European Journal of Clinical Nutrition (2002) 56, Suppl 4, S2S15

2002 Nature Publishing Group All rights reserved 09543007/02 $25.00

www.nature.com/ejcn

Fermented milks: a historical food with modern

applications a review
AY Tamime1*
1

Dairy Science and Technology Consultant, Ayr, Scotland, UK

Background: This paper was presented at the symposium which was organized by Instituto Danone Mexico in May 2001, and it
provides an overview of the current scientific knowledge on fermented milks concerning the historical developments,
manufacturing stages, classification of such products, and nutritional aspects. Particular attention has been paid to the
human health benefits associated with the consumption of these products, the use of probiotic starter cultures and their
industrial applications, and the significance of using a trained sensory panel for the evaluation of probiotic fermented milks made
with different commercial blends of starter cultures. The paper also highlights the future research areas for the exploitation of
starter microflora (Lactobacillus, Bifidobacterium and Enterococcus species) in fermented milk products.
Conclusion: This review indicates that the complex metabolism of the starter cultures is well established; however, more
information is still needed on specific microbial metabolites such as polymerization of milk sugars for the production of
exopolysaccharides and the modification of the milk peptides and secretion of bacteriocins. More clinical studies are still
required to establish the functional health benefits of probiotic fermented milks to humans.
Sponsorship: The visit to Mexico City was supported by Instituto Danone Mexico.
European Journal of Clinical Nutrition (2002) 56, Suppl 4, S2 S15. doi:10.1038/sj.ejcn.1601657
Descriptors: review; fermented milks; starter cultures; probiotic microflora; health benefits

Introduction
Fermented milks are widely produced in many countries.
This type of process is one of the oldest methods used to
extend the shelf-life of milk, and has been practised by
human beings for thousands of years. The exact origin(s) of
the manufacture of fermented milks is difficult to establish,
but it is safe to assume that it could date to more than
10 000 y ago as the way of life of humans changed from food
gathering to food producing (Pederson, 1979). This change
also included the domestication of certain mammals such as
the cow, sheep, goat, buffalo and camel; it is most likely that
the transition occurred at different dates in different
countries. However, archaeological evidence of certain
civilizations (Sumerians, Babylonians, Pharos and Indians)
suggests that they were well advanced in agriculture and in
the production of fermented milks.

*Correspondence: AY Tamime, Dairy Science and Technology Consultant,

24 Queens Terrace, Ayr KA7 1DK, Scotland, UK.
E-mail: adnan@tamime.fsnet.co.uk

It is likely that the origin of these products was the Middle

East and the Balkans, and the evolution of fermented milks
through the ages could be attributed to the culinary skills of
the inhabitants living in these regions. Todays fermented
milk products are manufactured in many countries, and the
stages of manufacture, which is still a complex process,
combine the art of such ancient craft and science (microbiology and enzymology, physics and engineering, and
chemistry and biochemistry) together.
This article summarizes the research findings on yoghurt
and probiotic fermented milk products made from cows
milk over the past century.

Diversity of fermented milks

Fermented milks are manufactured throughout the world,
and approximately 400 generic names are applied to traditional and industrialized products (Kurmann et al, 1992), but
in actual essence the list may only include few varieties. In
the 1980s, Kurmann (1984) attempted, in part, the classification of fermented milks into a family tree (see Figure 1;
Bylund, 1995), which was based primarily on the optimum

Fermented milks a review

AY Tamime

S3

Figure 1

The family tree of fermented milk types. Adapted from Kurmann (1984).

growth requirements of the starter cultures (ie mesophilic

and thermophilic microflora).
Nevertheless, taking into account the microorganisms
that dominate the product, including their principle
metabolites, Robinson and Tamime (1990) proposed a
scheme for the classification of fermented milks as follows:

lactic fermentations (a) mesophilic type, eg cultured

lk, ta tmjo
lk and la ngofil; (b) thermobuttermilk, filmjo
philic type, eg yoghurt, Bulgarian butter-milk, zabadi,
dahi; and (c) therapeutic or probiotic type, eg acidophilus
milk, Yakult, ABT, Onka, Vifit); products within this
group constitute by far the largest number known
worldwide;
yeast lactic fermentations (kefir, koumiss, acidophilus
yeast milk); and
mould lactic fermentations (villi).

Tamime and Marshall (1997) have detailed the manufacturing stages of these types of fermentations. However, the
so-called fermented milk drinks and=or beverages including
the carbonated products should be classified separately
rather than being known as fermented milks; such an
approach will minimize confusion among consumers. Nevertheless, some closely related products are manufactured from
fermented milks by de-wheying, and some examples are
labneh, skyr and ymer. The different methods available to
manufacture concentrated fermented milks are as follows:

(a) cloth bag or Berge system; (b) mechanical or nozzle

separators; (c) ultrafiltration (UF); and (d) product formulation (Tamime & Robinson, 1999).

