EVALUATION OF SELECTED PHYSICO-CHEMICAL PROPERTIES OF TVAROG PRODUCED FROM EXTENDED SHELF LIFE MILK (ESL)

Izabela Dmytrów¹, Anna Mituniewicz-Małek¹, Krzysztof Dmytrów², Józef Antonowicz^3
1 Department of Dairy Technology and Food Storage, West Pomeranian University of Technology, Szczecin, Poland
² Department of Econometrics and Statistics, Faculty of Economics and Management, University of Szczecin, Poland
³ Department of Environmental Chemistry, Pomeranian University, Słupsk, Poland

ABSTRACT

In this study, we analyzed physico-chemical indices of tvarog produced from extended shelf life milk (ESL). Two variants of tvarog were compared: (i) produced from milk pasteurized at a high temperature (control sample), and (ii) manufactured from ESL milk. The lactic acid cheeses produced under laboratory conditions were vacuum-packed in polyethylene foil and stored for 14 days at 5°C (± 1°C). The examined cheeses were assessed organoleptically, as well as determining their water, fat, calcium contents, titratable acidity, pH and hardness (TPA test). It was found that during storage the tvarog produced using ESL milk was characterized by better organoleptic attributes. The type of milk did not significantly influence the water content in tvarogs; an insignificantly higher water content was observed in the tvarog made from pasteurized milk. Tvarogs significantly differed in fat and calcium contents, and also titratable acidity. Higher hardness in the whole study period was a characteristic of the tvarog manufactured from ESL milk.

Key words: acid curd cheese, bacteriollogicaly, coagulated cheese, microfiltration, physico-chemical indices.

Abbreviations:
ESL – extended shelf life milk, ESLMC – extended shelf life milk cheese, MF – microfiltration, MFMC – microfiltered milk cheese, PMC – pasteurized milk cheese, RMC – raw milk cheese

INTRODUCTION

Nowadays, for hygienic reasons, cheeses are produced from pasteurized milk. Heating of the base milk is sometimes used by cheese makers to increase cheese yield (by thermal denaturation of serum proteins) and modification of milk constituents' functionality [11]. However, food scientist have been looking for alternative processing techniques that ensure the safety of dairy products without simultaneous changes in composition of the base milk. Microfiltration (MF) is favored since it reduces the use of heat treatment, which is particularly beneficial for products which are denaturated via heat, without inducing significant changes in overall milk composition [2,15,28,40]. MF membranes of about 1.4 µm pore size can achieve the right balance between rejection of the bacteria with little or no rejection of other milk components, such as protein, lactose and ash components. The low-bacteria milk not only aids in extending the shelf-life of pasteurized milk but also improves the keeping quality of cheeses made from the milk [22]. There has been a lot of work regarding the influence of different factors (e.g. coagulants, thermal treatment of milk) on characteristics of several varieties of ripening cheeses. In compliance with them, the final quality of cheese is effected, i.a. by  treatments to raw milk such as pasteurization, irradiation and microfiltration [21,23,39]. There has been no information to date in literature on possibility of application of ESL milk in a bacteriollogicaly coagulated and non-ripening cheese (tvarog) production. The objective of this study was to investigate whether pasteurization connected with microfiltration of milk used in tvarog production leads to significant improvements in selected characteristics of the final product.

