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1 Influence of diff erent typ es of fermentation- produced chymosin on quality of soft cheeses D S Myagkonosov, I T Smykov, D V Abramov, I N Delitskaya All-Russian Scientific Research Institute of B utter- and Cheesemak ing, Uglich,

Yaroslavl Region, Russian Federation

E-mail: mail@vniims.info

Abstract. The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content, proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe. The main proteoly tic agent in soft cheeses is the m ilk-clotting enzym e (MCE). Increasing the shelf life of cheeses can be achieved by using MCE types having low proteolytic activity (PA). We have studied the effect of MCE based on different types of fermentation- produced chymosin: Chy-max® Extra (bovine chymosin), Chy-max® M (camel chymosin), Chy-max® Supreme ("modified" chymosin) on the dynamics of proteolysis in soft cheeses and related changes in the structure of cheeses during their storage. All 3 types of studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose of MCE also increases the rate of proteolysis in cheeses. To increase the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is promising to use MCE with low PA based on camel chymosin (Chy-max® M) and "modified" chymosin (Chy-max® Supreme). 1. Introduction Soft chee ses combine the prope rties of a healthy and nutritious food prod uct a nd are widely represented among the assortment of cheeses in countries with the developed cheesemaking process.

The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content,

protein proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe, acquiring a

viscous consistency that sticks to the knife, as well as taste defects (bitterness). The main proteolytic agent in soft cheeses is the milk-clotting enzyme (MCE), up to 30% of which

is retained in the cheese mass [1]. An increase in the shelf life of cheeses can be achieved by using

technological methods aimed at reducing the proteolytic activity of MCE in cheese: the use of MCE types with low proteolytic activity (PA) [2, 3, 4] and a decrease in the dose of MCE when using coagulation conditions that compensate for the decrease in the dose of MCE due to an increase in its activity (an increase in the coagulation temperature and a decrease in the pH of milk) [5]. Since MCE is added into milk in terms of the number of units of milk-clotting activity (MCA), the resulting indicator of nonspecific PA is the MCA/PA ratio. Fermentation-produced chymosins has the highest MCA/PA ratio among all types of MCEs used in the cheesemaking industry [6]. The group of fermentation-produced chymosins includes c hymosin of various origins, which, a ccording to th e MCA/PA values, are arranged i n the fol lowing sequence: b ovine chy mosin < ca mel chymosin <

2modified chymosin. The effect of bovine and camel chymosin on proteolysis in cheeses has been

extensively studied. The obtained results show a clear decrease in the level of proteolysis and an

increase in the shelf life of cheeses when replacing bovine chymosin with a camel one [7, 8]. Modified

chymosin-based MCE was recently put on the market under the brand name "Chy-max® Supreme" by Chr Hansen A/S in 2019. The modified chymosin was obtained by artificial modification of the amino acid composition of natural chymosin (presumably, camel chymosin), which made it possible to lower

the level of its PA [9]. By now, there is very little scientific data on the use of Chy-max Supreme in

cheese production. The influence of MCE based on different types of fermentation-produced chymosin on the

dynamics of proteolysis in soft cheeses and the related changes in the structure and taste of cheeses

during storage has been investigated. The research results are of practical interest in terms of

improving the technology of soft cheeses and increasing their shelf life. The value of this work is

confirmed by the reliability of the received results using a method of experimental design and

mathematical analysis of experimental data. The scientific level of obtained data correspond to the modern level of Russian science [10, 11].

2. The purpose of the study

The purpose of this study is to increase the shelf life of soft cheeses by regulating the intensity of

proteolytic processes at the stage of cheese storage due to a reasonable choice of milk-clotting enzyme

according to the ratio of its milk-clotting and general proteolytic activity.

3. The object of the study

Soft Italian cheese "Crescenza" was chosen as the object of research, a technology feature of which is

the use of starter culture from thermophilic lactic acid microorganisms and a high coagulation

temperature (38 °C). Such coagulation temperature is close to the optimum activity of chymosin (~45

°C), which contributes to an increase in the activity of MCE and makes it possible to use lower doses

of MCE without prejudice to the duration of milk coagulation and the quality of the resulting cheese.