Patterns of consumption
Until the 1950s, production and=or consumption of yoghurt
(ie natural type) was confined to communities in the Middle
East, the Balkans, India, Eastern Europe, to ethnic groups
living in different parts of the world, and to those who
perceived that the product was beneficial to health. However, consumers attitudes towards yoghurt have changed,
possibly for the following reasons: (a) as refrigeration became
widespread worldwide, the product became widely distributed and readily available on the market; (b) the introduction
of a new generation of yoghurts (eg addition of fruits and
sugar) gave the product an entirely fresh image and it
became an inexpensive snack or dessert; and (c) the advent
of incorporation of probiotic bacteria into the product have
enhanced the health benefits of fermented milks.
Consumption figures reflect the expanding markets in
some selected countries between 1970 and 1999 (Table 1).
Until the early 1990s, fermented milks were classified as
buttermilk, yoghurt and others according to the statistical
data published by the International Dairy Federation, and
such information provided consumption preferences of consumers in different countries; the current approach of
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AY Tamime

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Table 1 Per capita annual consumption (kg=head) of milk drinks
and fermented products including yoghurt
Country

1975

1980

1990

1999

Australia
Austria
Belgium
Canada
Chile
Czech=Slovakia
Denmark
Federal Germany
Finland
France
Iceland
Israel
Italy
Japan
Netherlands
Norway
Poland
Spain
Sweden
Switzerland
UK
USA

1.0
7.9
11.8
5.1
a
NR
3.9
36.1
16.5
32.6
9.6
1.7
14.1
NR
2.5
24.7
9.8
3.2
3.4
24.1
16.4
1.7
9.9

1.8
9.8
7.7
2.3
1.4
7.3
26.7
10.1
41.0
9.3
5.7
14.3
1.3
2.4
27.3
10.1
2.0
6.0
23.5
14.8
2.8
3.1

3.5
10.4
8.4
3.7
3.9
NR
21.6
14.2b
38.3
16.4
24.6
NR
4.0
7.8
32.5
14.9
NR
8.0
29.1
19.0
4.4
3.5c

5.8
15.1
20.3
4.2
6.7
20.0
29.8
25.5b
NR
27.4
NR
29.3
NR
NR
NR
19.9
7.9
15.4
30.2
NR
NR
NR

NR not reported.
Data includes German Democratic Republic.
c
Data for 1993.
Data compiled from Tamime and Robinson (1999) and IDF (2000).
b

per capita consumption figures covers milk drinks and fermented milks including yoghurt. It could be argued, however, that this classification of these products may not
provide a clear picture or patterns of consumption. Furthermore, in some countries, the consumption of buttermilk is
not properly defined because:

traditional=natural buttermilk is the by-product of butter

making from ripened or cultured butter;
cultured buttermilk is the fermentate of skimmed milk
followed by the addition of butter flakes; and
sweet buttermilk is not fermented; hence the data for
buttermilk in such statistical tables have to be assessed in
a cautious manner.

It is safe to conclude that fermented milk products that

fall within the category of lactic fermentations (mesophilic
type) are widely consumed in northern European countries,
whilst the yeast lactic fermented milks are popular in the
Russian Federation, eastern European countries and Mongolia. Conversely, natural yoghurt is popular in countries
between the Balkans and the Indian sub-continent, while
the fruit flavoured and=or flavoured products are normally
consumed in the rest of the world.
Little data is available on the world production figures of
probiotic-fermented milks but during the last decade production has increased dramatically in some selected countries.
For example, the French market of fermented milk
containing Bifidobacterium spp. and Lactobacillus acidophilus
European Journal of Clinical Nutrition

increased from 1500 to > 91 000 tonnes between 1986 and

1990 (Renard, 1998).

Definition and terminology of fermented milks

including yoghurt
The existing legal standards or the provisional regulations of
yoghurt in many countries classify the product on the basis
of chemical composition or fat content (full, semiskimmed=medium or skimmed=low); the reviews by Robinson and Tamime (1976), Pappas (1988), Anonymous (1989,
1996), FAO=WHO (1990), and Tamime and Robinson (1999)
are recommended for further reading.
The International Dairy Federation (IDF, 1992a,b) published general standards of identity of fermented milks that
could be briefly defined as follows: Fermented milks are
prepared from milk and=or milk products (eg any one or
combinations of whole, partially or fully skimmed, concentrated or powdered milk, buttermilk powder, concentrated or
powdered whey, milk protein (such as whey proteins, whey
protein concentrates, soluble milk proteins, edible casein
and caseinates), cream, butter or milkfat all of which
have been manufactured from raw materials that have
been at least pasteurized) by the action of specific
microorganisms, which results in a reduction of the pH
and coagulation. It is of interest that in some countries the
fortification of milk components with dried dairy ingredients during the manufacture, of yoghurt for example, is
against the existing legal standards.
Other IDF specifications include the following aspects: (a)
the starter cultures shall be viable, active and abundant
( 107 cfu=g) in the finished products at all times of sale for
consumption; (b) these products may or may not be homogenized, must at least be pasteurized, contain certain permitted additives (optional), and have a shelf-life up to 30
days at 4 7 C; (c) post-fermentation heat treatment shall
not be allowed; and (d) de-wheying after fermentation is
only permitted during the manufacture of concentrated
fermented milk products such as labneh, ymer, skyr and
stragisto. Furthermore, the identity of the starter cultures is
also detailed and, in the case of yoghurt, Streptococcus thermophilus (formerly known as S. salivarius subsp. thermophilus)
and Lactobacillus delbrueckii subsp. bulgaricus are normally
used because of the associative growth that exists between
these two microorganisms, whilst in some countries, Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis (ie
non-traditional organisms) are sometimes mixed with the
starter culture. However, the name(s) of the so-called bio,
therapeutic and=or probiotic fermented milks is not well
established and should be addressed because consumers
and=or some manufacturers in different countries consider
all these products to be yoghurt (for more details refer to
subsequent section).
The milk fermentates by mesophilic microorganism
include the products that are grouped under lactic and
yeast lactic fermentations. The starter microorganisms of