MATERIAL AND METHODS

Research material consisted of tvarogs produced under laboratory conditions using two different kinds of milk: (i) homogenized (15MPa/55°C) and pasteurized at high temperature (85°C/15s) and (ii) microfiltered (50°C, 7.2 ms^-1, 0.5bar, membrane pores 1.4 µm) and pasteurized (72°C/15 s), according to Manufacturing Instruction "Non-maturing acid curd cheeses" No. 342/88. The base milk had been produced at a local creamery and transported, immediately, in a cooled condition to the Dairy Technology Department. The contents of fat, proteins and lactose in the ESL milk were: 2%, 3.1% and 4.5%, respectively, while pasteurized milk differed subtly only with respect to contents of proteins (3%). The experimental cheeses (pasteurized milk cheese – PMC and ESL milk cheese – ESLMC respectively) were obtained using lyophilized and concentrated aromatic lactic culture type B for direct vat inoculation (DVI) offered by ROSSELL Inc. Institute (Canada). Activity of applied starter was consistent with producer's declaration and was 500u/5000 l (30°C). The applied inoculum culture contained the following bacteria Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp.lactis var. diacetylactis, Leuconostoc cremoris. Both cheese variants were produced with identically maintained production technology parameters. Production of tvarog was started by adding starter (2.5%) to the base milk (pasteurized and ESL) and previously heated at 23°C. Then cheeses were left at this temperature until the formation of the curd (approx. 10 hours). Mature curd (pH 4.6) was delicately heated in order to separate from tank walls and then cut into cuboids with dimensions 120 x 120 mm. After averting the curd was warmed and was delicately stirred and its temperature was raised with velocity of 1°C/1 min. Warming was ended after the temperature was 45°C in the centre. Obtained slurry (after straining out the whey by the sieve) was split into single-use PE sacks. Obtained samples was pressed by 1h with 1 kg load per 1 kg of tvarog. The final cheeses, with a mass of about 0.15 kg each, were vacuum-packed using a vacuum pressure of 15 mbar for 2.5 s and a 400 mbar 'soft-air' option. The applied foil was 40 ľm PE/PA foil with EVOH. The packed cheese (60 wedge-shaped portions) was kept at 5°C ± 1°C for 14 days. Analyses were performed on the day of production and on the 1^st, 3^rd, 7^th, 10^th and 14^th days of storage. The tvarog samples were each subjected to an organoleptic assessment using a five-point scale following the recommendation of [12,25,26]. The structure, consistency, colour, taste and aroma of the cheeses were determined by a 9-person panel trained in sensory evaluation of cheeses. Samples for the analyses were collected randomly. Examination was carried out in a room free from foreign odours, where each of the tasters had a separate test stand and distilled water for mouth rinsing. In the examined tvarogs, according to Polish Standard [24], measured titratable acidity (°SH) and pH. Additionally, water and fat contents were determined at the beginning, in the middle and at the end of the experiment [24]. In milk used for the production of tvarog and in the final cheeses calcium content was quantified. The mineralization of the obtained samples was carried out using a monomode reactor Maxidigest MX 350 Prolabo. The calcium content in the mineralized samples was determined using atomic emission flame spectrometry [9]. A lanthanum buffer was added to samples and standards. Recovery of standards was used as a control method. To this end, a series of repetitions were performed for the middle standard and were subjected to identical processing to the analyzed samples. The mean recovery of the analyzed samples did not exceed 4.9%. The experimental cheeses also underwent a rheological analysis, which was based on the evaluation of their hardness. This analysis was made by means of a double compression test (TPA) using a TA.XT Plus texture analyzer manufactured by Stable Micro System [27]. The samples were penetrated with an aluminium cylinder with a diameter 6mm to a depth of 20 mm and a speed of 5 mm·s^-1 and force 1 G. The obtained results being the arithmetical means from 4 repetitions, and in the case of hardness, 12 repetitions were subjected to statistical analysis in a Microsoft Excel 2000 software package using the Student's test, Cochrane-Cox test and Shapiro-Wilk test for dependent and independent means. The tests were performed at a significance level α = 0.05.

RESULTS AND DISCUSSION

The organoleptic assessment score showed the influence of the kind of the base milk on the organoleptic attributes of cheeses. The examined tvarogs were characterised by appropriate attributes with the largest differences being found in the taste and aroma and the structure and consistency of analysed samples (Fig. 1).

