minus fat in cream 30.00 Kg/hL x 1.00 hL = 30.0 Kg
Weight of fat in standardized milk 350.0 Kg - 30.0 Kg = 320.0 Kg
Net volume of milk 100.00 hL - 1.00 hL = 99.0 hL
Weight of protein 3.10 Kg/hL x 99.0 hL = 306.9 Kg
Protein/fat ratio 306.9/320.0 = 0.960
(v) Adjust the final weight for the quantity of cream removed. If you wish to fill the vat completely sum the vat
capacity and the initial estimate of the cream to be removed and recalculate the required amount of cream.
Approximate total volume of fresh milk: 100.00 hL + 1.01 hL = 101.01 hL.

Weight of cream to be removed 1.00% of 101.01 hL = 1.01 hL
Final volume of standardized milk 100.00 hL - 1.01 hL = 99.99 hL
Standardization by Adding skim milk
The following calculation is based on the assumption that the protein content of the skim milk is the same as the
protein content in the skim portion of the fresh milk to be standardized. This is exactly true only when the skim
milk is derived from the same source as the fresh milk.
(i) Use a Pearson square to determine the relative proportions of skim milk and milk required to yield a fat
content of 3.23% as calculated in Step B above.
Skim milk 3.5 - 3.23 = 0.27 Parts

0.10 Kg/hL Skim Milk
Standardized Milk
3.23 Kg/hl
Unstandardized Milk 3.23 - 0.10 = 3.13 Parts
3.50 Kg/hL Unstandardized Milk
Total Parts 0.27 + 3.13 = 3.40
This means that 0.27 parts of skim are required for 3.13 parts of milk where the total mixture consists of 0.27 +
3.13 = 3.40 parts. On a percent basis, the mixture is:
Skim milk 100 x 0.27/3.4 = 7.9%

Unstandardized Milk 100 x 3.13/3.4 = 92.1%
(ii) Calculate the amount of skim and fresh milk required.
Weight unstandardized milk 92.1% of 100 hL = 92.10 hL.

Weight of 0.1% skim milk 7.9% of 100 hL = 7.90 hL.
(iii) Check:
Weight of fat in unstandardized milk 3.50 Kg/hL x 92.10 hL = 322.4 Kg

Weight of fat in skim milk 0.10 Kg/hL x 7.90 hL = 0.80 Kg
Total fat 322.4 Kg + 0.8 Kg = 323.2 Kg
Weight of Protein 3.10 Kg/hL x 100 hL = 310.0 Kg.

Protein/fat ratio 310.0/323.2 = 0.959
Addition Of Cream
The natural P/F of milk is higher in low fat milk. In practice, this means that when the milk fat is less than 3.0%,
it may be necessary to add fat to obtain P/F = 0.96 and make a full fat cheese with F/DM = 50%. When the
required F/DM is less than 50%, it is unlikely that fat would have to be added to the milk. The natural P/F is also
high in the fall and early winter so fat may have to be added for full fat cheese at these times. Some cheese such
as double cream Blue or double cream Havarti may also require addition of fat. Given the fat content of
available cream, a Pearson's square can be used to calculate the amount of cream required in a similar manner to
the examples given above.
General Guidelines for Standardization

Determine the fat and protein content of milk accurately and daily.
Measure milk volume or weight accurately and keep accurate records.
If powder is being added, use only high quality, low temperature, antibiotic free powder of known protein
content. Low temperature powder is required to ensure that excessive denaturation of whey proteins in the
powder will not impair milk coagulation and/or cause texture defects in the cheese. To ensure low
temperature powder, ask your supplier to certify a whey protein nitrogen index (WPI) greater than 6.0.
Effects of heat treatment on coagulation and cheese quality are discussed in Chapter 8.
Weigh accurately the weight of powder or skim milk added or the weight of cream to be removed.
Determine the composition of the standardized cheese and if necessary adjust the proportions of fat and
protein in the cheese milk on succeeding days.
If bulk starter is being added reduce the amount of protein added by the amount of protein in the culture.
The maximum recommended level of skim milk solids in cheese milk is 11%. Normal milk contains about
9% skim solids so the maximum level of additional skim solids is 2%. If standardization requires more it
is recommended to standardize by removing fat or adding skim milk rather than by adding skim milk
powder. Another alternative is to add some powder and then complete standardization by removing cream
or adding skim milk.
Without sophisticated metering equipment it is difficult to obtain exact standardization. Provided you have
a milk analyser, you can do a final check of milk composition after the milk is in the vat and then 'fine
tune' the P/F ratio by adding skim solids or cream as required.
It is not possible to predict the exact composition of the finished cheese. However, when manufacturing
conditions and milk composition are the same from day to day, it is possible to predict the composition of
cheese with greater accuracy and the proportions of fat and protein in the cheese milk can then be 'fine-
tuned' accordingly. It is, therefore, important to keep accurate records.
Be careful to use the correct units when calculating and weighing and metering. See Section 2.
Section D: Acidification and Coagulation

Cultures
General Functions of Cheese Cultures
Lactic acid bacteria and other microorganisms are present as 'contaminants' in cheese milk and further
environmental contamination takes place during cheese manufacture. Provided the milk is not chilled, it is
possible to make cheese without any additional cultures, but normal practice is to add domestic cultures for the
manufacture of cheese from both raw and pasteurized milk. Culture, then, refers to prepared inocula of bacteria,
yeast and moulds which are added to cheese milk and cheese. In the broadest terms cultures have two purposes
in cheese making: (1) to develop acidity; and (2) to promote ripening. Lactic acid cultures contribute to both of
these functions, while numerous special or secondary cultures are added to help with the second function.
Development of Acidity
Raw milk at warm temperature
will support a variety of micro-
organisms in succession as the pH
changes over time (see illustration
on the right). In controlled
conversion of milk to fermented
dairy products, a primary
component of fermentation is
development of acidity by lactic
acid bacteria. Acid development in
cheese making is absolutely
essential to cheese flavour, cheese
texture and cheese safety. Acid is
required to:
Assist coagulation. Lower

pH results in faster
coagulation and in acid
coagulated cheese is the
only factor which induces
coagulation.
Promote syneresis. This is a most critical means of controlling moisture content. Acidity (specifically
reduced pH) causes the protein matrix in the curd to contract and squeeze out moisture. That process of
contraction is called syneresis.
Prevent growth of pathogenic and spoilage bacteria. Proper rate and extent of acid development is the
most important principle with respect to quality and safety of natural cheese. I would argue that with the
exception of noncultured cheese varieties such as ricotta, proper culture growth and acid development is
equal in importance to pasteurization with respect to safety.
Develop cheese texture, flavour and colour. The following general associations are relevant to most cheese
varieties.
high pH produces soft, soapy, fruity and bitter cheese
low pH produces cheese with brittle texture and mottled colour
Assist curing
Growth factors produced by lactic cultures are required for other non-starter microorganisms which
contribute to the desired flavour and body of cheese
Enzymes (both lipases and proteases) produced by lactic cultures contribute to interior ripening of cheese
and are important to both flavour and texture development.
Special or secondary cultures are responsible for eye development, surface ripening etc. See Section 7.5.
General characteristics of lactic acid cultures

Lactic acid cultures are often called starters or referred to by the acronym 'LAB' which stands for lactic acid
bacteria. The following lists identify and briefly describe some properties of LAB. LAB are:
Non-motile gram+ bacteria

Non spore forming
Catalase and nitrate negative
Micro-aerophilic which means they tolerate only low oxygen concentrations
Not psychrotrophic which means that cold storage rapidly depletes their numbers and encourages the
growth of spoilage bacteria as described in Raw milk quality.
Cocci (spherical cells) 1 m in diameter OR rods (rod shaped cells) 1 m wide and 2 to 3 m long.
Classification of Lactic Acid Cultures

Classification of lactic cultures, is confusing, because many LAB have been renamed. Table 7.1 lists the old and
new Latin names for some common lactic cultures.
It is helpful to categorize lactic cultures according to general technological and growth characteristics. From that
perspective, LAB are grouped by four criteria, namely:
Principal metabolites (end products of fermentation)

Optimum growth temperatures: meso- versus thermophilic
Starter composition:
Pure defined strains
Mixed defined strains
Pure (single) strains
Forms of inoculation
(I) Principal metabolites: homo- versus heterofermentative

Homofermentative means that lactic acid is the principal metabolite without production of gas (CO2) and
flavour compounds.
Heterofermentative means that lactic acid is the principal end product of fermentation but technologically
significant amounts of one or more of the following metabolites are also produced.
Carbon dioxide (CO2 ) which causes the small gas holes in Havarti, Gouda and other cheeses. Gasiness in
most cheese varieties is a defect.
Short chain fatty acids such as acetic acid and propionic
Acetaldehyde, a principal component of yoghurt flavour
Diacetyl, a principal flavour note in sour cream, butter milk, Dutch cheese and Havarti cheese
Ethyl alcohol
(II) Optimum growth temperatures: meso- versus thermophilic
Mesophilic cultures as the name implies prefer medium range temperatures, rather than cold temperatures
(psychrophilic) or hot temperatures (thermophilic).
Optimum growth range for mesophyllic cultures is 30 - 35C.