Fermented milks a review

AY Tamime

the former group belong to the genera of Lactococcus(Lac. lactis

subsp. lactis, Lac. lactis subsp. lactis biovar diacetylactis and Lac.
lactis subsp. cremoris) and Leuconostoc (Leu. mesenteroides
subsp. cremoris and Leu. mesenteroides subsp. dextranicum),
and they are normally used as mixed cultures. However, the
microflora of the latter products is complex and not always
constant; numerous species of lactic acid bacteria, yeasts and
moulds have been found, for example, in the kefir grains.
Tamime and Marshall (1997) have detailed the organisms that
have been identified by many researchers in many countries.
Traditionally, many products are made from natural
yoghurt in the Balkans and the Middle Eastern countries
and these can be briefly described as follows: (a) dilution of
the fermentate with water for the production of drinking
yoghurt; (b) concentration of the yoghurt by de-wheying for
the manufacture of strained yoghurt and yoghurt cheese; the
latter product is formed into small balls and preserved in
olive oil; (c) cultured butter and=or ghee can be made by
churning full-fat yoghurt, and the aqueous phase is either
utilized as drinking yoghurt or mixed with parboiled cracked
wheat which is later sun-dried and milled into powder (such
product is known as Kishk); and (d) surplus of yoghurt is
preserved by drying or gently heating yoghurt for few hours
over low and smokey fires (the end product is known as
smoked yoghurt). Nevertheless, at present the classical
approach to categorize yoghurt products is based on its
physical characteristics as shown in Table 2 and, in particular, yoghurt is further sub-divided into different groupings
based on the following aspects:

legal and=or proposed standards classify the product, for

example, on the fat content (full, medium or low);
physical nature of the product (ie set or stirred; drinking
yoghurt is normally considered as stirred-type of low
viscosity);
flavours of yoghurt are divided into plain=natural,
fruit or flavoured; the latter two products are normally
sweetened;
probiotic, bio and=or therapeutic yoghurts contain
non-traditional organisms beside Lb. delbrueckii subsp.
bulgaricus and S. thermophilus (see Table 5);
miscellaneous yoghurts include pre-fermentation processing (lactose hydrolysis or replacement of the animal fat
with vegetable oil) or post-fermentation processing (vitamin fortification or heat treatment).

Table 2 Proposed scheme for the classification of

yoghurt products
Category

Physical state

Yoghurt products

I
II
III
IV

Liquid=viscous
Semi-solid
Solid
Powder

Yoghurt
Concentrated=strained
Frozen
Dried

After Tamime and Robinson (1999).

Reproduced by courtesy of Woodhead Publishers, UK.

S5

Technology of manufacture
Currently, the technological aspects of fermented milks are
well established and extensively reviewed (Rasic & Kurmann, 1978; IDF, 1984, 1986, 1988, 1992c; Chandan,
1989; Tamime & Marshall, 1997; Tamime & Robinson,
1999). The principal manufacturing stages and=or processes
for all fermented milk products (set- or stirred-type) have
many features in common, which can be briefly described
as follows:

Preparation of the milk base

The fat and the non-fat solid (SNF) contents of the milk are
standardized and fortified to ensure compliance with existing legal standards. The fat level may range between < 0.5
and  5.0 g=100 g, whilst the SNF is increased to  14 g=
100 g (of which 5 g=100 g is protein). The fortification of the
latter fraction(s) enhances the viscosity of the product, and
the methods available include the addition of powders or
concentration by evaporation or membrane filtration. It is
recommended that recombination of powders is carried out
at  40 C, and followed by filtration (to remove any
undissolved or scorched particles) and de-aeration (to
remove the air which is incorporated during recombination
stage). The presence of air in the milk base does not provide
the appropriate growth conditions of Lactobacillus acidophilus and bifidobacteria. It also increases heat-exchanger
fouling, and affects the characteristics of the gel, especially
the products fermented with mesophilic lactic starter
cultures.

Homogenization
Advantageous physical chemical changes caused by homogenization of the milk base occur during the manufacture of
fermented milk, which may include: (a) a reduction in the
diameter of fat to < 2 mm, these small globules being less
inclined to coalesce into larger units and rise to the surface;
(b) the milk becomes whiter in colour due to enhanced light
reflectance and scattering; (c) an increase in viscosity of the
product due to interaction and=or adsorption of the fat
globules onto the casein micelles; and (d) a decrease in
syneresis of the gel due to increase in its hydrophilicity
and water-holding capacity as a result of the interaction(s)
of the proteins.
A typical operating pressure used during the processing
of the milk base ranges between 15 and 20 MPa at 60 90 C,
and the homogenizer (single-stage type) is placed upstream
during the production of fermented milk. However, during
the manufacture of buttermilk, homogenization of the
milk base takes place on the downstream after the heat
treatment stage and, after acidification, the gel is homogenized at 5 10 MPa and 20 C; alternatively, two-stage
homogenization of the milk base may be used (Tamime
& Marshall, 1997; Tamime & Robinson, 1999; Tamime
et al, 2001).
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Heat treatment
Different temperatures are used for the heat treatment of the
milk base during the manufacture of fermented milk, and
can vary depending on the time and temperature combinations. Some examples are: (a) 85 C for 30 min; (b) 90 95 C
for 5 min; and (c) 105 C for 10 s. Thermal treatment of the
milk base induces numerous physical and chemical changes
that are complex and multi-functional; the topic has been
extensively studied (see the reviews by IDF, 1995; Tamime &
Marshall, 1997; Tamime & Robinson 1999), and in the
context of fermented milks the effects of heat treatment are:

destruction and=or elimination of pathogens and other

microorganisms present in the milk base and, hence, the
processed milk provides a good growth medium for the
starter cultures;
changes in the physical chemical properties of the
milk constituents, mainly the denaturation of the blactoglobulin, which forms a complex with k-casein;
such interaction minimizes syneresis and improves the
firmness of the gel; the protein=fat interaction in milk
should not be overlooked;
production of components that are stimulatory to the
starter cultures.

