An Investigation of Effects of Whey Protein Hydrolysate on Yogurt Starter Cultures and Probiotic Bacteria in Ayran

Bu calismanin amaci, peynir alti suyu (whey) protein hidrolizatin (WPH) ayrana eklenmesinin probiyotik bakterilerinin (Streptococcus thermophilus, Lactobacillus delbrueckii spp. bulgarikus, Lactobacillus acidophilus ve Bifidobacterium lactis) aktivitesinin ve gelisimlerinin arttirilmasinin arastirilmasidir. Fermentasyon oncesi ve sonrasinda 7., 14. ve 21. saklama suresince, WPH ve probiyotiklerin %0, %0.25, %0.5 ve %1 konsantrasyonlarinda probiyotik bakteri buyumesi uzerine etkileri calisilmistir. WPH ve probiyotiklerin hedef mikroorganizmalar ve biyokimyasal degiskenler uzerinde onemli etkilere (p<0.05) sahip oldugu belirlenmistir. WPH ve probiyotiklerin eklenmesi fermantasyondan sonra 21’inci saklama gunune kadar kontrole kiyasla onemli olcude (p<0,05), probiyotiklerin buyumesini arttirdigi gorulmustur. Farkli WPH duzeylerini iceren numuneler calisma suresi boyunca kontrole kiyasla anlamli derecede (p<0,05) daha yuksek titre edilebilir asitlik seviyesine sahip oldugu belirlenmistir. WPH %1 duzeyinde karistirilmasi, kontrol numunelerine kiyasla en yuksek toplam kati ve protein icerigine sahip oldugu tespit edilmistir. WPH iceren numuneler viskoziteyi ve Hunter renk parametrelerini azaltmistir (p<0.05). Sonuclar WPH ayran probiyotik bakterilerin buyumesini ve besinselligini arttirmada buyuk bir potansiyele sahip oldugu gorulmustur.


INTRODUCTION
The growing request for a healthy diet is caused by developing innovation and new product stimulation in the food industry (Champagne et al., 2018). Nowadays fermented milk and products participate in health with the natural food and improve the intestinal flora with existing lactic acid bacteria. Fermented milk products such as yogurt, ayran, kefir, etc. are supplement-strong foods that are considered to be one of the most popular fermented milk products around the world (Shangpliang et al., 2018, Turkmen et al., 2019. The general utilization of milk products, especially, probiotic milk products achieves perfect evaluation of food long-term because of the great impacts over the health authenticated by the records of nutritionists and specialists (Kerry et al., 2018). Probiotics are live microorganisms food supplements, can benefit the health of users by preserving, or beneficent their intestinal microorganism balances (Kerry et al.. 2018(Kerry et al.. , Şanlier et al., 2019. As well as the increase rate of probiotics in fermented milk products is a typical and common issue with a mercantile importance (Korbekandi et al., 2011).
Currently, the species of Lactobacillus and Bifidobacterium are extremely utilized as a part of probiotic fermented milk products and commonly connected with the gastrointestinal tract (Turkmen et al., 2019). Lactic acid bacteria (basically Lactobacillus) and Bifidobacteria are the essential operators of the probiotics in the practical nourishment industry (Kerry et al., 2018, Turkmen et al., 2019. They provide for the human body pioneer benefits such as energizing of the immune system (Perdigon et al., 1992), protection from many infections (De Macias et al., 1993), improving digestion and absorption (Michael et al., 2010). Probiotic bacteria are related to various health advantages and the most important issue for probiotic is viability and survival in a specific concentration in the gastrointestinal tract, stay survive in the different conditions (Gerez et al., 2012, Turkmen et al., 2019 and improve the microbial balance of the intestinal medium. Whey protein (WP) is a group of globular protein isolated from whey that can be used as a nutritional supplement (Wirunsawanya et al., 2018). Whey protein hydrolysate (WPH) is utilized generally by dairies, bread shops, confectionaries, meat preparing, canned products, and refreshment foundations for their different capacities in sustenance quality and solidness (Wirunsawanya et al., 2018). The WPH is predigested, that's mean the tall chains of amino acids organized in isolates and concentrates, will be solved to a simple amino acid. Also, WPH has the fastest absorption capacity; release of amino acid through the bloodstream and may carry to greater muscle proteins when intake after the exercises than other kinds of WP. So, the WPH is utilized in the multiplication of body-building. Many studies proved the possibility of WP can promote the survival of probiotic and culture bacteria (Wirunsawanya et al., 2018, Turkmen et al., 2019. Whey protein hydrolysate is added into yoghurt to improve the growth of yoghurt cultures and probiotics in milk supplemented (McComas Jr andGilliland, 2003, Krunić et al., 2019).
Ayran is one of the most important fermented milk drinks and common in many countries in Asia, the Middle East, and known especially as a traditional drink in Turkey. Ayran usually prepared in-home by adding water to yoghurt or industrially by adding yoghurt starter culture like Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus into milk (TFC, 2009). Whenever, ayran is prepared from yoghurt, is produced by mixing of yoghurt with water (between 30-50%) and is salt (0.-1%) (Köksoy and Kılıç, 2003).
Effect of supplement with nutrient addition on the viability of probiotic bacteria in fermented milk is an important issue through the fermentation and storage. Considering this, the addition of WPH and probiotics to the fermented milk will promote the ability of these probiotics, will increase their functional characteristic and will increase the effectiveness of the product by promoting the viability of the probiotics. Due to containing a high level of nutrients, WPH and probiotics added into ayran can improve functionality. The present study was aimed (i) to investigate enhancement probability of growth and activity of probiotic bacteria (Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, S. thermophilus and Bifidobacterium lactic) by adding of WPH and probiotics into ayran before and after the fermentation and 7 th , 14 th and 21 th days of fermentation; (ii) to improve the chemical characteristics of ayran such as pH and acidity, reduce the decline in pH values through the incubation time and at the end of storage time; (iii) to enhance protein content, and (iv) to follow color and viscosity changes were studied during the storage period of ayran.