Fig. 1. Results of score organoleptic assessment for tvarogs stored at 5°C±1°C

As the storage time passed, a gradual deterioration occurred in organoleptic attributes in the samples analysed. The evaluating test panel selected the tvarog produced using ESL milk (ESLMC) as the most desired one. Mc Sweeney et  al. [19] had compared ripening PMC and cheese produced using microfiltered and pasteurized milk and assumed that the taste in both samples did not differ significantly, which indicates the prominent role of milk flora in flavour development in raw milk cheese (RMC). However, Beuvier et al. [3] found that PMC (Swiss-type) was slightly more bitter and more acid than ESLMC. Bachmann et al. [1] affirmed that microfiltration and pasteurization caused a reduction in the aroma note of cheeses. The analysis of a chemical composition of experimental samples showed that kind of the base milk did not influence water content of tvarogs (Table 2). In spite of this, over the whole analyzed period, ESLMC showed a lower water content. For PMC the mean water content was 72%, and for the tvarog made from ESL milk 71%. The plot of curves describing changes in water content for both variants of the examined samples was practically identical and the observed fluctuations were statistically insignificant  (Fig. 2, Table 3).

Table 1. Results of statistical analysis for titratable acidity, pH and hardness of tvarogs

	Titratable acidity (oSH)		pH		Hardness (N)
	pasteurized milk cheese (PMC)	ESL milk cheese (ESLMC)	Pasteurized milk cheese (PMC)	ESL  milk cheese (ESLMC)	pasteurized milk cheese (PMC)	ESL milk cheese (ESLMC)
Day of production
S2	0.6667	2.8889	0.0003	0.0001	3.2005	7.2105
test	Student's t		Student's t		Student's t
t	7.250		0.274		20.271
tα	2.776*		2.306		2.306*
After 3rd day of storage
S2	1.5556	2.0000	5.6E-05	0.0002	10.8943	9.4404
test	Student's t		Student's t		Student's t
t	5.500		0.825		13.923
tα	2.776*		2.306		2.306*
After 7thday of storage
S2	23.1947	0.6667	0.0008	0.0005	34.0551	28.9026
test	Cochrane-Cox		Student's t		Cochrane-Cox
t	3.708		0.112		15.185
tα	2.776		2.306		2.306*
After 10th day of storage
S2	0.8889	2.6667	2.4E–05	2.4E–05	8.1156	8.6240
test	Student's t		Student's t		Student's t
t	10.000		17.321		13.923
tα	2.776*		2.306*		2.306*
After 14th day of storage
S2	0.6667	0.8889	0.001	9.6E–05	8.1156	76.6374
test	Student's t		Cochrane-Cox		Cochrane-Cox
t	7.559		0.943		12.141
tα	2.776*		2.306		2.306*

S2-variance of the analysed indicator. * – on the basis of appropriate test rejection of null hypothesis (on significance level α=0.05) – differences are significant. t – value of Student's-t or Cochrane-Cox statistics. t – critical value

Table 2. Results of statistical analysis for fat, water and calcium contents in tvarogs (t-Student's test)

	Fat content (%)		Water content (%)		Calcium content (mg100cm-3)
	pasteurized milk cheese (PMC)	ESL  milk cheese (ESLMC)	pasteurized milk cheese (PMC)	ESL  milk cheese (ESLMC)	Pasteurized milk cheese (PMC)	ESL  milk cheese (ESLMC)
Day of production
S2	0.3889	0.0556	2.6179	2.4335	28.500	4.567
t	4.596		0.520		5.215
tα	2.776*		2.776		2.776*
After 3rd day of storage
S2	–	–	–	–	7.738	2.491
t	–		–		5.053
tα	–		–		2.776*
After 7thday of storage
S2	0.6667	0.1667	9.8739	1.3674	13.206	2.852
t	2.824		0.928		6.155
tα	2.776*		2.776		2.776*
After 10th day of storage
S2	–	–	–	–	7.740	2.490
t	–		–		5.365
tα	–		–		2.776*
After 14th day of storage
S2	0.3889	0.0556	0.3131	5.5938	7.742	2.490
t	2.828		2.650		5.365
tα	2.776*		2.776		2.776*