Starter culture
The heated milk base is cooled to the desired incubation
temperature of the product; some examples are: (a) yoghurt
at 40 45 C for up to 3 h, at 30 C for 18 h or  5 h when
using direct-to-vat inoculation (DVI) starter culture; (b) probiotic products at 37 C for a few hours or up to 5 7 days
depending on the organism(s) used; (c) buttermilk at 20
30 C for up to 10 20 h; and (d) kefir at 22 C for 16 24 h
depending on the type of kefir grains used (Tamime &
Marshall, 1977; Wszolek et al, 2001).
Gel formation in milk is mediated by acid coagulation
due to the activity of the starter cultures and their enzymes
to hydrolyse the lactose and to a lesser degree the proteins.
The mechanisms involved of casein dissociation and aggregation during acid-induced gelation of milk are pH-, ion
concentration- and temperature-dependant, and these
aspects of gelation are similar for set- or stirred-type fermented milks; in the former type, the milk is incubated in
the retail container, whilst for the stirred product the milk
is incubated in large fermentation tanks.

Cooling and miscellaneous handling

Cooling is only method used to control the metabolic
activity of the starter cultures and their enzymes in order
to retain an abundant count of these organisms in fermented milks. The primary objective is to cool the product to
 5 C, and the process of cooling commences at  4.6 pH,
which is carried out using a one- or two-phase cooling. The
latter method is widely applied during the manufacture of
European Journal of Clinical Nutrition

fruit-flavoured fermented milk products; the first stage of

cooling, for example for yoghurt, reduces the temperature
from 45 to 20 25 C prior to the addition of fruit and packaging, and the second phase of cooling takes place in the cold
store (chill tunnel is optional), where the product is cooled to
< 10 C (Tamime & Robinson, 1999; Tamime et al, 2001).

Historical background on probiotic fermented milks

The gross chemical composition of fermented milks provides
a useful indication of the potential nutritional value of these
products. The main components are protein, fat,
carbohydrate, minerals and vitamins. However, the bioactive
peptides (eg casomorphins, a- and b-lactorphin, immunopeptides, lactoferricin or phosphopeptides), which are
claimed to be health-enhancing components, should not
be overlooked (Meisel & Bockelmann, 1999). The beneficial
health properties of these products and their use in the
treatment of body ailments dates back to few thousands
years ago; they have been also mentioned in Biblical scriptures, and some ancient scientists have prescribed them as
medicine for curing metabolic disorders of the stomach and
the intestine (Oberman & Libudzisz, 1998). However, the
health aspects of fermented milks including yoghurt became
apparent in the late 1800s and the early part of the 1900s; in
part, this could be attributed to the scientific observations
and=or explanations by Tissier and Moro (Rasic & Kurmann,
1983) and Metchnikoff (1910). Tisser had isolated Bacillus
bifidus communis (currently known as Bifidobacterium bifidum)
and this microorganism was predominant in the faeces of
breast-fed infants, whilst Moro around the same period
isolated Bacillus acidophilus (currently known as Lactobacillus
acidophilus) from the stools of breast-fed infants. Metchnikoff
(1910) put forward the beneficial effects of lactic acid bacteria (Bulgarian bacillus, which was later designated Lactobacillus bulgaricus and currently known as Lb. delbrueckii subsp.
bulgaricus), that were present in yahourth, and postulated
the longevity-without-aging of the Caucasians with high
consumption of such product.
As a consequence, the views and=or observations of Tisser,
Moro and Metchnikoff caused a considerable flurry of interest among scientists and=or doctors regarding the beneficial
role of certain microorganisms (eg Bifidobacterium spp. and
the yoghurt starter cultures) that can colonize and possibly
displace toxin-producing organisms that are present in the
intestinal tract of humans. These aspects have initiated
scientific studies on the human health benefits associated
with lactic acid bacteria in fermented milks, and it was not
until the early part of the last century that Lb. acidophilus was
identified as being able to colonize and proliferate in
humans rather than Lb. delbrueckii subsp. bulgaricus and
S. thermophilus (Rettger & Cheplin, 1921; Rettger et al,
1935). Research studies in this field have been enormous
over the past years, and the knowledge acquired in terms of
human health benefits of fermented milks has helped, in
part, to increase the consumption of these products in many

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AY Tamime

S7
countries. Nevertheless, who could have imagined in the
1950s that these products would be used successfully for the
treatment of certain human diseases in the 1980s or 1990s?
(See also Shortt, 1999.)

Probiotic microorganisms
The current definition of probiotics, as agreed by European
scientists, is a live microbial feed supplement that is
beneficial to health (Salminen et al, 1998a). However, the
microflora of the human intestinal tract in both infants and
adults is highly complex and many bacterial species have been
identified; the topic has been extensively researched, and the
following selected reviews provide relevant information with
specific emphasis on probiotic microorganisms and their
health applications to humans (Robinson, 1991; Fuller,
1992, 1997; Sanders, 1994; Salminen et al, 1996a, 1998b, c;
Marshall & Tamime, 1997a; Holzapfel et al, 1998; Salminen &
von Wright, 1998; Dunne et al, 1999; Hirayama & Rafter, 1999;
Lee et al, 1999; Tannock, 1999; Fuller & Perdigon, 2000;
Salminen, 2001). Periodically, Danone Vitapole Recherche
publishes monographs (Capron et al, 2000; Malagelada et al,
1999; Denariaz et al, 1999; Gibson et al, 2000; Hartley et al,
2001) and World Newsletter (23 issues have been published)
updating the health aspects associated with fermented milks.
As mentioned earlier, lactic acid bacteria, yeast, moulds or
combinations of these are widely used in the production of
fermented milks and cheeses. The characteristics of these

