Article

DOi: 10.5504/bbeq.2012.0055

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MODELING THE RIPENING DEGREE AND BIOREACTOR DESIGN OF TULUM CHEESE RIPENING BY PENICILLIUM ROQUEFORTI IMMOBILIZED IN SOLID-STATE BIOREACTOR
Ahmet Erdoan1 and Yakup ermurat2 1 Ataturk University, erzurum technical Vocational School, erzurum, turkey 2 Ataturk University, hinis technical Vocational School, hinis, turkey correspondence to: Yakup ermurat e-mail: yakupermurat@gmail.com

ABSTRACT

Modeling the ripening degree and bioreactor design was done for tulum cheese fermentation in batch-packed solid state fermentation (SSF) processes by using toxic and nontoxic strains of Penicillium roqueforti. Water soluble protein and ripening degree were evaluated to determine the bioreactor performances. The calculated kinetic constants and the statistical analysis showed that the batches of SSF reactor ripened with nontoxic strains of P. roqueforti at 12 C gave the highest values of soluble protein and, consequently, ripening degree and highest productivity yield. The highest productivity yield of ripening degree was recorded in SSF reactor with a lipid content of 6.45%, acidity of 0.64%, pH of 6.29 and nontoxic strains of P. roqueforti at 12 C. Biotechnol. & Biotechnol. eq. 2012, 26(4), 3129-3131 Keywords: modeling, ripening degree, Penicillium roqueforti, solid state fermentation operation costs through high yield overall productivity. long fermentation time and unsolved engineering problems in the scaling up of the SSF processes are the major disadvantages and negatively influence the overall performance of SSF reactors (4). tulum cheese is a type of blue cheese which is produced by using P. roqueforti in an SSF process. the fungal SSF process of tulum cheese ripening relies on the breakdown of protein macromolecules into amino acids releasing unique flavors. The aim of this study was to model the changes in water soluble protein (PS) and make statistical analysis of the ripening degree to determine the bioreactor performances for tulum cheese fermentation in batch-packed solid state fermentation (SSF) processes by using toxic and nontoxic strains of Penicillium roqueforti.

Introduction

Ripening grade is used as a parameter of maturation grade in cheese production to measure the breakdown of protein macromolecules into peptides derived from casein hydrolysis and amino acids products of proteolysis which are soluble in the aqueous phase of cheese as well as extractable with water (1, 7). Proteolysis is affected by the ripening period and technological parameters, such as curd preparation, cheese composition, starter type and ripening conditions, have direct and indirect influence on cheese ripening (9). Time, temperature, moisture, salt content, acidity and ph are key controlling parameters of the tulum cheese ripening process which affect the microbial growth and metabolic activities like enzymatic and nonenzymatic decomposition of the cheese constituents. the strain type and quantity of fungi most of all influence the product yield and quality. The ripening degree related to water soluble protein (PS) shows the level of substrate conversion into product, and thus, the overall productivity of the bioprocess and performance of the bioreactor. Solid-state fermentation (SSF) is a ripening process that has been defined as processing of a low-moisture solid substrate for fermentation of valuable bioproducts. Various types of SSF bioreactors have been used traditionally and industrially due to their easy and low cost operation and productivity to produce cheese types with exceptional taste and flavor (2, 3, 5, 6). The SSF process is mainly used to cultivate fungi in tight contact with the insoluble substrate concentrations for fermentation (8). Fungi are the most commonly used microorganisms in SSF processes for the production of fermented products. like other SSF processes, fungal SSF processes involve cheap Biotechnol. & Biotechnol. eq. 26/2012/4

Materials and Methods

one-kilogram capacity plastic jars were used as ripening bioreactors. tulum cheese samples were placed inside the bioreactors by pressing to achieve controlled and limited oxygen diffusion and were left for ripening. tulum cheese samples were mixed separately with toxic and nontoxic P. roqueforti strains which were isolated and identified by liquid substrate Yeast-extract-Sucrose (YeS) broth fermentation culture trials at 5 C and 12 C. The cheese-filled plastic jars used as batch filled solid state bioreactor had their opening holes covered with cotton cloth to allow moisture to disperse from the cheese matrix to the gravel bath. the jars were placed on the gravel bath upside down; then the cheese was left for ripening for 4 months at 5 C and 12 C. Physical mixing or shaking, aeration or heating were not applied to the bioreactor. Volume, time, pressure and temperature were kept constant during the ripening and changes of moisture, salt content, acidity and ph were monitored. Water soluble protein 3129

(PS) and ripening degree measurements were determined by observation of protein amount. Fig. 1 shows a drawing of the bioreactor system used for tulum cheese ripening in batch packed solid state fermentation.

the ripening processes were assumed to be mostly enzymatic and zero order reaction due to high substrate amount. eadiehofstee plot of MichaelisMenten kinetic model was used for the determination of Vmax and Km values. the MichaelisMenten equation is rearranged for linearization of the data and for determination of Vmax (y intercept) and (Vmax /Km) (x intercept) by eadiehofstee plot (V) vs. (V/[S]):

the eadiehofstee plots in Fig. 3 were used for the estimation of Vmax and Km values for water soluble protein changes, which are given in Table 1.
Eadie-Hofstee of Michaelis-Menten Zero Order Kinetics for (P S) % changes 1.8 1.7 1.6

Fig. 1. Solid state fermentation bioreactor used for cheese ripening.