Acid production is slow or absent at temperatures less than 20C.
Growth is inhibited at temperatures greater than 39C.
Generally any cheese which does not require high temperatures to dry the curd will utilize mesophilic
cultures. These include Cheddar, soft ripened cheese, most fresh cheese, and most washed cheese.
Include both homo- and heterofermentative cultues (Table 7.1)
Thermophilic cultures are defined by their ability to grow at temperatures above 40C. With respect to cheese
making their important characteristics are:
Optimum growth in the range of 39-50C

Survive 55C or higher
Minimum growth temperature is about 20C below which cell counts decrease rapidly, so, bulk
thermophilic cultures should not be stored at temperatures <20C.
Thermophilic starters are normally mixtures of cocci and rod cultures which at the time of inoculation are
about equal in numbers. That is, the initial inoculum is 50% cocci and 50% rods.
Rod/cocci blends grow together in a relationship referred to as 'mutualism' where the overall growth rate
and acid production is faster than either culture on its own. The rods produce amino acids and peptides
which stimulate the growth of cocci, and the cocci produce formic acid which is required by rods.
The balance between the rods and cocci can be controlled by temperature and pH
The cocci prefer higher temperatures (optimum about 46C) than the rods (optimum about 39C).
The rods are more acid tolerant than the cocci, so, normally the cocci develop the initial acidity and
out grow the rods. But, as the acidity increases the rods begin to grow faster than the cocci.
Some thermophilic rod cultures have the ability to ferment galactose as well as glucose which is desirable
in some cheese, especially Mozzarella.
Although yoghurt cultures which include both rod and cocci, produce acetaldehyde which is the principal
component of the characteristic yoghurt flavour, none of the thermophilic LAB are considered
heterofermentative (Table 7.1).
(III) Starter composition:

Pure defined cultures are single strain cultures selected from natural mixed populations for specific
properties such as proteolytic characteristics or resistance to phage (bacterial viruses).
May be rotated to avoid phage infection
Have the advantages of uniform rate of acid development and uniform flavour profiles
A mixed defined culture is a blend of single strain cultures.
May be rotated to avoid phage infection
Has the advantages of uniform rate of acid development and uniform flavour profiles
Mixed cultures are nonspecific blends of cultures, some what like a natural eco system
Normally have complex systems of phage resistance
Mixed mesophilic starters are still common, but thermophilic starters are usually mixed defined
cultures.
Disadvantage is nonuniform rates of acid development from vat to vat and nonuniform flavour
profiles.
(IV) Forms of Inoculation
Cultures can be carried and prepared for cheese milk inoculation in one of three general formats:
Traditional starters which need several scale up transfers. This system requires some microbiological
facilities and expertise and is only feasible for very large plants or perhaps for smaller plants which use
mixed strain cultures.
Bulk set culture. In this system, the culture supplier does all the purification and transfer work, and
delivers a bulk set culture which is used to inoculate a bulk culture, which in turn is used to inoculate the
cheese milk. Bulk cultures are the norm in medium to large plants because the cost savings are significant.
Direct to the vat cultures require no scale up at the cheese plant. Concentrated cultures ready to inoculate
the cheese milk are supplied directly by the culture supplier.
Summary: technological properties of lactic acid

cultures
In addition to properties mentioned above, the following lists includes other technological properties of
importance to cheese making. Note that many of these technological characteristics are encoded on extra-
chromosomal genetic material called plasmids. Plasmids have the disadvantage of being unstable so
characteristics encoded on plasmids are also unstable. The advantage is that plasmids can be transferred to other
bacteria so microbiologists can readily transfer technological properties from one LAB to another.
Lactose metabolism. Most but not all LAB are able to metabolize lactose.
Galactose metabolism. The ability to ferment lactose is important for late acid development in Italian
cheese and to control browning on Mozzarella cheese.
Proteolytic characteristics which determine cheese flavour development.
Resistance to phage (bacterial viruses).
The ability to metabolize citrate which is associated with flavour development (diacetyl or butter milk
flavour) and gas formation.
Production of bacteriocins, that is, antibiotics produced by bacteria against other bacteria.
Resistance to bacteriocins
Antibiotic resistance.
Secondary Cultures
In addition to lactic acid cultures many special or secondary cultures are used to promote specific ripening (both
flavour and texture) characteristics.
Large holes: Propioni bacterium freudenreichii subsp. shermaniee

White moulds: Penicillium camembertii, P. caseiocolum, and P. candidum
Blue/green moulds: Penicillium roqueforti, Penicillium glaucum
Smears:
yeasts and moulds.
Various coryneform bacteria including Brevibacterium linens, several species of micrococci, and
several species of Staphylocci.
Ripening adjuncts:
Bacterial or yeast cultures added in addition to the regular lactic acid cultures.
Attenuated cultures which are not intended to grow but only to contribute their enzymes.
Species of Lactobacilli and pediococci which are intended to grow during cheese ripening and
contribute enzymes.
Culture Production, Distribution and Storage

Commercial culture preparation
Genetic techniques offer much opportunity to develop cultures with specific technological characteristics.
However, at the commercial level, culture preparation is relatively simple.
Lactic cultures are grown in buffered media to facilitate maximum growth without acid inhibition
The cells are concentrated by centrifugation
The cell concentrate is fast frozen or freeze dried (lyophilized). Frozen (-40C) or lyophilized cultures can
be stored for several months without substantial loss of activity. Lyophilized cultures usually require a
longer "lag time", i.e. time between inoculation and rapid cell growth.
Culture Practice in the Cheese Plant

Direct to the vat cultures need only be stored under prescribed conditions and opened and delivered to the vat
under aseptic conditions. The following comments relate to the preparation of bulk culture at the cheese plant.
Culture preparation should take place in a separate culture room which is kept at positive air pressure with
hepa-filtered air (0.2 m filter).
All surfaces in the culture room must be of a material that can be sterilized.
Use sterile pipettes and sanitize surfaces and equipment with 200 ppm chlorine.
Alternative culture media are:
Milk, but care must be taken to avoid rancid milk, mastitic milk, milk containing antibiotics, and
milk with high bacteria counts.
10 -12% reconstituted skim milk powder is adequate provided that the powder is tested and certified
antibiotic free.
Whey and reconstituted whey powder may be used, but may not achieve the same cell counts as
skim milk (due to less buffer capacity).
A number of commercially prepared culture media are available. Most of these are based on milk
protein powders.
Culture media may be buffered with phosphates to increase cell counts but some cultures
particularly Lactobacillus. bulgaricus appear to be inhibited by phosphates.
Addition of phosphates also confers phage resistance because phosphates bind calcium, and phage require
calcium to attach themselves to the bacterial cells.
Calcium reduced skim milk powder and addition of anhydrous ammonia have also been used to inhibit
phage in bulk cultures
Culture media should be heated (at >88C for 1 h) to destroy bacteria and some inhibitory substances.
Heating also reduces the redox potential (lowers oxygen concentration) which encourages the growth of
LAB.
Optimum pH endpoint before cooling is between 4.5 and 5.0. At pH less than 4.5 some cultures will pass
from growth (log) phase to stationary phase and will be less active when added to the cheese vat.
Cell count can be increased by:
Internal pH control using buffered media
External pH control by adding sodium hydroxide or ammonium hydroxide to maintain pH at 5.0 -
5.5.
Generally cultures should be cooled to 4C after the desired minimum pH and cell counts are obtained.
However, the optimum storage temperature depends on the particular culture. Consult with the culture
supplier. For example, some thermophilic cultures should not be cooled below 20C. Storage time should
be as short as possible, but I am aware of plants which successfully use a single bulk set culture for a week
before making a new batch.
Bacteriophage (bacterial viruses)