S2-variance of the analysed indicator. * – on the basis of appropriate test rejection of null hypothesis (on significance level α=0.05) – differences are significant. t – value of Student's-t or Cochrane-Cox statistics. t – critical value

Table 3. Results of statistical analysis of mean values for dependent samples (t-Student's test )

		Pasteurized milk cheese (PMC)	ESL milk cheese (ESLMC)
Titrable acidity (°SH)	t	6.364	7.216
	tα	4.303*	4.303*
Active acidity (pH)	t	0.785	3.752
	tα	4.303	4.303
Water content (g·kg-1)	t	0.532	3.917
	tα	3.182*	3.182*
Fat content (g·kg-1)	t	0.245	4.899
	tα	4.303	4.303*
Hardness (N)	t	10.493	9.231
	tα	4.303*	4.303*
Calcium content (mg·100cm-3)	t	0.695	1.131
	tα	4.303	4.303

* – statistically significant differences at p ≤ 0.05. t – value of Student's-t or Cochrane-Cox statistics. t – critical value

Fig. 2. Changes of water content in tvarogs stored at 5°C±1°C

This study also showed that the examined tvarogs significantly differed in fat content. ESLMC had the highest fat content during the analyzed period (Fig. 3, Table 2). Mean calcium content for pasteurized milk was 138.32 mg·cm^-3 and for ESL milk 147.65 mg·cm^-3. Both tvarog variants had a stable calcium content (Table 3), with a higher calcium concentration observed in the cheese produced from milk subjected to microfiltration and mild pasteurization (ESMLC) (Fig. 4, Table 2).

Fig. 3. Changes of fat content in tvarogs stored at 5°C±1°C

Fig. 4. Changes of calcium content in tvarogs stored at 5°C±1°C

The obtained results were confirmed in available references. In the study carried by Śmietana et al. [36], aimed at assessing the quality of tvarogs produced with the use of a fully automated technological system, water content ranged from 71% to 72%. Additionally, no significant changes were stated in the chemical composition of tvarogs stored under cooling conditions. Bachmann et al. [1] in their investigation focused on the evaluation of the consistency of the three treatment effect on model cheeses produced in six laboratories made from (i) raw (ii) pasteurized and (iii) microfiltered milk, concluded that mean moisture content was significantly influenced by pasteurization of the skimmed milk. According to them the increased moisture content of pasteurized cheese could be associated with the binding of whey protein to the casein micelle. Rosenberg [31] mentioned that the water-holding capacity of curd increases when milk is processed by MF connected with pasteurization.

The observed differences in calcium concentration in the examined samples were most likely caused by different heat treatment parameters, which may result in modifications of the mineral equilibrium of milk [16]. Milk heating decreases the amount of soluble (ionic) calcium and magnesium, which are precipitated as phosphate and citrate salts [17]. Law [13] and Singh et al. [34] showed, to the contrary, that the pattern of release of calcium from micelles during acidification remained largely unaffected by any previous heat treatment of milk.

The cool storage of the examined samples in this study was associated with changes in the titratable acidity of all variants of tvarog (Fig. 5). In both tvarog variants, titratable acidity decreased significantly (Table 3). The observed difference in the potential acidity of tvarogs were statistically significant, with higher values in ESLMC (Table 1), which showed that a more intensive fermentation processes took place there. The mean potential acidity of ESLMC was 53.87 °SH, for PMC it was 43.91 °SH. The changes in pH found in both tvarogs variants proved to be statistically non-significant (Table 3). Although PMC had a higher pH in the examined period, the statistical analysis of the obtained results confirmed the lack of significance of the observed differences (Fig. 6, Table 1).

