The effect of proteolytic activity of starter cultures on technologically important properties of yogurt

Abstract In this study, the effects of proteolytic activity of yogurt starter bacteria on physicochemical and technological properties of yogurt were investigated. Moreover, impact of proteolytic activity and production of exopolysaccharide (EPS) on the performance of each strain were screened. In order to compare the textural properties of yogurt samples, four parameters were evaluated: syneresis, water‐holding capacity, cohesiveness, and hardness. Results showed that strains with high proteolytic activity had lower acidifying activity during fermentation and storage. Samples containing EPS‐producing starter cultures had low proteolytic activity except samples K, L, and M. These differences related to nature and characteristics of each strain. Counts of starter cultures in samples produced using strains with high proteolytic activity were higher than other samples. Textural analysis data showed significant differences (p < .05) among strains in the four tested parameters. Strains with high proteolytic activity showed lower texture properties than other samples. Evaluation of sensory characteristics also showed samples prepared using strains with low or medium proteolytic activity and produced with EPS‐producing strains have higher overall acceptability than other samples. Accordingly, microbial, physicochemical, and sensory properties of produced yogurts confirm that proteolytic activity is one of the most effective factors in quality of product and performance of each strain.

of this slight activity is a breakdown of only 1-2% of milk protein (Belkaaloul, Chekroun, Ait-Abdessalam, Saidi, & Kheroua, 2010). Due to proteolytic nature of Lactobacillus delbrueckii ssp. bulgaricus that leads to production of essential amino acids and because of symbiotic relationship of L. delbrueckii ssp. bulgaricus and S. thermophilus, these bacteria are able to grow in yogurt (Shihata & Shah, 2000). Therefore, type of strains and ratio of the two organisms used for inoculation are effective on degree of proteolysis in yogurt. These properties of starter cultures have been linked to its importance for texture, taste, and flavor development during fermentation and storage period.
During storage period, yogurt texture changes due to degradation of the protein network. It also contributes directly to taste and flavor by the formation of peptides and free amino acids as well as by liberation of such substrates for further catabolic changes and thereby formation of volatile flavor compounds. In terms of taste, proteolytic activity causes bilateral effect; peptides can taste bitter or delicious and amino acids can taste sweet, bitter, or broth-like (Zainoldin & Baba, 2012).
The study by Ramchandran and Shah (2009) showed that proteolytic activity has adverse effect on textural properties of yogurt.
Other researches also indicate that yogurts containing EPS-producing starter cultures have higher proteolytic activity (Peterson, Dave, McMahon, Oberg, & Broadbent, 2000;Ramchandran & Shah, 2010). Ramchandran and Shah (2010) stated that, proteolytic activity has improving effect on the survival of the starter cultures in the product. Slocum et al., (1988) investigated that proteolytic activity of yogurt culture influences the keeping quality; therefore, it is better to minimize proteolysis during the production and storage of yogurt.
With regard to the effect of proteolytic activity of yogurt cultures on the quality during storage and other technological properties of yogurt, one of the important aims in this study was to determine the proteolytic activity of yogurt cultures using the o-pthaldialdehydebased spectrophotometric method. Correlation of this characteristic with other technological properties of yogurts also was evaluated.
Moreover, effect of proteolysis on performance of starter cultures during fermentation and storage were estimated.

| MATERIAL AND METHODS
Agar and d-glucose were purchased from Merck (Merck, Darmstadt, Germany). Skimmed milk powder, whole milk powder, and cream powder were produced by Pegah Fars Company (Shiraz, Iran).

| Preparation of inoculums
The strains used in this study were isolated from traditional Iranian yogurt samples. These strains were isolated and identified by Rushanzadeh (2011) in Shiraz University, Iran.  (IDF 1997(IDF , 2003. For inoculation into milk, these were grown in specific medium broth until they reached late exponential growth phase (approximately, 10 8 cfu/ml overnight). The cells were harvested by centrifugation at (SW14R, Froilabo, Paris, France) 10,000g for 10 min at 4°C. The pellet was then washed twice with sterile distilled water. The resultant pellet was suspended in entire milk used for yogurt production (Lim, Suntornsuk, & Suntornsuk, 2009). For production control sample, we used Sacco 480 starter culture (blends of EPS + S. thermophilus and L. bulgaricus).