(d(P )/dt)

1.5 1.4 1.3 1.2 1.1 1 -0.8

Results and Discussion

Water soluble protein (PS) observations were plotted against time to see changes of protein breakdown and to define polynomial equations. Fig. 2 shows the plot lines of water soluble protein (PS) vs. time (t) (month) and the results verify that the amount of water soluble protein reached up to 8.69 % in the cheese sample ripened with nontoxic strains of P. roqueforti at 12 C. the levels of water soluble protein increased rapidly from the beginning of the ripening through the end of the ripening fermentation.
Water Soluble Protein (PS) % vs Time (t) (Month) 9

-0.7

-0.6

-0.5 -0.4 ((d(PS)/dt)/ (PS))

-0.3

-0.2

-0.1

Toxic Strain of Pr at 5C NonToxic Strain of Pr at 5C Toxic Strain of Pr at 12 C NonToxic Strain of Pr at 12 C

Fig. 3. eadie-hofstee plot of MichaelisMenten kinetic model for water soluble protein.

TABLE 1 Vmax and Km values for water soluble protein toxic strain of Pr at 5 C nontoxic strain of Pr at 5 C toxic strain of Pr at 12 C nontoxic strain of Pr 12 C
40

Water Soluble Protein (P ) %

Vmax -0.2111 -0.187 -0.13 -0.168

Km 1.79 1.67 1.73 1.57

4 Toxic Strain of Pr at 5C NonToxic Strain of Pr at 5C Toxic Strain of Pr at 12 C NonToxic Strain of Pr at 12 C

Box Graph of ANOVA for Ripening Degree (P S / PT)

35

Ripening Degree (P / P )

2 1

1.5

2.5

3 3.5 Time (t) (Month)

4.5

30

Fig. 2. Water soluble protein vs. time plot.

25

Bioreactions of the microbial conversions were modeled by using MichaelisMenten equation: , where: [P] is the concentration of the product P; [S] is the concentration of the substrate S; Vmax is the maximum velocity, Vmax = k2[E0]; [E0] is the initially prepared concentration of the enzyme; and KM is the Michaelis constant.

20

15

10 Toxic Pr at 5C NonToxic Pr at 5C Toxic Pr at 12C NonToxic Pr at 12C

Fig. 4. Box graph of AnoVA for ripening degree (PS/Pt).

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the ripening degree (PS/PT) is a dimensionless form of the water soluble protein (PS) and total protein (PT) ratio. the Biotechnol. & Biotechnol. eq. 26/2012/4

statistical analysis of the ripening degree results showed that the cheese ripened with nontoxic strains of P. roqueforti at 12 C was significantly different from that in the other experiments. A very low P-value (8.88502e-005) was found in the statistical analysis for the ripening degree which states that the differences between the results are highly considerable. the box plot in Fig. 4 shows that the ph of the nontoxic strain of P. roqueforti at 12 C was significantly different from that in the other experiments. The SSF reactor with a lipid content of 6.45 %, acidity of 0.6 4%, ph of 6.29 and nontoxic strains of P. roqueforti at 12 C offered the highest productivity yield in terms of ripening degree. the constants of the MichaelisMenten kinetic model were estimated for the water soluble protein changes in batchpacked tulum cheese. the batches of SSF reactor ripened with nontoxic strains of P. roqueforti at 12 C gave the highest values of soluble protein and gave the highest productivity yield. the statistical analysis of the results showed that the differences in the ripening degree values were found to be significant for the nontoxic strain of P. roqueforti at 12 C.

Conclusions

1. Fox P.F. (1988) J. Dairy Sci., 72, 1379-1400. 2. Hesseltine C.W. (1972) Bitechnology and Bioengineering, 14, 517-532. 3. Hlker U., Hfer M., Lenz J. (2004) Appl. Microbiol. Biotechnol., 64, 175-186. 4. Mitchell D.A., von Meien O.F., Krieger N., Dalsenter F.D.H. (2004) Biochem. eng. J., 17, 15-26. 5. Moo-Young M., Moriera A.R., Tengerdy R.P. (1983) in: Fungal Biotechnology (J.e. Smith, D.R. Berry, B. Kristiansen, eds.), edward Arnold Publishers, london, 117-144. 6. RaimbaultM., Alazard D. (1980) eur. J. Appl. Microbiol., 9, 199-209. 7. Rank T.C., Grappin R., Olson. N.F. (1985) J. Dairy Sci., 68, 801-805. 8. Tengerdy R.P. (1985) Trends Biotechnol., 3, 96-99. 9. Van der Berg G., Exterkate F.A. (1993) int. Dairy J., 3, 485-507.

REFERENCES

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