Bacteriophage are the stuff of a cheese maker's nightmare. Like all viruses, bacteriophage (hence forth
abbreviated to phage) are parasites, that is, part of their life cycle is dependent on the host bacteria. Here's a few
facts about their characteristics and how they can be controlled.
Extracellular phage, that is, phage particles existing separate from their bacterial hosts are called mature or
resting particles.
Resting particles are sperm shaped, < 1 micron in length.
Resting particles consist entirely of DNA (genetic material) and protein. The basic construction is a DNA
core enclosed in a protein sheath.
The basic life cycle, called the lytic cycle, is:

The phage attaches itself to the bacterial cell wall by its tail, bores a hole in the wall with the help of
enzymes and injects its DNA into the cell. The protein sheath remains outside the cell.
From the moment of invasion the bacteria begins to reproduce phage DNA and protein in addition
to its own.
Nucleic acid and protein strands assemble themselves into new phage particles which eventually
lyse the cell (break it open) to release the phage particles into the medium. A new generation of
resting phage are now available to repeat the lytic cycle
Sometimes infection occurs without lysis resulting in a lysogenic culture where infected cells survive and
reproduce infected daughter cells. Therefore, cheese cultures can exist in one of three states with respect
to phage sensitivity:
1. Insensitive due to inherent or acquired resistance.

2. Phage carrier (lysogenic). In this state the bacteria are resistant to another phage infection
3. Phage sensitive in which case the phage will grow quickly and may terminate the culture. Culture growth
will stop when phage levels reach 103 to 107 per ml.
Phage have a short latent period (reproduce as quickly as every 30 to 50 min) and a large burst size (each
lysed cell will release 50 to 100 new phage).
Phage are quite strain specific which is the reason for culture rotation. As many as 10 different cultures
may be rotated on a daily basis.
Culture failure due to phage can be recognized by normal acid development initially followed by a
decrease or termination of culture growth at a later stage. This is different than inhibition due to antibiotics
which can be recognized by no or slow initial growth; if inhibition is not severe, culture growth and acid
development by resistant strains or mutants may increase with time.
Summary of phage control measures

Use aseptic techniques with proper culture room.
Rotate cultures daily and/or use defined phage resistant strains.
Use phage resistant media for culture preparation.
Use direct-to-vat culture to avoid contamination during transfers.
Use a mixed strain culture of two closely related strains.
Remove and dispose of whey daily
Routinely check for presence of phage using a culture activity test with the culture currently in use and
some whey from the most recent vat
Coagulation
Milk Structure
Raw milk quality provided an introduction to milk chemistry. Now we look briefly at milk physics to help
understand how milk coagulation works. Refer to the figure on the right and review the following facts:
Milk is an emulsion with fat particles (globules) dispersed in an aqueous (watery) environment.
The fat globules do not coalesce and form a separate layer (oil off or churn) because they are protected by
a membrane layer which keeps the fat particles separate from the water phase.
The principal group of milk proteins, the caseins, are not soluble in water and exist in milk as small
particles (<300 nm) called micelles.
We can now define the following terms:
Milk: a dispersion of milk fat globules

(fat particles) and casein micelles
(protein particles) in a continuous phase
of water, sugar (lactose), whey proteins,
and minerals.
Milk Plasma: what is left after you

separate the fat globules; equivalent to
skim milk for practical purposes.
Milk Serum: what is left after you take

away both fat globules and casein
micelles; equivalent to cheese whey for
most practical purposes
Milk permeate: what is left after you

take away fat globules, casein micelles,
and whey proteins.
Coagulation is what happens when the

casein micelles stick together. Because
casein particles are hydrophobic (they
hate water) their natural tendency is to
aggregate (clump together). In normal
milk, aggregation is prevented by two
factors. If one of these factors is eliminated the micelles will aggregate and form a gel something like jello.
The first stabilizing factor is a 'hairy' layer of surface active protein, called kappa-casein (-casein), on the
surface of the micelle. This layer helps prevent the micelles from getting close enough to stick together.
The second factor is a negative charge on the micelles. At the pH of milk the micelles are negatively
charged so they repel each other.
So, basically there are two ways to coagulate milk; one is to remove the hairy layer from the micelles. That's
called enzymic coagulation. The other is to neutralize the negative charge on the micelle. That can be
accomplished by acidification or a combination of high temperature and acidification.
Enzymic Coagulation of Milk

The three stages of enzymic coagulation
(1) Primary Stage
In the first stage, the enzyme (rennet) cuts off a specific fragment of one of the caseins, namely, -casein. At the
natural pH of milk, about 80% of -casein must be cleaved to permit aggregation of the micelles to proceed.
(2) Secondary Stage
The next stage is the physical process of aggregation of casein particles (micelles) to form a gel. After losing its
water soluble tail, -casein can no longer keep the casein particles separated, so they begin to form chains and
clusters. The clusters continue to grow until they form a continuous, three dimensional network which traps
water inside, and forms a gel, something like Jell-o.
(3) The third stage refers to an ongoing development of the gel network. For some cheese the gel is cut as soon
as it is firm enough to do so. For others, like soft ripened cheese, cutting is delayed while the gel continues to
become firmer.
Effects of processing parameters on enzymic coagulation

Because rennet coagulation takes place in stages, it is necessary to understand the effect of processing on each
stage. We will focus mainly on only the first and second stages.
Effect of pH. Lower pH increases enzyme activity and neutralizes charge repulsion between micelles. Therefore,
both primary and secondary stages of coagulation proceed more quickly at lower pH.
Effect of Calcium . Calcium is not required for the primary stage (i.e., enzyme hydrolysis of -casein) but is
essential to aggregation of the casein micelles. At low levels of calcium the primary phase goes to completion.
Subsequently, instantaneous coagulation can be induced by adding sufficient calcium chloride.
Effect of temperature. The optimum coagulation temperature for most cheese is 30-32C, the exception is Swiss
which is set at 37C.
At temperature less than 30C the gel is weak and difficult to cut without excessive yield loss due to fines.
At temperatures less than 20C coagulation does not occur, but the primary stage goes to completion and
the milk will then coagulate quickly when warmed.
Effects of heat treatments.
Mild heat treatment such as pasteurization decreases the rate of the secondary stage. During heat treatment
calcium and phosphate move from soluble to colloidal (insoluble) form, so there is less calcium available
to assist with coagulation. This effect is reversed by cold storage or the addition of calcium chloride
Heat treatment in excess of pasteurization results in increased clotting time and a weak gel. High heat
treatments cause absorption of whey proteins onto the casein particles. The casein particles are then
unable to form a strong gel.
Effects of Homogenization: The following effects occur if the cheese milk is homogenized in its entirety. As
noted in Treatment of milk for cheese making, some of these results may be different if only the cream is
homogenized and then added back to the skim milk. Homogenization primarily affects the secondary phase of
aggregation. Some cheese quality effects are also noted.
Reduced aggregation of casein particles

Decreased syneresis
Finer gel network due to smaller fat globules
Improved texture of soft cheese
Fat recovery (i.e., percent transfer from milk to cheese) is increased (Note: the same is true for acid and
heat/acid coagulated cheese).
Hard cheese becomes rubbery
Makes cheese whiter because the yellow fat is masked by the artificial protein membranes on the
homogenized fat globules.
Coagulating Enzymes
The traditional enzyme is rennet (chymosin) which is derived from the abomasum of the milk fed calf. The
practice of cheese making probably began when somebody discovered that milk stored in bags made from calf
stomachs formed a sweet curd.
Other proteases which have been used for cheese making include:
Pepsins from the pig, cow and chicken