| Yogurt making
Yogurt mixes were made using mixtures of skim milk powder and cream powder (Pegah Fars Dairy Co., Iran) to prepare reconstituted milk (14% w/w), then homogenized and stored at refrigerator (4°C) overnight. The following day, it was pasteurized at 90°C for 5 min under agitation in a water bath followed by cooling to 45°C. The heating and cooling processes were carried out in a closed container to minimize losses due to evaporation. This was followed by inoculation with different strains of S. thermophilus (1 × 10 8 cfu/ml) and L. bulgaricus (1 × 10 8 cfu/ml). The inoculated milk was then mixed thoroughly and dispensed in 100 mL polystyrene cups, sealed with aluminum sheet, and incubated at 42°C until the pH dropped to 4.6 ± 0.1. The fermentation was stopped by transferring the samples immediately to refrigerator maintained at 4°C ± 1. The samples were kept there and at 7-day intervals (up to 28 days), were subjected for further use. Characteristics of isolates have been shown in (Table 1).

| Acidification activity of strains
During fermentation, acid-producing activities of all batches were recorded at 1 hr intervals at 40°C using a pH meter (model ST 300; Ohaus, Singapore, USA). All pH measurements were performed in triplicate (Ramchandran & Shah, 2010).

| Microbiological analysis
During the cold storage, starter culture plate counts were determined at 1, 7, 14, 21, and 28 days. S. thermuphilus colonies were enumerated in M17 agar after incubation aerobically at 37°C for 48 hr (IDF 1997(IDF , 2003; while L. bulgaricus were counted on MRS agar after incubation anaerobically at 37°C for 72 hr (IDF 1997(IDF , 2003. The results were obtained as the logarithms of the number of colony forming units per mL (log cfu/ml) of yogurt. The microbiological analyses were performed in two replicates.

| Determination of crude EPS content
Method of Ramchandran and Shah (2010)

| Evaluation of the proteolysis
pH of inoculated samples before incubation were reduced to 4.6 with glacial acetic acid followed by centrifugation at 4000g for 30 min at 4°C. The supernatants were filtered through 0.45 μm syringe membrane filter (Merck, Darmstadt, Germany). The serum of the experimental samples at days 1, 7, 14, 21, and 28 of storage were also centrifuged and filtered similar to the above. The filtered solutions were stored at −20°C until assayed (1-2 weeks).
Free amino acid content in filtrated samples represent the extent of proteolysis measuring by the o-phthaldialdehyde (OPA) at 340 nm within 2 min using spectrophotometer (UV 9200,Raylight,Beijing,China). The absorbance of the inoculated samples (before incubation) were deducted from the corresponding absorbance of yogurt samples to obtain the amount of free amino acids released as a consequence of the proteolytic activity of the starter cultures during fermentation and storage (Ramchandran & Shah, 2010).

| Changes in spontaneous whey separation
Set-style yogurt samples were prepared in conical centrifuge tubes (25 g). Then, syneresis was evaluated by centrifuging at 500g for

| Measurement of water-holding capacity
Samples (50 g) from each batch were weighed in centrifuge tubes and incubated at 40°C, after which the set gels were stored during storage period at 4°C. The tubes were centrifuged at 3000g for 10 min at 4°C.
The whey was separated, then weighed and results expressed as the weight percentage of serum released by centrifugation (Riener, Noci, Cronin, Morgan, & Lyng, 2009).   Bayarri, Carbonell, Barrios, and Costell (2011) with scores between 1 and 9 (1 = dislike extremely, and 9 = like extremely) for flavor, body and texture, appearance, and color. Panelists evaluated all yogurt samples after storage for 7 days at 4°C.