Table 3 Examples of probiotic micro-organisms that

are used during the manufacture of fermented milks
Genera
Lactobacillus

Pediococcus
Bifidobacterium

Lactococcus
Enterococcus
Saccharomyces
a

microorganisms have been recently reviewed by Tamime

(2002), and only the relevance of probiotic microflora in
fermented milks will be discussed. It is evident that these
bacterial species have to withstand the acidic and bile salts
conditions in the gastro-intestinal tract of humans when
compared with traditional dairy starter cultures, and reach
and=or colonize the large intestine. Researchers in different
countries have identified many probiotic microorganisms,
and Table 3 illustrates some examples.
The recent taxonomy and physiology of probiotic lactobacilli species have been reported by Klein et al (1998), and
these species belonged to: (a) Lb. acidophilus group; (b) Lb. casei
group; and (c) Lb. reuteri=Lb. fermentum group; most Lb.
acidophilus strains used in the manufacture of probiotic
fermented milks have been identified as Lb. johnsonii or
Lb. gasseri; both species are members of the Lb. acidophilus
group. Hence, the issue of health-promoting properties of
such products is questionable, and it cannot be assumed that
all strains of Lb. acidophilus have or possess the desirable
properties of probiotic microorganisms (Salminen et al,
1996b; Hamilton-Miller & Gibson, 1999; Ouwehand et al,
1999); furthermore, not all the commercial probiotic fermented milk products disclose the strain or species used or
the actual numbers (Hamilton-Miller, 1999; Sanders & Huis
int Veld, 1999). In some instances, researchers have referred
to Lb. rhamnosus GG as Lb. acidophilus, Lb. casei subsp. casei
or Lb. paracasei subsp. paracasei.
Until the mid 1980s, the genus Bifidobacterium was classified as Lactobacillus spp. and currently 30 different species of
bifidobacteria have been identified. The six organisms that
have been used in dairy products are shown in Table 3 but,
according to Klein et al (1998), most of the strains used in

Microbial species
Lb. acidophilus
Lb. acidophilus strains LC1,
La5, La1, La7, Gilliland
Lb. casei strains Shirota, GGa
or LGG,a Imunitass, NCC208
Lb. rhamnosus strain GG
Lb. johnsonii
Lb. helveticus
Lb. delbrueckii subsp. bulgaricus
Lb. gasseri
Lb. plantarum
Lb. paracasei subsp. paracasei
and subsp. tolerans
Lb. reuteri
P. acidilactici
B. bifidum, breve, longum,
adolescentis, infantis,
lactis, animalis
Lc. lactis subsp. lactis
E. faecium, faecalis
b
S. boulardii

This strain could be confused with Lb. rhamnosus GG due

to latest nomenclature classification.
b
Most likely application is during the manufacture of kefir.
Data compiled from Tamime et al (1995), Tamime and
Marshall (1997), Shortt (1997), Lee et al (1999) and
Sanders and Huis int Veld (1999).

Table 4 A summary of some health-promoting activities in

humans associated with probiotic fermented milks
Action=effect

Alleged health benefit

In digestive tract

Active against Helicobacter pylori

Enhanced lactose digestion
Stimulation of the intestinal immunity
Stabilization of Crohns disease
Stimulation of intestinal peristalsis

On intestinal
microflora

Improves balance between

microbial populations
(eg increase in faecal bifidobacteria)
Decrease in faecal enzyme activity
Colonisation of the intestinal tract
Reduced carrier time for Salmonella spp.

On diarrhoea

Prevention=treatment of
acute and of Rotavirus diarrhoeas
Prevention of antibiotic-induced diarrhoea
Treatment of relapsing Clostridium difficile
diarrhoea

Other effects

Improved immunity to disease

Suppression of some cancers
Reduction in serum cholesterol
Reduction in hypertension

Adopted from Tamime and Robinson (1999).

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probiotic fermented milks are B. animalis, although they
are declared on some labels as B. longum. B. animalis was
renamed B. lactis in the late 1990s (Meile et al, 1997), and
now it is re-classified as B. animalis (Cai et al, 2000). In my
view, the term bifidus should not be used because it is old
terminology (Lactobacillus bifidus), and can cause some confusion to the reader.
The 16S rRNA sequencing within the genus Entrococcus
has revealed the presence of three species groups: E. faecium
and E. durans are found in the first group, whilst E. faecalis
forms an individual line of descent. However, the pathogenicity of these organisms should not be overlooked when they
are used in probiotic fermented milk products.
Limited data are available on the use of the probiotic yeast
(Saccharomyces boullardii) in fermented milks (LourensHattingh & Viljoen, 2001a), and the most likely application
is Kefir and Koumiss making. Other non-lactic acid bacteria
that have been used in probiotic products around the world
include Bacillus cereus, Escherichia coli and Clostridum
butyricum (Shortt, 1999).