Microbial proteases (Mucor miehi, Mucor pusillus, and Endothia parasitica).
Synthetic chymosin by recombinant DNA techniques using strains of Eshericia coli or Klaveromyces
lactis or Aspergillus niger as host organism is now available. The transferred genetic material exists in the
host cell in the form of a plasmid and is used as a template for the production of an enzyme identical to
chymosin.
Requirements of suitable coagulating enzymes
Suitable ratio of clotting to proteolytic activity (C/P). This ratio is dependent on the specificity of the
enzyme for the Phe105-Met106 bond of -casein. Most rennet substitutes are more proteolytic than rennet
(i.e., low C/P) and cause diminished yields of casein and fat, and bitterness during ripening
Proteolytic specificity. Structure and flavour of ripened cheese depends on the type of proteolysis caused
by the coagulant during cheese curing. The exception is in cheese such as Swiss or Parmesan where most
of the rennet activity is destroyed by the high cooking temperature. During ripening chymosin breaks
down one of the caseins, namely s1-casein much more than other caseins.
High pH optimum. Rennet activity is stable and able to coagulate milk at the normal pH of milk although
its activity increases with decreasing pH. Most pepsins and microbial proteases are denatured at the pH of
milk which has been a major difficulty in developing rennet substitutes.
Denaturation temperature is important for two reasons:
Ripening due to coagulating enzymes is not desirable in cooked cheese such as Swiss and Italian
types. Rennet is eliminated during the high temperature cook in these cheeses but microbial
coagulants are not.
The coagulant must be eliminated by pasteurization to prevent proteolysis in products made from
whey. Some microbial rennets survive pasteurization.
Distribution between curd and whey. Only 0-15% of rennet remains in the curd, but small amounts of
residual rennet are significant to ripening of aged cheese. The most important factors which determine
rennet retention are:
Cooking treatments.
As noted above, rennet does not survive in high temperature cooked cheese varieties.
In low cooked cheese such as Cheddar, variations in cooking temperature and time influence rennet
activity during aging.
The pH at draining. Rennet is more soluble at low pH and, therefore, the amount retained in the curd
increases with decreasing pH at draining. Retention of microbial rennets in the curd is independent of pH
at draining.
Changing rennet sources may also influence rennet retention and cheese ripening. Different rennets with
the same coagulating properties may have different thermal tolerances and different proteolytic
characteristics.
Standard and consistent activity. Single strength rennet is standardized so that 200 ml coagulates 1,000 kg
of milk in 30 - 40 min. Typical commercial rennet preparations are about 50% chymosin (calf rennet) and
50% bovine pepsin, so there is much opportunity for variation. Commercial calf rennet preparations are
about 94-96% chymosin. Using recombinant rennet it should be possible to produce commercial rennet
preparations which are more consistent with respect to all of the properties listed above, including
proteolytic specificity and heat tolerance.
Acid coagulation
Acid milk gels can be formed by lactic bacteria or the use of acidifying agents such as glucono-delta-lactone
(GDL is slowly hydrolysed to gluconic acid in the presence of water). Acid coagulation is used in the production
of cottage cheese, bakers cheese and quark as well as other fermented milk products such as yoghurt,
commercial butter milk, kefir etc. In the case of cottage cheese and quark a small amount of chymosin may be
used (2 ml/1,000 hl) to make the curd more elastic and less subject to breakage (dusting).
Heat-Acid coagulation
This process permits recovery of caseins and whey proteins in a single step. The basic principle is that whey
proteins which are normally acid stable, become sensitive to acid coagulation after heat treatment. This principle
is exploited in the manufacture of ricotta cheese, Paneer and Channa, and in the manufacture of "co-precipitated"
milk protein concentrates. The basic process for heat-acid coagulation is:
Heat milk or milk-whey blends to at least 80C for at least five minutes to completely denature (unfold) the
whey proteins and encourage association of whey proteins with casein micelles.
Continue heating and acidify slowly with gentle agitation. The caseins and whey proteins will coagulate
together and form either sinking or floating curds.
Section E: Manufacture, Ripening, Process Control

and Yield Efficiency
Cheese making step by step
This chapter describes the principal steps involved in cheese manufacture. Tables 9.1 and 9.2 are sample process
(make) and quality sheets.
Ripening the Milk

This term is a little confusing because it is also used to describe the ripening or aging of cheese. Here, ripening,
refers to the practice of giving the culture time to begin acid production before the rennet is added. This is done
for two reasons:
To ensure the culture is active before the milk is renneted. It is impossible to inoculate after the milk is set.
Normally, 45 - 60 min is sufficient to decrease pH by 0.01 units or increase TA by 0.005 - 0.01%
Development of acidity aids the coagulation process, especially the secondary stage.
In some varieties such as brine brick and Swiss, low amounts of culture are used and renneting proceeds with
little or no prior ripening.
Setting the Vat

Handling Rennets
Repeatable performance depends on accurate measurement. For most varieties the quantity of rennet is
selected to set the milk to a firm coagulum in 30 - 40 min. Measure the rennet accurately and monitor to
ensure that coagulation rate is uniform from day to day.
Rennet must be diluted (about 20 times) in water and well mixed when added to ensure uniform
distribution.
Use nearly the same dilution each time to improve the consistency when adding the diluted rennet to the
vat.
Watch out for chlorine. It is imperative that the dilution water contains no chlorine. Only 2 ppm of
chlorine will destroy 40% of rennet activity in 3 minutes. Similarly, do not sanitize the container used for
the rennet with chlorine.

Another water quality issue is pH. Typically hard water also has pH greater than 7.0 which also decreases
rennet activity.
Finally, dilute immediately before adding the rennet to the vat. After the brined rennet is diluted in water,
its activity declines quickly.
Optimizing setting parameters

Milk preparation was discussed in Treatment of milk for cheese making. Here are the principal
considerations:
Pasteurization temperature: higher temperatures increase yield by increased recovery of whey
proteins, but a suggested maximum with respect to curd quality is 75C, 16 s.
Temperature history: if the milk is pasteurized and immediately sent to the setting vat, it will be
necessary to adjust the mineral balance by adding calcium chloride.
The jury on selection of coagulant always seems to be out. I tentatively suggest that microbial coagulants
are not advisable for high temperature varieties for reasons of heat stability, and not advisable for other
varieties unless other setting and conditions are under tight control. The preferred choices, then, are rennet
and recombinant rennet.
The amount of rennet must be carefully determined. Because rennet is costly, it is desirable to minimize its
use, but this can be false economy if curd properties are compromised. Poor setting means increased
losses of both fat and protein as fines.
Temperature control must be accurate and uniform through out the vat, because both the enzyme activity
and the subsequent process of micelle aggregation are extremely temperature sensitive. Inaccurate or
nonuniform temperature during setting will result in local areas of under or over set curd which in turn
causes loss of fines during cutting.
Soft curd results from:
Over heat treatment
Low setting temperature
Homogenization
Colostrum or mastitic milk
Firm curd results from:
High calcium
Low pH
Standardisation to high protein content.
Cutting The Curd