| Statistical analysis
The majority of experiments were performed in triplicate. All data were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test. Statistical significance (p < .05) was assessed using the SAS 9.1 software (SAS Institute, North Carolina), Pearson correlation test was also employed. Nonparametric data were analyzed using Kruskal-Wallis test. p values <.05 were considered statistically significant. Analysis was performed using a SPSS package (SPSS 16 for windows, SPSS Inc, Chicago, IL).

| Postacidification and titratable acidity
The results of postacidification (pH) and titratable acidity during the shelf-life of the yogurts are presented in Table 2. After 28 days of cold storage, pH dropped to pH 4.07-4.38 and titratable acidity varied from 109 to 126 g/100 g. These acid-production trends during storage are similar to other research (Çeilik, 2007). However, some studies showed this trend because of producing some metabolites become reversed (Ramchandran & Shah, 2009

| Changes in the counts of yogurt bacteria
The changes in the viable counts (log cfu/ml) of S. thermophilus and L. bulgaricus during refrigerated storage of yogurt are presented in Table 3. During the shelf-life, counts of two starters were stable and ranged, as an average, from 7.30 to 9.55 log cfu/ml. Although there are different standards for count of starters, acceptable count is about 10 7 cfu/ml (Ramchandran & Shah, 2010). This confirms that the native starter cultures isolated from indigenous yogurt remained viable in the product until the end of storage (28 days) which is satisfactory for the yogurt production. In general, the counts of strains with high proteolytic activity (109 and 110p of L. bulgaricus in combination with three strains of S. thermophilus) were higher (p < .05) than others. In fact, continued proteolysis (Table 4) improve survival of the starters in the product by providing the essential growth factors in the form of peptides and amino acids (Ramchandran & Shah, 2010). In all samples, counts of two starters showed a reduction at day 14, 21 or at end of the storage, but counts of starter cultures remained stable in yogurts made using starter with high proteolytic activity throughout the storage period. This confirms the protective effect of proteolysis on survival of the starter cultures. Another possible reason could be attributed to slightly lower pH and higher acidity (   Amatayakul, Halmos, Sherkat, and Shah (2006) have also found that concentration of EPS in yogurt made using ropy starter cultures increased during storage.

| Changes in crude EPS concentration
They also have stated that this was not found in yogurt made using capsular EPS-producing starter cultures, in which the EPS concentration remained constant during storage at 4°C.
Variations in the method of estimating the EPS, differences in the types of EPS, as well as strain variations could be the possible reasons for the differences observed (Ramchandran & Shah, 2010).
The concentration of EPS in yogurts made using EPS starter cultures ranged from 30 to 60 mg/100 g. The amount of extracted curd EPS in Amatayakul et al. (2006) were also in these same ranges. It is interesting to note that a low concentration of EPS (10-20 mg/100 g) was found in yogurt produced with non-EPS-producing starter cultures.
The low amount of EPS, detected in the yogurt made with non-EPSproducing starter cultures, might be due to the residue of lactose remaining after the purification (Amatayakul et al., 2006). According to the results, EPS-producing strains have lower proteolytic activity. For example, L. bulgaricus strains 109 was non-EPS-producing starter cultures and showed high proteolytic activity. Also, L. bulgaricus strains 88s and 122 were EPS-producing starter and showed lower proteolytic activity. Nevertheless, EPS-producing starter cultures such as L. bulgaricus strains 110p and 96 in combination with three strains of S. thermophilus showed higher proteolytic activity than other EPSproducing starter cultures. Peterson et al. (2000) reported that EPS + starter culture has more proteolytic activity.

| Changes in extent of proteolysis
The results shown in Table 5

| Whey separation and water-holding capacity
The percentage of spontaneous whey separation and water-holding capacity of samples is given in to metabolic activity of starters and reduction in pressure of protein network. Ramchandran and Shah (2009) reported syneresis decreased until 14th day, indicating a rapid recovery of structure after the destruction of structure, however after 14 days this trend reversed and syneresis increased that represents the disintegration of the structure during storage. Actually, the sudden increase in syneresis is related to high proteolytic activity of isolates; therefore, leading to disintegration  the first day and decreased to 2.71-3.25% during 28 days of storage). These samples also showed highest water-holding capacity (63.50-67.11% on the first day and increased to 73.19-77.60% during 28 days of storage). Therefore, these results confirm that yogurts prepared using cultures with low proteolytic activity had better waterholding capacity and thereby lower syneresis.