Table 5

I. Fermented milks
ABT, Biogarde1, Philus

ACT4
Vifit, Gefilus

SymBalance
Actimel
Biola
Gaio

Country

Microorganismsa

Germany
UK
Denmark
Sweden
Austria
Germany
UK
Netherlands
Belgium
Finland
Switzerland
France
Norway
Denmark
UK
Germany
Finland
Sweden

Lb. acidophilus B. lactisb or bifidum S. thermophilus

II. Fermented milk beverages

Onaka He GG
Japan
Yakult
Japan
Brazil
Some Asian countries
Caldus
Japan
Vitagen
Malaysia
Singapore
Aktifit
Switzerland
LC1 or LC1 GO
Many European countries
a

It is well established that the probiotic microorganisms

shown in Table 3 have therapeutic properties, and when
they are added with traditional starter cultures during the
manufacture of fermented milks (see later), such products
have certain health-promoting activities in humans; Table 4
attempts to highlight the possible advantages. However, upto-date reports on some of the clinical=nutritional studies on
humans using probiotic fermented milks and their successful
applications in the treatment of certain human diseases,
include the following: (a) adherence of probiotic microorganisms to human intestinal cells; (b) decreasing faecal
mutagenecity; (c) improvement of constipation; (d) production of bacteriocins; (e) effects on superficial bladder cancer;
and (f) effect on the human salivary microflora (MattilaSandholm & Salminen, 1998; Salminen et al, 1998b; MattilaSandholm et al, 1999a,b; Ouwehand et al, 1999; Petti et al,
2001). Some selected studies on nutritional aspects relating
to humans have been reported by Oberhelman et al (1999),
Alander et al (1999), Lee et al (1999), Kar (1999), Ashar and

Some examples of probiotic fermented milk products available in different countries

Product

BRA

Therapeutic properties of bio-fermented milks

Similar to ABT Lb. casei

Yogurtc B. bifidum Lb. acidophilus Lb. rhamnosus GG

Lb. casei Lb. reuterix Lb. acidophilus Bifidobacteria

Yogurt Lb. casei strain Imunitasse
B. lactis Lb. rhamnosus Lb. acidophilus
S. thermophilus E. faecium

B. infantis Lb. reuteri Lb. acidophilus

Yogurt Lb. rhamnosus GG
Lb. casei strain Shirota

B. longum Lb. acidophilus

Lb. acidophilus
Similar to ABT Lb. rhamnosus GG
Lb. acidophilusg or Lb. helveticus

Lb. Lactobacillus; S. Streptococcus; B. Bifidobacterium; E. Enterococcus.

Currently it is reclassified as B. animalis (Cai et al, 2000).
c
Yogurt Lb. delbrueckii subsp. bulgaricus S. thermophilus.
d
Yogurt starter cultures have been found in the product rather than bifidobacteria (Sanders & Huis int Veld, 1999).
e
Lb. casei Lb. delbrueckii subsp. bulgaricus were the only organisms identified in the product (Sanders & Huis int Veld, 1999).
f
Lb. rhamnosus was the only organism present in the product (Sanders & Huis int Veld, 1999)
g
Lb. johnsonii S. thermophilus were found in the product (Sanders & Huis int Veld, 1999).
Data compiled from Tamime et al (1995), Tamime (1998), Sanders and Huis int Veld (1999) and Lee et al (1999).
b

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Prajpati (1999), Arunachalam (1999), Barone et al (2000),
Heyman (2000), Arunachalam et al (2000), Ahn et al (2000),
Chiang et al (2000), Meydani and Ha (2000), Armuzi et al
(2001), Collins (2001), Volpe et al (2001), Kawase et al (2001),
Perdigo n et al (2001) and Reid et al (2001).
Another aspect, which has links with gut health, involves
prebiotics; they are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and=or activity of one or limited number of
bacteria in the colon, that have the potential to improve the
host health (Gibson & Roberfroid, 1995). Most potential
prebiotic food ingredients are carbohydrate-based components such as oligosaccharides (Hartemink, 1999; Gibson &
Angus, 2000). However, a mixture of probiotic microorganisms and prebiotic food ingredient is known as synbiotic.

Commercial probiotic fermented milk products

Some of the critical aspects that can affect the quality of
fermented milks are: (a) good make which is attributed to

Table 6

fast acid development using traditional starter cultures; acid

development of probiotic starter cultures is poor in comparison with Lb. delbrueckii subsp. bulgaricus and S. thermophilus;
(b) stable product after manufacture where few undesirable
microorganisms can withstand the rapid acid development
or initiate growth at pH 4.6; and (c) the lactic and sour
flavour is the characteristic flavour acidity of majority of
traditional types of mesophilic and thermophilic fermented
milks, and the action of acid on the milk proteins (mainly
the casein) is important in texture formation of the product.
However, the heterofermentation by the bifidobacteria
results in lactic and acetic acid production; the latter acid
has a harshness which is undesirable for milk products
(Marshall & Tamime, 1997b).
As a consequence, the majority of commercially available probiotic fermented milks in different markets of the
world (ca 80 100) are made with a mixture of non-traditional and traditional starter cultures, and detailed examples have been reported by Hoier (1992), Tamime et al
(1995), Tamime & Marshall (1997), Holzapfel et al (1997)

Some selected bacteriocins produced by dairy starter cultures and probiotic microflora

Genera=microorganisms
I. Lactococcus
Lac. lactis subsp. lactis

Bacteriocins

Molecular mass range (kDa)

1.3 > 10

Lac. lactis subsp. lactis biovar diacetylactis

Lac. lactis subsp. cremoris

Lactostrepcin, Nisin, Lacticin, Bacteriocin,

Dricin, Lactococcin
Bacteriocin, Lactococcin, Lactocin, Diacetin
Lactococcin, Bacteriocin, Diplococcin

II. Leuconostoc
Leu. mesenteroides subsp. mesenteroides

Mesenterocin, Leucocin

2.5 < 10

III. Streptococcus
S. thermophilus

Bacteriocin, Thermophilin

4 > 100

IV. Lactobacillus
Lb. acidophilus

3.4 5.8
3.4 8.5

Lb. johnsonii
Lb. gasseri
Lb. reuteri
Lb. casei
Lb. plantarum
Lb. rhamnosus
Lb. delbrueckii subsp.
bulgaricus and subsp. lactis