Proper cutting is extremely important to both quality and yield. Improper cutting and handling the curd results in
the loss of fines, that is, small curd particles which are not recovered in the cheese. Unlike whey fat, fat trapped
in fines; is not recovered by whey cream separation. Therefore, both fat and protein losses occur when shattered
curd results in fines too small to be recovered in the cheese.
Determination of curd cutting time
Both early cutting when the curd is fragile and late cutting when the curd is brittle cause losses of fines. Several
means are used to determine cutting time.
Manual testing. The curd is ready to cut if it breaks cleanly when a flat blade is inserted at 45o angle to the
surface and then raised slowly.
Several mechanical devices based on oscillating viscometry, thermal conductance and sonication have
been tested experimentally.
Some plants cut by the clock. This may be OK as long as all conditions are uniform from day to day (is
that every true??) and adjustments are made for any change in milk composition or properties.
If setting temperature is high as for Swiss types, the curd firms rapidly and cutting must begin early when
curd is still somewhat soft to prevent over setting. Agitation should begin immediately to prevent matting.
Curd size
Curd size has a great influence on moisture retention. Hence, there is an obvious relationship between cheese
moisture and the prescribed curd size:
High temperature and low moisture varieties such as Italian hard cheese require the smallest curd. Cutting
continues until the curd cutting is the size of rice grains.
Medium moisture cheeses like most washed varieties and Cheddar are cut to Omega cm cubes.
High moisture varieties like soft ripened cheese are cut with 2 cm knives or the curd is simply broken
sufficiently to be dipped into forms.
Small curd size will result in greater fat and SNF recovery because large curds tend to get crushed resulting in
the loss of 'fines'. Smaller curds will also dry out faster and, therefore, other factors such as cooking temperature
and stirring out may have to be adjusted according to curd size.
Manual cutting
Manual cutting is done with cutting harps, made by stretching stainless steel wire over a stainless steel frame.
Total cutting time should not exceed 10 minutes (preferably less than 5 minutes) because the curd is continually
changing (becoming overset) during cutting. The knives should be pulled (not pushed) quickly through the curd
so has to cut the curd cleanly.
Automated cutting
With mechanical knives, curd size is determined by the design of the vat and agitators, the speed of cutting (rpm)
and the duration of cutting. In Double 'O' vats for Cheddar and American varieties, cutting is normally at a speed
of about 4 rpm for 7 - 13 minutes, corresponding two a total of 30 to 50 revolutions. It is important that the
knives are sharp and cut the curd cleanly rather than partially mashing the curd or missing some pieces
altogether.
There is evidence (Johnston et al 1991, J. Dairy Res. 58:345) that curd particle size at draining in mechanized
Cheddar cheese is influenced by cutting time, cutting speed, and subsequent agitation such that:
Short cutting times and low rpm result in small particle size at draining and larger losses of fines.
With increasing cutting time (more total revolutions), curd particle size at draining reaches a maximum
which corresponds to a maximum in fat recovery.
Further increased cutting time causes decreased curd size at draining with little effect on fat recovery.
Healing
Curd should be agitated gently or not at all after cutting to prevent formation of fines. The exterior of the freshly
cut curd is fragile so some time is needed for the edges to close up (heal) and prevent the loss of fat and protein
to the whey.
An index of cutting quality
The loss of fines is best monitored by accurate analysis of whey fat content. Whey fat for Cheddar types should
be <0.3%;. Efficient operations may achieve levels near 0.2%.
Cooking
The combination of heat and the developing acidity (decreasing pH) causes syneresis with resulting expulsion of
moisture, lactose, acid, soluble minerals and salts, and whey proteins. It is important to follow the cooking
schedule, closely. Cooking too quickly causes the curd to shatter more easily and forms a tough exterior on the
curd particles which prevents moisture release and hinders development of a smooth texture during pressing.
Draining
Most cheese is drained in the range of whey pH 6.1-6.4 (curd pH 6.0 - 6.3). Draining time should be uniform at
about 20 min to prevent variation from vat to vat. Cheddar types may be stirred out 1 to 3 times as required to
obtain required curd moisture.
Washing
Lactose content can be adjusted by moisture removal (syneresis), fermentation, or leaching with water. By
leaching lactose with water it is possible to make a high moisture cheese (such as brine brick or Muenster) and
still achieve a final pH of about 5.0 - 5.2. The temperature of the wash water will determine the moisture content
of the curd. Sometimes relatively hot water (eg., Gouda) is used to dry the curd and develop its texture.
Traditionally washing was accomplished by removing Omega to 2/3 of the whey and replacing it with water and
agitating for about 15 min. This process results in the dilution of large amounts of whey which must be
reconcentrated or dumped. It also creates problems where curd tables have less capacity than setting vats. The
solution is to remove more whey and add less water.
Curd Handling
Most brine or surface salted varieties are dipped directly into the forms or pressed under the whey. In the
absence of salt, the curd is fused to form a smooth, plastic mass. The hoops are turned at regular intervals to
promote uniform drainage, symmetrical shape, and a smooth finish.
Some varieties such as Gouda and Swiss are pressed under the whey before draining. This encourages formation
of smooth texture and prevents incorporation of mechanical openings in the cheese due to trapped air or pockets
of whey.
For Cheddar, American, and Pasta Filata varieties the curd is kept warm in the vat or drain table and allowed to
ferment to pH 5.2 -5.4. Pasta Filata varieties are then worked in warm water while Cheddar and American
varieties are salted in the vat.
Pressing
Pressing varies from little or none for soft cheese up to 172 kPa for firm Cheddar cheese. The warmer the curd,
the less pressure required. Mechanical openings may be reduced by vacuum treatment before, during or after
pressing.
Salting
Almost all cheese is salted by one of three methods: before pressing as in Cheddar and American varieties,
surface salting after pressing, or brine salting.
Purposes of Salting
Promote further syneresis
Slow acid development
Check spoilage bacteria. Lactics are more salt tolerant than pathogens and spoilage bacteria.
Promote controlled ripening and flavour development.
Salty flavour
Brine salting:
Concentration 16 - 25% NaCl

Time:
20 kg cheese, 5 days or sometimes several weeks
3-5 kg, 24 h
250 - 350 g, 1 - 4 h
New brine should be treated with about 0.1% of CaCl2 to prevent conversion of calcium and hydrogen
caseinate to sodium caseinate. The latter has high water holding capacity, so the cheese takes up water
from the brine and the cheese surface becomes soft and slimy.
Brine pH should be adjusted to the pH of the cheese. Normally a pH of 5.2 - 5.6 is adequate.
If the pH is too high, ion exchange causing sodium caseinate is encouraged.
If the pH is too low, there is insufficient Ca/Na exchange and the cheese is too hard and coarse.
Brine must be cleaned regularly by filtration, preferably microfiltered. UV sterilization combined with
filtration is also used.
Brine must be continuously agitated to prevent density fractionation (lower concentration brine on top)
and dilution of the brine around the cheese.
If cheese is floated rather than immersed in the brine, the exposed surface of the cheese should be dry
salted.
Vat salting
For vat salted cheese, uniform salt content depends on accurate estimate of the weight of unsalted curd,
accurate weighing of salt, and consistent processing conditions.
Salt uptake is:
Increased by increased acidity (lower pH) at salting.
Decreased by increased time between milling and salting due to healing of the cut surfaces on the
curd particles.
Increased by increased curd moisture content.
Decreased for larger curds.
For Cheddar and American varieties the salt content as a percent of moisture (S/M) should be greater than
4.0%.
Ripening and packaging