| Texture analysis
In order to compare the textural properties of the samples, tow parameters were evaluated: hardness and cohesiveness. Texture profile analysis results of the experiments are shown in Table 7. As can be observed in this table, the yogurts made using native strains displayed better textural properties. Analysis variance of textural data showed that there were significant differences (p < .05) among strains for the two tested parameters. Textural studies conducted by many authors suggested that syneresis, texture, and viscosity of fermented milks were affected by milk composition and type of culture (Chammas, Saliba, Corrieu, & Béal, 2006;Marshall & Rawson, 1999). Since milk composition was kept constant in this study, the differences observed within the cultures were due to the strains.
Hardness of all samples except samples K and L increased during storage (p < .05), but in samples K and L, it increased until 21st day of storage then decreased in the last week. Samples produced by strains with low proteolytic activity showed more hardness than other samples.
For example, samples made using L. bulgaricus 88s in combination with three strains of S. thermophilus had higher hardness than others (p < .05).
Among all samples, hardness of sample C was higher than others and Overall among all samples, samples made using strains with higher proteolytic activity showed lower textural characteristics than others. Buffa, Morais, Jiménez-Belenguer, Hernández-Giménez, and Guamis (2005) also reported that high proteolytic strains are not always the most suitable for use as starter cultures, since excessive proteolysis can cause uncontrolled production of bitter peptides and other undesirable compounds, or even excessive casein hydrolysis resulting in a too-soft final product. However, Ruas-Madiedo, Alting, and Zoon (2005) asserted that proteolytic activity of the strains did not seem to play any significant role. Figure 2 presents the sensory evaluation values of the samples. There were different scores, but without significant difference (p > .05) after storage for 7 days. Samples produced using strains with high proteolytic activity had lower score of flavor, taste, and textural properties than others samples. EPY in terms of sensory characteristics were similar with NEPY but had better textural properties. In general, overall acceptability of EPY prepared using strains with low or medium proteolytic activity was higher than that for NEPY and strains with high proteolytic activity.

| Correlation between proteolytic activity and technological parameters
The statistical relationship among proteolytic activity and technological parameters of acidification activity, textural properties, and content of exopolysaccharide were examined using Pearson's correlation procedure, for each combination of bacterial species. Result of correlations is shown in Table 8. Proteolytic activity was correlated to pH (r = −.87 to 0.96) and acidity (r = .80 to .96). In this study, there was also a correlation between the proteolytic activity and texture properties such as syneresis (r = −.65 to .94), water-holding capacity (0.84 to 0.98), and hardness (0.76 to 0.97). According to the results of the correlation analysis, proteolytic activity of strains is not correlated well with the EPS content, viable counts of two starter cultures and cohesiveness.
F I G U R E 2 Sensory evaluation values of yogurts after 7 days of storage in 4°C. Means (n = 13). For details of samples, see Table 1

| CONCLUSION
The findings of this study illustrates the considerable difference among technological characteristics of yogurts made from each native strains. All isolates showed medium or high acidifying activity during incubation and storage at 4°C. During shelf-life, counts of two starter cultures were stable and ranged, as an average, from 7.30 to 9.55 log cfu/ml. Strains were EPS-producing culture and with low or medium proteolytic activity such as L. bulgaricus strains 88s and 122 have displayed better textural parameters. These strains also showed low syneresis and high WHC than other strains. Cohesiveness values in the majority of samples displayed no significant difference during storage. The strains with high proteolytic activity showed low acidifying capacity during incubation and storage and counts of these strains were higher. Textural parameters were negatively affected by the high proteolytic activity. Samples containing EPS-producing starter culture displayed low proteolytic activity except samples K, L, and M showed these strains would be good candidates as starter culture for using in the industrial. However, their potential use depends on further assessment of their aptitude for producing of fermented products in industrial scales and their preservation by freezing and spray drying.