Lactacin, Acidophilin, Acidophilicin,

Acidocin, Acidolin, Bacteriocin
Lactacin
Bacteriocin, Gassericin
Reutericyclin, Reutericin
Caseicin
Plantaricin
Bacteriocin
Bulgarican
Lacticin, Lactobacillin

0.2 50

V. Enterococcus
E. faecalis
E. faecium

Enteroccin
Enterocin

VI. Bifidobacterium
Bifidobacterium spp.
B. longum
B. bifidum

Bacteriocin
Novel protein
Bifidocin

3.3 104

VII. Pediococcus
P. acidilactici

Pediocin

2.5 4.6

2.5 > 300

4.5 5.6
0.4 > 200
 41
1 5.9
< 1.0 14
a
NR
NR
7.2
NR

a
Data not reported.
After de Vuyst and Vandame (1994), Ali et al (1995), Maisnier-Patin et al (1996), Dave and Shah (1997), Marshall and Tamime
(1997a), McAuliffe et al (1998), Papathanasopoulos et al (1998), Anderssen et al (1998), Kawai et al (1998), Suma et al (1998), van
Reenen et al (1998), Yildirim and Johnson (1998), Farias et al (1999), Remiger et al (1999), Fujiwara et al (1999), Zhu et al (2000),
Ganzle et al (2000).

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Fermented milks a review

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Bacteriocin production

and Tamime (1998) (see Table 5); hence, probiotic microorganisms are used as therapeutic adjuncts in fermented
milk products (see also the review by Lourens-Hattingh &
Viljoen, 2001b).

Bacteriocins are antibacterial substances produced by

certain groups of microorganisms including probiotic and
lactic acid bacteria; they are also known as colicin-like
inhibitory substances. Incidentally, bacteriocins are usually
segregated from antibiotic compounds, and such naturally
produced metabolites inhibit the growth of undesirable
microorganisms in food and dairy products. In the case
of lactic acid bacteria, the bacteriocins produced could be
used to replace added chemical preservatives and, hence,
the shelf-life of the product could be extended by natural
means; however, these inhibitors can also affect certain
strains of starter cultures.
At present more than 70 different types of bacteriocins
have been identified as produced by lactic acid bacteria.
Table 6 summarizes some selected characteristics (for further
information refer to de Vuyst & Vandamme, 1994; Holzapfel
et al, 1995; Desmazeaud, 1994; Nes et al, 1996; Richard, 1996;
Marshall & Tamime, 1997a).

Exopolysaccharides (EPS) production

Microbial EPS material is synthesized by certain strains of
lactic acid bacteria, and it can enhance the rheological
properties (ie minimize syneresis, improve the texture and
modify the structure) and sensory perception (ie firmness
and creaminess) of fermented milks and other dairy products. The topic has been extensively reviewed (IDF, 1998;
Cerning & Marshall, 1999), and illustrations of the microstructure of both mesophilic and thermophilic fermented
milks have been reported by Tamime and Marshall (1997)
and Tamime and Robinson (1999).
Quantitative and qualitative properties of EPS material are
influenced by the following aspects: (a) microbial strain; (b)
composition of the growth medium and=or growth factors;
and (c) processing conditions that include the growth temperature and time of fermentation. In 2001, the scientific
papers presented at the first Symposium on EPS from Lactic
Acid Bacteria provided an up-to-date review of this field,
including their applications in the dairy industry and the
structures of EPS (Monsan et al, 2001; de Vuyst et al, 2001;
Boels et al, 2001; Jolly & Stingele, 2001; Degeest et al, 2001;
Duboc & Mollet, 2001; Marshall et al, 2001). However, some
of these EPS materials contain gluco- and=or fructo-oligosaccharides and may generate short-chain fatty acids upon
hydrolysis in the intestinal tract by the colonic microflora,
and may have potential health (eg an anti-tumor effect,
cholesterol-lowering effects or immune-modulators effects)
and nutritional benefits as a prebiotic to the intestinal
microflora.

Table 7

Partial characterization of the quality of

probiotic fermented milks
Many diversified probiotic dairy products are currently available to consumers in many markets, and some examples are:
pasteurized liquid milk, cheeses, ice-cream, milk powder feed
for infants and fermented milks (Tamime & Marshall, 1997).
However, fermented milk products are the most popular
vehicle used in the industry for the implantation of the
probiotic microflora in humans. In the mid 1990s, a collaborative research programme was established between the
Scottish Agricultural College (SAC) and the Hannah Research
Institute (HRI) to evaluate the quality (chemical composition, microbiological analysis, rheological properties and
sensory perception) of different types of newly developed

The microorganisms used in commercial mixed starter cultures

Bifidobacterium spp.

Code

Supplier

AB
AC=BL
DVB-100
MSK 2
e
ABT 1
ABT 3
e
MSKB 2
MY 087f

Chr. Hansen
Rhodia
SBId
Visby
Chr. Hansen
Chr. Hansen
Visby
Rhodia

Typea

bifidum

lactisb

FD
FD
FD
DF
FD
FD
FD
FD

longum

infantis

P
P
P
P

P
P
P

FD freeze dried, DF deep frozen.