Ripening processes: chemical and physical changes
Cheese ripening is basically about the breakdown of proteins, lipids and carbohydrates (acids and sugars) which
releases flavour compounds and modifies cheese texture. The biochemical and biophysical processes involved
have only partly been elucidated. Here we include only a few practical principles of ripening.
General Principles
Ripening varies from nil for fresh cheese to 5 years for some hard ripened cheese. Like a good wine, a
good aged cheese should get better and better with age.
Ripening processes are broadly classified as interior and surface ripened.
Cheese which depend mainly on interior ripening (most hard ripened cheese such as Cheddar and
Italian types) may be ripened with rind formation or may be film wrapped before curing. Having
said that, I hasten to add, that traditional Italian types are always rind ripened. Cheddar and
American varieties are the only ripened cheeses which (in my view) are not drastically altered by
film wrapped curing.
Cheese which depend mainly on surface ripening include smear ripened and mould ripened
In the broadest terms there are three sources of cheese flavour:
Flavours present in the original cheese milk, such as natural butter fat flavour and feed flavour.
Breakdown products of milk proteins, fats and sugars which are released by microbial enzymes,
enzymes endogenous to milk, and enzyme additives.
Metabolites of starter bacteria and other microorganisms. These include products from catabolism
of proteins, fats and sugars.
Flavour and texture development are strongly dependent on:
pH profile
Composition
Salting
Temperature
Humidity
EXPERIENCE.
As a general rule factors which increase the rate of ripening increase the risk of off flavour development,
and reduce the period of time when the cheese is saleable.
Protein Breakdown (Proteolysis)
Natural degradation of protein is called 'putrefaction' and results in 'rotten potato' type odours, especially if high
quality proteins such as animal proteins are involved. That's because animal proteins contain the essential sulfur
amino acids. These 'putrefactive' components are also the stuff of which good flavours are made. Protein
degradation during cheese curing is a directed process resulting in protein fragments with desirable flavours.
Some off flavours associated with undesirable or excessive protein breakdown in cheese are bitter,
stringent, putrid and brothy.
Protein breakdown causes shorter body which is less rubbery, less elastic, more meltable. For example,
flavour and texture development in Cheddar are mainly dependent on protein breakdown and much less
dependent on fat breakdown.
Protein breakdown involves three general types of processes:
Proteases break proteins into smaller peptides, some of which are flavour compounds. For example,
bitter and brothy flavoured peptides are well known to occur in cheese.
Peptidases further break down peptides to amino acids.
Further catabolism of amino acids by cheese microorganisms produces aldehydes, alchohols,
carboxlic acids and sulfur compounds, many of which are flavourful.
The amino acid, tyrosine, forms crystals in aged cheese such as Parmaggiano regiano, which are readily
detected on the palate.
Fat Breakdown (Lipolysis)

Dairy fat is a wonderfully rich source of flavours, because it contains an extremely diverse selection of fatty
acids. In particular, butter fat is the only natural fat which is rich in short chain fatty acids. Butyric acid for
example is a potent flavour compound. As with all potent flavours the trick is to add just the right amounts in
balance with other flavours. Here are a few principles:
Dairy fat without any ripening during cheese making is an important contributor to cheese flavour and
texture:
Fresh dairy fat has the well known 'buttery' flavour associated with extremely low levels of free
fatty acids.
Fat also acts as a flavour reservoir, so hydrophobic (fat soluble) flavours derived from protein
breakdown are stored in the fat and released during mastication in the mouth.
Finally, fat is an important component of cheese softening and melting.
The fat derived flavours associated with cheese ripening result from the release of fatty acids by lipolysis
and further modification of fatty acids by microorganisms to other compounds.
Varieties traditionally made from goats' milk have higher levels of lipolysis.
Blue moulds are generally quite lipolytic
Lactose
Milk contains no starch or fibre or any sugar other than lactose so all carbohydrate compounds in cheese are
derived from lactose or produced by microorganisms. Relative to fat and protein lactose contributions to flavour
are minimal. Here's a few principles:
At Day 1 following cheese manufacture most of the milk sugar has been removed in the whey by or by
fermentation, that is converted to lactic acid by the cultures.
Residual lactose depends on the type of cheese and other factors. For examples:
High salt in the moisture phase of Cheddar slows lactose metabolism so lactose content is .3 to
.7%% at one day after manufacture and slowly declines to less than 0.1%.
Residual lactose in Camembert cheese is used by Penicillium camemberti so it decreases quickly,
especially on the surface, when the mould begins to grow.
In well drained cheese such as Swiss types, lactose is completely used up in a few hours.
In washed cheese varieties, lactose not leached by washing is quickly used up by the culture,
especially for Dutch type cheese where salting is delayed. In Colby, early vat salting reduces the
rate of utilization of residual lactose.
Many organisms, including yeasts and moulds in mould and smear ripened cheeses utilize lactate and
produce various flavourful compounds.
Calcium salts of lactic acid may form white precipitates on the surface of aged cheese.
Principal Ripening Agents

Milk Enzymes
Plasmin: A milk protease which survives pasteurization and breaks down caseins during cheese ripening.
Particularly important in Swiss type cheese.
Inhibited by Beta-lactoglobulin, so it has minimal activity in cheese made from ultrafiltered milk.
Lipoprotein lipase is the principal milk lipase
Inactivated by low heat treatment but is important to flavour development in raw milk cheese
Milk Coagulant
Each milk coagulant has its own proteolytic profile (see section on coagulants).
Purified extracts produce more consistent flavours but lack character.
For aged cheese no enzyme other than calf rennet and recombinant calf rennet has proven fully acceptable.
Rennet and recombinant rennet actively break down alpha-casein but do not break down beta-casein in
cheese.
Lactic Cultures
During the early days and weeks of ripening, LAB numbers decrease while the numbers of nonstarter
bacteria decrease. For example, in Cheddar cheese, LAB counts reach a maximum (up to 500 million per
gram) within 3-4 days and then decrease to about 20 million at 4 weeks. However, the dying cells release
enzymes which continue to ripen the cheese.
Lactic cultures contribute to proteolysed flavours but are minimally lipolytic
Heterofermentative cultures ferment citrate as well as lactose and contribute both flavour (diacetyl) and
carbon dioxide for small eye development
Secondary Cultures
In Swiss types, carbon dioxide production by Propionibacterium is encouraged by exposure to 200C for
about 3 weeks after brining and drying off in the cold room.
For smear ripened cheese, Brevibacterium linens , coryneform bacteria, and yeasts are encouraged by high
humidity (90-95%) and washing to discourage moulds
Penicillium sp. for Camembert, Brie and Blue types require 85-90% humidity and air circulation to
provide oxygen
Non-starter Microorganisms
Microorganisms present in the milk due to environmental contamination are important contributors to milk
ripening. Some important facts are:
Bulk cooling and storage of raw milk selects for cold tolerant (psychrotrophic) bacteria (see Process and
quality control procedures).
Heat treatment selects for thermal stable spore forming bacteria
Non-starter bacteria commonly present in heat-treat Cheddar include Lactobacillus sp. and Pediococci sp.
Many other bacteria and yeasts may be present and may or not grow depending on complex symbiotic
relationships with other bacteria.
Heat treat is really a process of standardizing the nonstarter microorganisms, namely, eliminate proteolytic
psychrotrophic bacteria but retain a range of useful ripening microbial agents.
Non-starter bacteria in cheese milk can be reduced by microfiltration.
Added Ripening Agents
Addition of lipases as noted earlier is common for Italian and other cheese varieties. The principal areas of
continuing development are:
Accelerated ripening agents for all ripened cheese, especially Cheddar

Ripening agents for low fat cheese, again especially Cheddar.
The principal approaches are:
Direct addition of single enzymes of dairy or non-dairy sources
Enzyme cocktails which are mixtures of proteases and lipases. Other than in the preparation of
enzyme modified cheese pastes, enzyme cocktails have had limited commercial success.
Enzyme capsules which release trapped enzymes during ripening.
Attenuated (freeze shocked or heat shocked) proteolytic cultures
Genetically modified cultures hold lots of promise for future success.
Culture adjuncts such as Lactobacillus helveticus in Cheddar cheese hold much promise to replace
the normal diverse microflora of raw milk.
Cheese Composition for Optimal Curing

Cheese composition is critical to yield optimization,
and both flavour and texture development. This section
gives some detail on several critical composition
parameters, with special reference to Cheddar cheese.
New Zealand export Cheddar cheese is all graded by
composition analysis as indicated in Figure A on the
right. Figure B on the right indicates the ranges which
are typical of good Canadian Cheddar.
MNFS
Moisture: higher moisture means faster ripening
which means more potential for off flavours and
over ripening.
water activity (aw) decreases with age because
ripening results in many soluble breakdown
products of acids, sugars, proteins and lipids
fresh Cheddar aw = 0.98 which is conducive to
most bacteria
aged Cheddar aw as low as 0.88 which is too low
for most bacteria
MNFS is a better index of cheese ripening
potential than % moisture
Optimum MNFS depends on expected date of
maturity and curing temperatures:
examples for Cheddar: 100C, 6-7 months MNFS = 53%
100C, 3-4 months MNFS = 56%
MNFS is controlled mainly by pH at dipping and cooking treatments. Subsequent curd treatment such as
cheddaring and salting also influence MNFS
MNFS is also influenced by FDM. Other conditions being kept constant, MNFS increases with increasing
FDM, because fat inhibits syneresis.
S/M
Determines rate of acid development during pressing and early curing and, therefore, influences the
minimum pH
Affects bacterial profile, eg., high S/M will discourage contaminating bacteria such as coliforms.
Critical to rate of proteolysis and the type of protein derived flavours
Acceptable range is broad (3.6 - 6.0), fortunately because S/M varies widely even within a single cheese.
Salt uptake is affected by quantity of added salt, size of curds, moisture content of curds, and acidity
FDM
Higher fat restricts syneresis, so MNFS tends to increase with FDM