In the cheese production, 3 brands of MCE were used on the basis of fermentation-produced chymosin of bovine, camel and modified chymosin. The doses of MCE in the experiment were selected based on

the analysis of scientific literature from the technical documentation for MCE, as well as the results of

their own research by the authors of this article [12].

4. Materials and methods

4.1 Materials

The studies used cow's milk from one supplier-manufacturer - AgriVolga LLC (Yaroslavl Region, Uglichsky District, Burmasovo village). In the production of cheeses, we used direct-to-vat starter culture of Streptococcus thermophilus based on the bacterial concentrate STI-12 and STI-14 (Chr Hansen A / S, Denmark) and MCE brands (Chr Hansen A / S, Denmark): - Chy-max® Extra (a fermentation-produced bovine chymosin with a nominal strength of 600 international milk clotting units (IMCU)/ml, - Chy-max® M 1000 (a fermentation-produced camel chymosin with a nominal strength of 1000

IMCU/ml,

- Chy-max® Supreme (a fermentation-produced chymosin with a nominal strength of 1000

IMCU/ml.

All used reagents have an analytical grade.

4.2 Determination of general proteolytic activity milk-clotting enzymes

The determination of the total proteolytic activity was carried out in accordance to Anson method in modification described in the Russian state standard method (GOST 34430-2018).

34.3 Cheese Manufacture Three laboratory-scale 12 l-capacity cheese vats with water jacket were used for cheese manufacture.

The 11 kg portion of whole milk was weighted with the accuracy of ± 1 g and transferred into each of

the cheese vats. The typical "Crescenza" cheesemaking process according to Alinovi et al. [2] was applied. For the standardization technological process stage duration and composition of produced cheeses, the time to ready a curd for cutting was determined with special measuring equipment based on a hot-wire method [13]. After complete molding and salting stage, cheeses were packed on a Henkelman Boxer 42 machine (Henkelman Vacuum Systems, The Netherlands) under modified atmosphere (N:CO

2 70:30) into bags

made of Amivak CH-B polymer film (Atlantis-Pak, Russia) and sent for ripening at a temperature of 3

± 1 °C for 6 days to complete the whey drainage and allow curd structuring. Until 7, 14 and 21 days of

storage the cheeses were unpacked and after collection of samples were repacked and stored under same conditions.

4.4 Compositional analysis and proteolysis determination

Chemical analyses of cheeses were carried out during the 1st, 2nd and 3rd wk. of storage. The dry matter content was measured by heating it in a drying oven at 102 °C (IDF 4). The fat content of milk and cheese samples was determined by the van Guilk method (ISO 3433:2008). The protein content was determined by the Kjeldahl method (IDF/RM 25). Salt was measured by titration with AgNO

3 by the Mohr method [14]. The cheese's pH was monitored by mixing 10 g of grated

cheese with 10 ml of distilled water and measuring the pH value of the resultant slurry using a digital

pH meter (model Testo 206-pH2, Testo SE & Co. KGaA, Germany). The degree of proteolysis in cheeses was expressed as a percentage of the absolute content of soluble nitrogen from the absolute content of total nitrogen. Determination of the mass fraction of

nitrogen in water-soluble extract from cheese was carried in accordance with Kuchroo and Fox

method in the modification described in [15].

4.5 Rheometry

A study of the rheological properties of cheeses was carried out based on Weissenberg rheogoniometer model R-19 (Sangamo Weston Controls Limited, Great Britain) by the method of small amplitude

oscillatory shear testing (mode: rotation with controlled shear rate). In provided tests, a reference

oscillation with an amplitude of 1.1*10 -3 rad was applied at a frequency of 3.16 Hz. Measuring

system: cone-plate (radius R = 25 mm, cone angle α = 2°, gap = 87 μm). The measurement

temperature was 21 ± 1 °C.

Based on determined primary viscoelastic terms (the storage, G', loss moduli, G"), complex

modulus (G*) along with phase angle tangent (tan δ) were calculated [16].

4.6 Microscopic techniques

The microstructure of the cheeses was investigated by the method of light microscopy in oblique

lighting mode, on micro-sections of cheese with a thickness of 100 ± 10 μm. Image correction was

carried out using Digital Photo Professional software v.4.5 (Canon Inc., Japan).