Currently it is reclassified as B. animalis (see text).
c
Lb. delbrueckii subsp. bulgaricus.
d
SBI Systems Bio-Industries Ltd.
e
The starter cultures produces exopolysaccharide (EPS) material
f
This is a yoghurt starter culture, which is used as a control.
The symbol (P) denotes the presence of the microorganism in the starter culture.
b

European Journal of Clinical Nutrition

Lactobacillus spp.
acidophilus

bulgaricusc

Streptococcus
thermophilus

P
P
P
P
P
P

P
P
P
P
P
P
P

Fermented milks a review

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S11

Figure 2 Sensory space maps for probiotic fermented milks, factor

scores on first two dimensions are shown. For starter cultures symbols
refer to Table 7. After La Torre (1997).

and B. infantis 410 B. longum 2 and 913 were obtained from

Chr. Hansen, Rhodia and Visby, respectively) were used to
manufacture fermented milks. The quality of the products
made with the NCFB strains did not satisfy the minimum
requirements, the pH values were borderline (eg between 4.8
and 5.4), excessive whey separation was evident, the coagulum was extremely week and, in some cases, the odour and
taste were unpleasant. However, slight improvement in the
quality of the product was evident, especially when the milk
bases were fortified with Raftaline1 or Jerusalem artichoke,
but the overall pHs ranged between 4.79 and 5.91 after 24 h
of incubation (with the exception of strains of B. longum BBL
and 2; pH  4.6). As the results were not satisfactory, it was
decided that commercial mixed blends of starter cultures
containing lactic acid bacteria and different Bifidobacterium
species would be used (see Table 7).
The quality of the fermented milks was superior to those
made with single strains of bifidobacteria, and the results
could be summarized as follows:

fermented milk products (eg vegetable oil yoghurt, kishk,
yoghurt made with fat substitutes, kefir and probiotic fermented milks). In this paper, some aspects of the quality of
probiotics fermented milks will be reported, and for further
details refer to La Torre (1997), Hartley et al (2001) and La
Torre et al (2002).
In the first instance, single strains of Bifidobacterium spp.
(B. adolescentis NCFB 2230, B. bifidum NCFB 2715, B. breve
NCFB 2258, B. infantis NCFB 2205 and B. longum NCFB 2259,
were obtained from National Collection of Food Bacteria;
B. longum BB 46 and B. bifidum BB11 B. longum BBL and BL

Figure 3

Acidification of the milk base (total solids, protein and fat

were 14.5, 5.5 and 1.5 g=100 g, respectively) ranged
between 6 and 10 h or 13 and 22 h for pH  4.4.
Organic acid profiling indicated that the starter cultures
produced appreciable quantities of lactic acid, and only
two cultures (AB and AC=BL) produced the high levels of
acetic acid (3229 and 2794 mg=g, respectively; figures are
averages of three trials and of fresh and stored products).
The viable cell counts in fresh and stored products for
bifidobacteria varied (eg two cultures (AB and AC=BL)
showed an increase, three cultures (DVB-100, ABT 3 and
MSKB 2) remained constant, and two cultures (ABT 1 and
MSK 2) exhibited a slight decline); Lb. acidophilus
increased during production but, in some products (DVB100, MSK 2, ABT 1 and 3 and MSKB 2), the counts were

Interpretation of sensory results for fermented milk products. After La Torre (1997).

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Fermented milks a review

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S12

decreased after storage; S. thermophilus counts of all starter

cultures showed a remarkable increase during production
and survival during storage.
Syneresis values of fermented milks ranged between 2.5
and 3.0 ml for fresh products and decreased to 1.5 2.5 ml
after 20 days storage at 5 C; the rate of decrease was
dependent on the starter culture used.
The firmness measurements of all fermented milks
increased over time, and the rate was independent
of the starter culture used; after the storage period, the
firmness values ranged between 1.8 and 2.5 N for the EPS
cultures MSKB 2 and ABT 1, respectively.
Sensory analysis indicated the following: (a) acid,
creamy and=or other characters (for the odour, flavour
and after-taste attributes) were significant (P < 0.001); (b)
the flavour character other was identified to the presence of a high level of acetic acid produced by two
starter cultures (AB and AC=BL); (c) analysis of variance
accounted for over 95% of the first three factors, explaining 52.0, 30.7 and 12.9%, respectively; and (d) sensory
space maps were constructed that grouped the starter
cultures into different clusters based on the identified
significant attributes (see Figure 2). Factors 1 and 2 clearly
separated the cultures AB, AC=BL and MSK 2 from the
others, and cultures MY 087 and MSKB 2 were slightly
separated from the grouping of ABT 1, ABT 3 and DVB100 cultures. Separation on factor 1 was mainly associated with differences in acid character, whilst factor
2 resolved the fermented milk products in terms of
the contrast between creamy=sweet odour and other
(ie vinegar) flavour (see Figure 3).
It is possible to conclude that the level of metabolites
(mainly acetic acid) produced by the bacterial strain(s)
can influence the organoleptic assessment.

Conclusion
It is evident that fermented milks can be successfully produced using different blends of lactic acid bacteria and
probiotic microorganisms. Over the past century, voluminous scientific knowledge has been well established regarding the technological aspects of fermented milks, including
the physiology of starter cultures and related probiotic
microflora. However, over the coming years the possible
research areas may include the following aspects:

appropriate international definition(s) of yoghurt and

other fermented milks including probiotic products;
more properly designed clinical studies to establish the
proper health benefits to humans; in vitro results cannot
be found always in vivo, and observations reported in
animals cannot be translated directly to humans; there
are problems in generalizing the results given the large
number of types of microorganisms used and, in some
instances, the count of probiotic microorganisms in clinical studies is not reported; clinical studies should be

European Journal of Clinical Nutrition

conducted on humans of different races in different

countries to properly substantiate the health benefits to
humans in general;
how the starter culture polymerizes lactose in milk to
produce exopolysaccharide material;
what induces the microorganisms to cyclize the
peptide(s) to secrete bacteriocins and what their mechanism(s) for controlling pathogens in the gut is (are);
the pathogenicity of certain cultures requires further
clearance.

Acknowledgements
The author is grateful for the invitation to participate in the
conference and the funding provided by Instituto Danone
Mexico.

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