Fat shortens and softens cheese texture because the fat globules physically disrupt the protein matrix.
Adjusted by milk P/F (See Treatment of milk for cheese making)
pH
The pH profile is the single most important trouble shooting tool. Critical points are: cutting, draining,
milling, 1 day and 7 days
Most cheese including Cheddar should reach a minimum pH of 5.0 to 5.1 during the first week after
manufacture; obtaining a final pH in this range is greatly helped by increased buffer capacity of milk
proteins in the pH range 5.4 - 4.8.
Factors determining the pH at one day are amount of culture, draining pH, washing, curd treatment such
as cheddaring and salting.
Draining pH is most important to cheese texture and also determines residual amounts of chymosin and
plasmin in the cheese.
pH increases with age due to release of alkaline protein fragments. This is especially true of mould
ripened cheeses. Camembert pH increases from 4.6 to 7.0, especially on the surface.
Increasing pH during curing encourages activity of both proteases and lipases.
Temperature of Curing
Cheddar types: 4 - 10C, 8-10C is the recommended range. It is desirable to initiate ripening for several
weeks at 4-6C and then increase the temperature to 8 - 10C. Low temperature initially, minimizes early
growth of starter and non-starter bacteria and reduces the risk of too rapid ripening and off flavour
development. It also minimizes the risk of the minimum pH reaching levels below 5.0.
Most European varieties are stored at 10 - 15C for initial ripening and then 4C until consumed.
Surface ripened varieties are ripened at 11 - 15C.
Humidity of Curing
Surface ripened cheese also require adequate air circulation to provide sufficient oxygen for moulds and yeasts.
Humidity requirements in general are:
Washed bacterial surface ripened: 90-95%

Fungal flora: 85-90%
Dry rinds: 80-85
Ripening Treatments
According to the type of surface characteristics, cheese treatments are grouped as follows:
Ripened by surface moulds

Washed rinds with out (or with little) bacterial growth, e.g., St. Paulin types.
Washed rinds with smear, e.g., Muenster types and Oka
Dry rinds which may be coated with oil or butter to prevent cracking and desiccation, e.g., Edam,
Scamorza, and Parmesan.
Waxes and resins which may be applied by dipping, brushing or spraying. These provide good protection
but are more permeable than plastic films, so it is still desirable to maintain 85% RH to prevent drying.
Rindless cheese which are cured in moisture and gas impermeable film or in large blocks (eg., 640 lb
Cheddar)
Waxes and films may be treated with anti-mould agents such as pimaricin, sorbic acid and propionates to prevent
mould growth.
Packaging
Vacuum and/or gas flush (N2 and CO2) in gas and moisture proof film are common.
Vacuum alone is not recommended because complete evacuation of oxygen is difficult and small unsightly
mould spots often appear.
Gas flush with CO2 or blends of CO2 and N2 effectively prevent mould growth.
CO2 is water soluble so it is absorbed into the water of the cheese and the package becomes tight.
N2 which is not water soluble is useful for applications, such as shredded cheese and cheese curd,
where a loose package is desired.
High density plastic (rigid containers) are used for fresh cheese such as cottage.
Oxygen permeable wrap such as grease proof paper and foil-laminated but unsealed wraps, are preferred
for surface ripened soft cheese.
Process Control
This Chapter will not be discussed during the short course lectures because most of its contents are covered in
other Sections or in the cheese make procedures. It is included here as a summary of important process control
principles.
The Objectives of Cheese Manufacturing

To maximize returns, the cheese maker must obtain the maximum yields which are consistent with good cheese
quality. For example, water and salt are cheaper than milk fat and protein, but you can only have so much cheese
moisture and salt---more on cheese yield in Yield efficiency. With respect to consistent production of high
quality cheese the objectives of the cheese maker are to:
1. Develop the basic structure of the cheese.

2. Obtain cheese composition required for optimum microbial and enzyme activity during curing. Optimum
composition mainly means optimum levels of moisture, fat, pH (lactic acid), minerals, and salt.
For example, the characteristic texture of Swiss cheese is largely determined at the time when the curd and whey
are transferred to the press table. At this time the basic structure (i.e., the manner in which the casein micelles
and fat globules are arranged) and chemical composition (especially mineral content) is already determined. You
can not take Swiss curd at this stage and make Cheddar cheese. On the other hand it is possible to produce both
Feta and a Brie type cheese from the same curd.
Moisture Control
cheese making is a process of removing moisture from a rennet coagulum or an acid coagulum consisting
of fat globules (unless the milk is skimmed) and water droplets trapped in a matrix of casein micelles
cheese is, therefore, a concentrate of milk protein and fat.
most cheese making operations are related to this process of removing water from the milk gel by the
process of syneresis
syneresis = to contract; refers to contraction of the protein network with the resulting expulsion of water
from the curd
the water and water soluble components are literally squeezed out of the curd
this liquid, (whey) contains water, sugar, whey proteins, lactic acid and some of the milk minerals
the final moisture content, therefore, to a large extent determines the final pH of the cheese because it
determines the residual amount of fermentable lactose in the cheese
at the same time other factors such as the amount and rate of acid development and the temperature and
time of cooking, determine the amount and the rate of syneresis
pH Control
with respect to cheese quality and safety, the most important process control factor is the development of
acidity
increasing acidity causes:
syneresis (due to reduced charge repulsion on casein micelles) and moisture expulsion
solubilization of calcium phosphates
disruption of casein micelle structure with alterations in curd texture
reduced lactose content by fermentation to lactic acid
acid development occurs mainly within the curd because most bacteria are trapped in the gel matrix during
coagulation
final pH (acidity) is dependent on the amount of acid developed during manufacture and the residual
lactose which will ferment during early curing and cause further acid development
the residual lactose content is mainly determined by the moisture content, washing which removes lactose
by leaching, and the extent of fermentation
ability of culture to ferment galactose is also important
both the rate of acid development and the amount of acid development (as measured by final pH) are
important
eg., final pH of Swiss is the same as Cheddar but Cheddar cheese reaches pH 5.2 after about 5 hours while
Swiss cheese requires about 15 h to reach this pH
it is important to maintain uniform rate of acid development; if acidity develops too slow or too fast,
adjust the amount of culture rather than changing cooking time or temperature
pH at draining largely determines the mineral and residual sugar contents of the cheese and from the
sugar, the final pH
salting reduces the rate of acid development, and, therefore, the time and amount of salting is important to
the pH at 1 day and 7 days following manufacture.
Mineral Control
loss of calcium phosphate determines extent of casein micelle disruption--hence it determines basic cheese
structure; the important parameter is the ratio of Ca to casein or Ca to SNF which is easier to measure
(See Table 1.1)
in Swiss (high Ca, about 750 mM Ca/kg SNF) micelle globular structure is intact while extensive
dissociation and disruption of submicelles is evident in Feta types (low Ca, about 400 mM Ca/kg SNF))
retention of calcium phosphate in the cheese also increases the buffer capacity of the cheese
pH at draining determines the solubility of calcium and phosphate when the curd is separated from the
whey
more Ca is retained at high draining pH as in Swiss cheese (pH 6.4 - 6.5) versus Cheddar 6.1 - 6.3
(See Table 1.1).
little Ca retained in Feta cheese which needs some explanation:
Feta is dipped into the forms early while the pH is still quite high. However, the moisture is also high because no
cooking has taken place. Therefore, the moisture is removed by syneresis as the pH decreases while the cheese is
in the forms. The net result is that a great deal of moisture (whey) is removed at low pH and most of the calcium
phosphate is removed with it. This is also true for other soft ripened cheese like blue and camembert.
Texture Control
untypical texture in a young cheese is a strong indication of probable flavour defects later; therefore, a
primary objective of cheese making is to develop the ultrastructure which will determine the proper
texture
conformation of the protein matrix is also influenced by pH--at lower pH micelles are disrupted, but the
proteins are tightly packed because of reduced charge repulsion; therefore, Feta is brittle while Camembert
is soft and smooth due to alkalinity contributed by ammonia during ripening
cheese drained at higher pH has higher calcium content and is firmer and more elastic
firmness is also affected by ripening agents (see 11.6 Flavour control)
other factors also play a role--salt, moisture, and fat, but none of these will alter the basic structure of the
protein matrix at the submicellar level.
Flavour Control
milk heating and clarification treatments which determine non-starter bacteria present in the milk
types of cultures and coagulating enzymes
all cooking and curd handling procedures have specific effects on the types of ripening agents (bacteria
and enzymes) which remain to ripen the cheese; especially in cheese such as Swiss where the composition
and functions of the culture are more complex
pH at draining again important because it determines the distribution of plasmin and rennin between the
curd and the whey
plasmin is the principal milk protease: it prefers neutral to slightly alkaline pH and is more soluble at low
pH; therefore, cheese which are dipped at high pH have higher retention and activity of plasmin (eg., in
Swiss protein breakdown during ripening is due to plasmin)
calf rennet is more soluble at higher pH but more active at lower pH; therefore, an acid cheese such as
Feta or Cheshire, has more rennet activity than Cheddar
the solubility of microbial rennets is independent of pH
For example ....