4.7 Experimental Design and Statistical Analysis

A full factorial randomized block design, which incorporated the 3 coagulant type (Chy-max Extra, Chy-max M and Chy-max Supreme) at 3 levels of the applied dose (1500, 2500 and 3500 IMCU / 100

kg of milk) and 3 blocks (replicate trials), was used for analysis of the response variables relating to

the cheese composition, proteolysis and rheological indices [17].

Experimental data were statistically analyzed using Statistica® software (ver. 5.5, StatSoft, USA).

The cheese composition at 7 d of storage was analyzed using one-way analysis of variance and

Tukey's paired comparison with a significance level of p =0.05. A split-plot design was used to

determine the effects of the coagulant type, applied coagulant dose, storage time and their interactions

4on the response variables (pH, proteolysis, rheological indices) measured at several time points (7, 14

and 21 day) during storage. The main-plot factors were coagulant type and coagulant dose, the sub- plot factor was storage time [17].

5. Discussion of the results

Levels of milk-clotting and general proteolytic activity of the examined MCE presented in Table 1. Table 1. Milk-clotting and general proteolytic activity of MCE

MCE brand

MCA (IMCU/g)* PA (PU/g) MCA:PA ratio Dosage proteolytic activity of enzymes added to milk (PU/100 kg of milk) at applied dose (IMCU / 100 kg of milk)

1500 2500 3500

Chy-max Extra 554 0.71 780 1.92 3.21 4.49

Chy-max M 904 0.68 1330 1.13 1.88 2.63

Chy-max Supreme 912 0.26 3500 0.43 0.71 1.00 * MCA was calculated from nominal activity expressed in IMCU/ml to activity in IMCU/g based on the

measured density of MCE that are equal: for Chy-max Extra - 1.083 g/ml, for Chy-max M 1000 - 1.107 g/ml,

for Chy-max Supreme - 1.096 g/ml. The data presented in Table 1 shows that all studied MCE samples have an interspecific difference by the magnitude of PA. In compliance with data, obtained by other authors [7, 8], bovine chymosin showed a much higher PA level than the one obtained for camel chymosin whereas camel chymosin has more proteolytic activity level than the one peculiar to a "modified type" chymosin [9].

Therefore, at the same dose of MCE in terms of MCA units, a different number of units of

proteolytic activity will be added into milk. Up to 30% of the activity of the added MCE will be

retained in the cheese mass, which theoretically should affect the proteolysis intensity in the cheeses

[1]. The composition of Crescenza cheeses made with different type and dose of milk-clotting enzymes

at beginning of storage (7 d-old) and pH at beginning and the end of storage are reported in Table 2.

Table 2. Chemical composition Crescenza cheeses made with different types and doses MCE

Treatment Parameter pH

Dose* Type** Dry matter (%) Fat (%) Protein (%) NaCl (%) 7 day 21 day

Low Extra 47.64±0.74 a 25.67±0.56 a 15.77±0.45 a 0.77±0.04 a 5.30±0.04 a 5.14±0.08 ab

M 47.91±0.37 a 25.67±0.64 a 15.81±0.29 a 0.80±0.04 a 5.28±0.07 a 5.13±0.07 ab

Supr 47.86±0.52 a 25.67±0.53 a 15.94±0.38 a 0.75±0.02 a 5.28±0.04 a 5.16±0.02 ab

Med Extra 48.27±0.67 ab 26.77±0.18 a 15.98±0.36 a 0.78±0.03 a 5.24±0.09 a 5.11±0.05 a

M 48.19±0.39 ab 26.40±0.63 a 15.90±0.24 a 0.81±0.02 a 5.24±0.08 a 5.17±0.06 ab

Supr 48.95±0.72 ab 26.77±0.24 a 16.30±0.42 a 0.77±0.03 a 5.26±0.06 a 5.19±0.09 ab

High Extra 49.28±0.43 ab 26.77±0.55 a 16.31±0.26 a 0.80±0.02 a 5.27±0.05 a 5.23±0.03 b

M 49.22±0.51 ab 26.40±0.49 a 16.24±0.37 a 0.77±0.05 a 5.27±0.02 a 5.21±0.06 ab Supr 49.61±0.67 b 26.77±0.74 a 16.52±0.33 a 0.80±0.04 a 5.28±0.08 a 5.22±0.04 b

Abbreviations are:

* Low - 1500 IMCU/100 kg, Med - 2500 IMCU/100 kg, High - 3500 IMCU/100 kg. ** Extra - Chymax Extra; M - Chy-max M; Supr - Chy-max Supreme. Values are means ± standard deviations (n = 3). a,b Means within the same column not sharing a common superscript differ (Tukey's HSD, p > 0.05).