Fig. 5. Changes of titratable acidity of  tvarogs stored at 5°C±1°C

Fig. 6. Changes of active acidity (pH) of tvarogs stored at 5°C±1°C

Variations in acidity occur in several processes in the dairy industry (e.g. during preparation of acid casein, yoghurt, fermented milk and cheese). Moreover, changes in pH and titratable acidity take place during cooled storage of final products. The decrease in pH is due to lactose degradation, casein dephosphorylation and calcium phosphate precipitation. The increase in pH (the decrease of titratable acidity) is due to lactic acid degradation and production of ammonia by microorganisms. These changes in pH are complex and not easy to interpret as they mainly depend on the product composition (especially mineral and protein content) [33]. Additionally, the ultimate pH of cheese results from the lactic acid produced (from starter cultures breaking down lactose) and moderated by the buffering capacity of the medium. Higher competition with other microorganisms, the presence of inhibitory substances, occurrence of bacterial phage or influences of other treatments could lead to slower culture development and lower acid production. Pandey et al. [23] tested pH change kinetics during various stages of cheddar cheese made from raw, pasteurized, microfiltered and high-pressure-treated milk. They observed a higher rate of change in pH for raw milk followed by pasteurized and high-pressure-treatment of milk. The MF milk showed the least pH change rate. These results contradict the work of Gay et al. [10] who found a slower acidification in microfiltered and pasteurized milk than raw goat's milk cheese. Premaratne & Cousine [29] found the differences in  titratable acidity of cheeses were explained by different levels of some group vitamins and amino acids in the retentates which can inhibit acidifying bacteria growth. Referring to results obtained by Thomas and Crow [38] increasing the cheese moisture content leads to higher levels of lactose in the cheese because water is  a solvent for lactose. Lactose is fermented to lactate, mainly L(+), at a rate dependent on the salt-in-moisture level in the cheese and the salt sensitivity of the starter culture strains used. Therefore a higher water content may be reflected by a higher potential acidity in the product. The results obtained in this work did not confirm the data of the aforementioned authors as the literature on the subject did not specify the plot of acidity for tvarogs produced from ESL milk. However, it is known that the acidity of the ready product is strictly related to the acidity of the raw material used in processing, and heat treatment used in the technological process. Differences in titratable acidity of the examined samples could have resulted from the 'acuteness' of base milk heat treatment. A change in the mineral equilibrium (transformation of phosphates from a soluble form into an insoluble one) contributes to an increase in milk's active acidity (pH) through the release of H+ ions [17].

The performed rheological analysis of the experimental cheeses showed the significant effect of the applied treatment of the base milk on the hardness of the tvarogs examined, with its higher values being obtained by ESLMC over the whole study period.