The following charts illustrate how a cheese maker may adjust process parameters to make cheese with different
moisture and ripening targets.
Yield efficiency
Distribution of Components During Cheese Making
TABLE 12.1. Distribution of milk components during cheese making (% by weight) and percent transfer from
milk to cheese.
Factors Affecting Yield

Milk casein is the principal yield determining factor. Casein contributes absorbed water and minerals as
well as its own weight. Cheese quality limits the ratio of moisture/casein, a ratio which corresponding to
MNFS.
Fat is also a principal yield component. Fat interferes with syneresis and, therefore, also contributes more
than its own weight, but if other conditions are adjusted to maintain constant MNFS, then fat contribution
to yield is dependent only on the conversion factor of fat from milk to cheese (i.e., fraction of milk fat
recovered in the cheese).
Cheese moisture. A 1% increase in Cheddar cheese moisture causes about 1.8% increase in cheese yield,
partly because more moisture means more whey solids and salt are recovered in the cheese (eg., given 90
kg cheese/1000 kg milk, a moisture adjustment to 36% would result in 91.6 kg cheese/1000 kg milk)
Cheese salt. An extra 0.1% salt means an extra 0.14% yield of Cheddar cheese if the moisture content is
increased accordingly.
Milk quality factors: somatic cell counts, psychrotrophic bacteria, protein quality etc. See Raw milk
quality.
Increasing time and temperature of milk pasteurization increases cheese moisture retention and the
recovery of whey proteins and soluble solids. There doesn't seem to be any consensus on how much is
desirable but it's safe to say that it depends on the type of cheese and the quality standards of the
manufacturer.
Process control parameters (See Cheese making step by step)
Careless cutting.
Heating too fast at early stages of cooking
Salting too soon after milling of Cheddar allows rapid salt uptake which in turn causes rapid
synerisis and increased solubility of casein. Yield is, therefore, reduced by losses of protein, fat and
soluble solids.
High temperatures during pressing cause loss of fat.
Proteolytic cultures or coagulating enzymes cause protein losses before and after cutting.
Washing removes soluble solids.
Working as in Mozzarella removes fat and soluble solids. Loss of soluble solids is minimized by
equilibration of the wash water with the cheese moisture.
Principles of Yield Optimization

With respect to yield the cheese maker's objectives are to:
1. Obtain highest MNFS (moisture in non-fat substance) consistent with good quality to maximize moisture
and the recovery of whey solids.
2. Standardize milk to obtain maximum value for milk components consistent with good quality (eg., adjust
P/F to maximize cost efficiency).
3. Minimize losses of fat and casein in the whey.
Yield Control
It is absolutely vital to be able to measure and maximize yield efficiency. This means maximizing the return (or
minimizing the loss in the case of lactose) from all milk components entering the plant. This includes obtaining
maximum returns for whey non-fat-solids, whey cream and cream skimmed during standardization. In general
the highest return for all milk components, is obtained by keeping them in the cheese, but this may not always be
the case.
Recovery of Milk Components

Yield efficiency can be determined by monitoring recovery of milk components and losses in the whey as
recommended by Gilles and Lawerence N.Z.J. Dairy Sci. Technol. 20(1985):205. By keeping accurate records of
all incoming milk components and their distribution between cream, cheese, whey cream and defatted whey it is
possible to determine the plant mass balance.

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8/23/2017 Cheese Making Technology eBook

Cheese Making Technology eBook

Section A: Getting Started

Family 1. Acid-coagulated Fresh Cheese

Varieties: Cottage, Quark and Cream

Family 2. Rennet-coagulated Fresh Cheese

Varieties: Queso Blanco, Queso Fresco, Italian fresh cheese, Halloumi

Curing: Consumed fresh and has a shelf life of only 2 - 4 weeks.

Family 3. Heat-Acid Precipitated Cheese

Family 4. Soft-Ripened Cheese

Curing Time: 2 - 8 weeks.

Family 5. Semi-hard Washed Cheese

Curing: 2 weeks - 9 months.

Family 6. Hard Cheese: Low temperature

Family 7. Hard Cheese: High Temperature

Other Technological Criteria

Species: cow, goat, sheep, buffalo, yak, other

Pfizer Cheese Monographs. C. Pfizer and Co., New York.

1. Italian Cheese Varieties

4. Ripened Semi-soft Cheeses

Centre For Dairy Research, Madison, WI. http://www.cdr.wisc.edu/

Agriculture Canada, http://res2.agr.ca/

Analytical Quality Control

Weigh 40 g cheese into a blender container

1. Balance, 1,000 g capacity

See discussion of pH and acidity in Section 3.5.

Apparatus and Reagents

For the 8.8 ml pipette, % Lactic acid = titre

For the 17.6 ml pipette, % Lactic acid = 0.5 x titre

For the 9.0 ml pipette, % Lactic acid = 0.98 x titre.

When using TA to monitor initial culture activity note that:

[H+] x [OH-] = 10-14

If [H+] = [OH-] the solution is neutral with respect to acidity.

pH 7.0 is neutral acidity [H+] = [OH-]

pH < 7.0 = acid condition [H+] > [OH-]

pH > 7.0 = alkaline condition [H+] < [OH-]

pH Versus Titratable Acidity

Babcock Methods for Milk Fat

Cream and Cheese

Skim milk, Buttermilk, Whey

1. Temper sample to 20C and mix gently.

Volhard procedure for salt determination

Apparatus and Materials

1. A torsion moisture balance

5. Burette graduated in ml and 1/10 ml, and burette stand

1. Prepare cheese sample as for cheese moisture test.

6.00 ml 0.1711 N silver nitrate combined with salt in cheese.

Therefore per cent salt by weight = 6.00/3.00 = 2.00

Culture Activity Test

Culture Activity Test

Bromocresol Purple (BCP) Phage Inhibition Test

BCP stock solution (1 g/100 ml water)

2. Add Whey to BCP Milk and Make Dilutions

3. Add Culture to Control and Whey Dilution Tubes

0 = No inhibition at any dilution

1 = Partial inhibition at 10-2 dilution

2 = Full inhibition at 10-2 dilution

3 = Partial inhibition at 10-4 dilution

4 = Full inhibition at 10-4 dilution

5 = Partial inhibition at 10-6 dilution

6 = Full inhibition at 10-6 dilution

An official method is described in a separate inhibitor policy document which states:

Growth Inhibition Assays