5At the beginning of the shelf life, there were no statistically significant differences in fat, protein,

sodium chloride and pH levels between the cheese options, but there was a trend towards an increase in the dry matter content in the cheeses with an increase in the dose of MCE used in the cheese

production. These differences were statistically insignificant, except for one case: the cheese options

produced with the maximum dose of Chy-max Supreme had a statistically significantly higher content

of dry solids than options of cheeses produced with minimal doses of coagulants. Data on the

composition of cheeses indicate that the type and dose of MCE affect the properties of cheeses already

at the stage of cheese production. Table 3. Mean squares and significance levels (in parentheses), and R

2 values for pH, proteolysis, and

rheological properties for Crescenza cheeses during 21 days of storage

Effect df pH

Degree of proteolysis

(%) Storage modulus G* (Pa) Loss tangent (tan δ) Type 2 0.004 (-) 67.007 (***) 71 086 530 (***) 0.22151 (***) Dose

2 0.015 (**) 20.673 (***) 10 168 610 (*) 0.01103 (-)

Age

2 0.063 (***) 140.266 (***) 1 037 347 000 (***) 1.11991 (***)

Type*Dose

4 0.002 (-) 2.202 (***) 9 041 086 (***) 0.02317 (**)

Type*Age

4 0.001 (-) 3.512 (***) 1 734 935 (-) 0.04677 (***)

Dose*Age

4 0.005 (-) 9.452 (***) 24 992 840 (***) 0.04467 (***)

Error

62 0.002 0.232 2 478 696 0.00443

R2 0.58 0.97 0.94 0.92

df - degrees of freedoms R2 - determination coefficient for the ANOVA model

Significance levels (in parentheses): -, no significant (P > 0.05); *, P < 0.05; **, P <0.01; ***, P < 0.001.

There was a statistically significant effect of the MCE dose along with the shelf life on the pH of

cheeses (Table 2, 3). The effect of these factors on the pH is associated with a decrease in the pH of

cheeses during storage, as a result of the conversion of milk sugar (lactose) into lactic acid by bacteria

of starter culture. The higher the moisture content in the cheeses, the higher the content of lactose,

which is dissolved in the aqueous phase. The extent decreasing the cheese pH is proportional to the

lactose content and consequently to the moisture content in the cheese. No significant differences in

moisture content between 7-day-old fresh cheeses made with different doses of MCE had consequences in significant differences in the cheese pH at the end of the storage period. This fact

explained the statistically significant dependence of decreasing pH of the cheese during the storage on

the MCE dose, applied at the production of these cheeses (Table 3).

The proteolysis degree of all cheese options increased significantly during the storage period

(Table 3, Figure 1). The main factors that affected the proteolysis degree in the cheese were coagulant

type, coagulant dose, storage duration, and the interactions between pairs of mentioned factors

(coagulant type and dose, coagulant type and storage duration, coagulant dose and storage duration). Based on such set of factors, the ANOVA model has appropriate fit with experimental data at R 2 = 0.97. Differences in the type and dose of MCE used in the cheese production lead to the differences in

the proteolysis rate of the cheeses during the shelf life. The greatest differences are noted between the

options of cheeses produced with the minimum dose of MCE with low PA (Chy-max Supreme) and the options of cheeses produced with the maximum dose of MCE with high PA (Chy-max Extra). The complex shear modulus (G*) characterizes the cheese consistency. An increase in the complex modulus increases the hardness of the cheeses, while a decrease in the complex modulus increases the

stickiness of the cheeses [18]. The loss tangent (tan δ = G"/G') denotes relative effects of viscous and

6elastic components in a viscoelastic behavior [16]. An increase in loss tangent indicates a change in

the character of the cheese from solid-like to viscous or liquid-like character. Figure 1. Changes in the degree of proteolysis of Crescenza cheeses made with different MCE doses during the storage period. Abbreviations are: Extra - Chy-max Extra; M - Chy-max M; Supreme - Chy-max Supreme. Values are means of 3 replicates; error bars indicate ± standard deviations.