Fig. 7. Changes of hardness of tvarogs stored at 5°C±1°C

The hardness of both acid curd cheeses (tvarogs) showed an increasing tendency in the whole study period and these increases were statistically important (Table 1). The type of milk treatment significantly influenced the hardness of the examined samples. It may be suggested that the texture parameters of cheeses produced from ESL milk changed in a slightly different way than cheese obtained from pasteurized milk. Additionally, ESMLC had a lower water content. Ziółkowski et al. [45] examined the durability of tvarogs cheeses produced according to modified production technology and packed with different methods (vacuum packing and thermo-shrinkable foil packing), and found an increase in the hardness of all analyzed samples during storage, which they explained by whey leakage. They also emphasized that the leakage was slight, and the differences in hardness were therefore minimal. According to Prentice [30] cheese rheological properties depend mostly on their protein and water content. This author states that when protein content increases in cheeses, their hardness increases as well. The study performed by Bonczar and Walczycka [4] showed that water content was negatively correlated with almost all texture parameters, including hardness. This means that the lower the cheese water content, the higher the hardness of this product. According to Law et al. [14] increasing the pasteurization temperature results in denaturation of whey proteins, and their probable interaction with casein micelles. The latter changes coincide with a reduction in syneresis and a higher content of cheese moisture [32]. The obtained results did not follow our expectations that a higher temperature pasteurization would increase the product's hardness. Dannenberg and Kessler [6] found that for heat treatment at temperatures higher than 70°C, the major whey proteins denature, namely lactoglobulin-β and lactalbumin-α. The temperature-induced conformational change of lg-β results in the exposure of both hydrophobic parts of the polypeptide and reactive thiol groups. These reactive thiol groups can form disulfide links with other reactive thiol groups or disulfide bridges as present in lac-α, lg-β, BSA,-κ and casein-αs₂ through thiol group / disulfide bond interchange reactions. During the heating of milk, mainly lg-β covalently interacts with casein-κ present at the exterior of the casein micelles. However, significant quantities of lactalbumin-α and the minor whey proteins also can interact with the casein micelles [5]. Additionally, soluble disulfide-linked whey protein aggregates are formed. So, heated milk is a complex mixture of native and denaturated whey proteins and casein micelles in which the denaturated whey proteins occur either as whey protein aggregates or as whey proteins (aggregates) associated with the casein micelles. Whey protein denaturation occurring during pasteurization of the base milk is generally recognized as a very important parameter for the textural properties of acidified milk products. The formation of disulfide bonds and post acidification of heated milk contributes to its mechanical properties. So, the gel hardness depends on the amount of reactive thiol groups present after heating the milk [43]. Thomann et al. [37] had examined the effect of homogenization, microfiltration and pH on curd firmness and syneresis of curd grains, and noticed that the curd firmness increased when the concentration factor of MF was increased and pH decreased, whereas homogenization of milk prior to MF decrease the gel firmness. An increase in the concentration factor of MF retarded the whey release. MF increased the number of structure-forming particles and after coagulation, more bond per time are linked within giving high curd firming rates [35,44]. This is also consistent with findings from Mishra et al. [20]. However, the influence of MF on curd firmness depends on the concentration factor. According to Tuinnier and De Kruif [41] heat treatment of milk prior to acidification of milk at temperatures ranging from 20°C to 40°C, changes the gelation properties markedly compared to those of unheated milk. Heat treatment has caused a shift in gelation pH towards higher pH values [42]. The final gel formed has an increased gel hardness, higher storage modulus and shows less susceptibility to syneresis [7].

CONCLUSIONS

1. The examined tvarogs were characterised by appropriate organoleptic attributes with the largest differences being found in the taste and aroma, and in the structure and consistency of analysed samples.

2. The evaluating test panel selected the tvarog produced using ESL milk as the most desired one.

3. Water content in the tvarog did not significantly depend on the type of milk. Its higher content was observed in tvarog produced from pasteurized milk. However, the observed differences were statistically insignificant.

4. Fat content in the tvarog significantly depend on the type of milk. Storage duration did effect fat content in the examined samples.

5. Tvarog produced from ESL milk had a higher calcium content.

6. Storage of tvarog was associated with a statistically significant decrease in titratable acidity. The influence of base milk treatment was also statistically significant.

7. The examined variants did not differ significantly in active acidity. The observed decrease in pH in PMC and the increase in ESLMC samples should be deemed statistically insignificant.

8. The type of milk treatment significantly influenced the hardness of the examined samples. The observed increase in hardness in both variants was statistically significant.

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Accepted for print: 01.07.2009

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Izabela Dmytrów
Department of Dairy Technology and Food Storage,
West Pomeranian University of Technology, Szczecin, Poland
Papieża Pawła IV/3, 71-459 Szczecin, Poland

Anna Mituniewicz-Małek
Department of Dairy Technology and Food Storage,
West Pomeranian University of Technology, Szczecin, Poland
Papieża Pawła IV/3 71-459 Szczecin, Poland
email: aniamalek4@wp.pl

Krzysztof Dmytrów
Department of Econometrics and Statistics,
Faculty of Economics and Management,
University of Szczecin, Poland
A. Mickiewicza 64, 71-101 Szczecin, Poland


Józef Antonowicz
Department of Environmental Chemistry,
Pomeranian University, Słupsk, Poland
Arciszewskiego 22b, 76-200 Słupsk, Poland

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