The coagulant type, coagulant dose, storage duration, and pair interactions of these factors

significantly affected the rheological indices of the cheese (Table 3). The selected set of factors of

ANOVA model has explained the principal part variation of G* (R

2 = 0.94) and tan δ (R2 = 0.92).

Age-related changes in rheological indices of the cheeses during the storage period depending on the used coagulant type and dose are presented in Figure 2 (complex modulus G*) and Figure 3 (loss tangent (tan δ)). Figure 2. Changes in the complex modulus (G*) of Crescenza cheeses made with different MCE

doses during the storage period. Abbreviations are: Extra - Chy-max Extra; M - Chy-max M;

Supreme - Chy-max Supreme. Values are means of 3 replicates; error bars indicate ± standard

deviations. The change in the rheological parameters of cheeses occurs as a result of the modification of the

structure and viscoelastic properties of the cheese mass under the proteolysis action. The higher the

dose of addition, as well as the proteolytic activity of the used MCE, the higher the proteolysis degree

and the greater the changes in the structure of the cheese occuring during its ripening and storage [1,

7].

Micrographs of the cheese structure, illustrated an effect of the proteolytic action of MCE

depending on the used type and dose on the protein matrix of the cheeses, are presented in Figure 4.

2468101214

7 d 14 d 21 d

Degree of proteolysis , WSN/TN (%)

Storage period (days)

1500 IMCU / 100 kg

ExtraMSupreme

2468101214

7 d 14 d 21 d

Degree of proteolysis , WSN/TN (%)

Storage period (days)

2500 IMCU / 100 kg

ExtraMSupreme

2468101214

7 d 14 d 21 d

Degree of proteolysis , WSN/TN (%)

Storage period (days)

3500 IMCU / 100 kg

ExtraMSupreme

0500010000150002000025000

7 d 14 d 21 d

Complex modulus, G* (Pa)

Storage period (days)

1500 IMCU / 100 kg

ExtraMSupreme

0500010000150002000025000

7 d 14 d 21 d

Complex modulus, G* (Pa)

Storage period (days)

2500 IMCU / 100 kg

ExtraMSupreme

0500010000150002000025000

7 d 14 d 21 d

Complex modulus, G* (Pa)

Storage period (days)

3500 IMCU / 100 kg

ExtraMSupreme

7The microstructure photographs, presented in Figure 4, show that cheeses with a low degree of

proteolysis are characterized by a structure of cheese grains with intense boundaries. Cheeses with a

high degree of proteolysis are characterized by a more homogeneous, finely dispersed structure, which

is associated with the disappearance of large grains as a result of their hydration. Differences in the microstructure of cheeses produced with different types and doses of MCE have

consequences in the difference in consistency of these cheeses. At the end of storage for 21 days, the

cheeses produced with the MCE of Chy-max Supreme had the densest and elastic consistency, while the cheeses produced with the MCE of Chy-max Extra had the most plastic consistency. The cheeses produced with MCE of Chy-max M occupied an intermediate position in consistency. Figure 3. Changes in the loss tangent (tan δ) of Crescenza cheeses made with different MCE doses during the storage period. Abbreviations are: Extra - Chy-max Extra; M - Chy-max M; Supreme - Chy-max Supreme. Values are means of 3 replicates; error bars indicate ±1 SD. Figure 4. Light micrographs of Crescenza cheeses made with different types and doses of milk-

clotting enzymes at the end of the storage period (21 d). Abbreviations are: Extra - Chy-max Extra; M

- Chy-max M; Supr - Chy-max Supreme. Bar indicates 50 μm.

6. Conclusion

We have studied the effect of MCE based on different types of fermentation-produced chymosin: Chy- max Extra (bovine chymosin), Chy-max M (camel chymosin), Chy-max Supreme ("modified"

8chymosin) in 3 doses on changing the consistency of the cheeses during the storage. All 3 types of

studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the

used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose

of MCE also increases the rate of proteolysis in cheeses. Analysis of the data shows that within the group of studied MCEs, the type of enzyme has a greater

effect on the proteolysis intensity in cheeses than the dose of its addition does. Therefore, to increase

the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is

promising to use MCE with low PA based on camel chymosin (Chy-max M) and "modified" chymosin (Chy-max Supreme).

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