Experimental design
Fresh milk was heated at 95 °C for 15 min. Heat treated milk was cooled down to the fermentation temperature of 40°C. Then yoghurt starter was inoculated as 4% (v/v) and divided into 10 groups (control, 3 groups for WHP (0.25 %, 0.5 %, and 1.0% (w/v)), 3 groups for probiotics (0.25 %, 0.5 %, and 1.0% (w/v)) and 3 groups for mix of WPH and probiotics). WPH and probiotics (Enterococcus feacium, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum contain 2.5x10 9 cfu/10g) were added in concentrations 0 (control), 0.25, 0.5, and 1.0% (w/v). Samples were produced by adding sterile water in 50% (v/v) and salt in the concentration of 0.5% (w/v) into the mixtures. All samples were mixed very well then were incubated at 40 °C. The fermentation process continued until reaching to pH of 4.4±0.02. After the fermentation, ayran samples were cooled down and keep at 4°C for 21 days in a refrigerator. The biochemical parameters such as the changes in the pH and acidity were measured through the fermentation, after fermentation and during the storage time after 7 th , 14 th and 21 st days. Probiotic bacteria were counted before and after the fermentation, and through 7 th , 14 th and 21 st days of the storage. For clear measurements, viscosity and Hunter Color Analyses were recorded after one-day of sampling.

Microbiological analysis
The numbers of bacteria were counted and expressed as log CFU/ml. Enumeration of bacteria in ayran containing 0, 0.25, 0.5, 1% (w/v) of WPH and/or probiotics were recorded before and after the fermentation and after 7 th , 14 th and 21 st days of the storage. All the samples were diluted by tenfold with peptone water (0.1% v/v) then pour or spread to agar after that were incubated. Finally, the colonies were counted. Growth of bacteria was recorded by the following method of enumeration of L. acidophilus (Dave and Shah, 1998) was completed by spreading on to MRS agar by adding 1% of maltose. The inoculated petri dishes were incubated at 37°C for 3 days after that the colonies were counted. S. thermophilus colonies were enumerated by the pour plate technique inoculation of appropriate dilution on M17 agar with lactose 1%. The Petri dishes were incubated at 45°C for 24 hour and colonies were counted. Lactobacillus bulgaricus colonies was counted by the pour-plate technique into MRS agar with fructose at 1%, petri dishes were incubated at 45°C for 3 days and counted. Enumeration of B. lactis was done by the pour technique in to MRS agar. Then petri dishes were incubated for 45°C-3 days and the colonies were counted.

Chemical analyses
Values of pH were measured using a pH meter every one hour through fermentation of samples containing 0.25, 0.5, 1% (w/v) of WPH and/or probiotics during the storage.
Total solid of samples were determined using an oven method proposed by TS1330 (Anonymous, 2006. Briefly, the samples were put into a beaker on a boiling water bath up to the excess water were removed from the samples then were cooled at room temperature. The empty dishes were heated in oven for one hour at 102-103°C. The 5 mL of sample was added and recorded the weight before drying, then put it in the oven at 102-103°C up to constant weight reached. Protein determination was carried out according to a Turkish standard method TS1330 (Anonymous, 2006). Briefly, 10 ml of milk, 0.5 ml of phenolphthalein, and 0.4 ml saturated potassium oxalate solution was mixed in a conical flask and waited for 2 min. After that, the mixture was neutralized with 0.1 M NaOH of a burette until getting a faint color. Then 2 ml of 40% formaldehyde solution was added and completed the titration until getting the same pink color like previously color and determine the level of 0.1 M sodium hydroxide wanted for the second titration.
The viscosity was followed for all ayran samples after 24 h of sampling time by a Brookfield viscometer (DV3T TM viscometer, USA) at 20 rpm in 150 mL beaker to taking the measurements of viscosity.
The color of ayran samples was recorded after the fermentation and through 7 th , 14 th and 21 st days of storage time by Hunter color equipment (Colorflex color measurement spectrophotometer, Hunter Associates Lab. Inc. Mumbai). For each sample was taken 10 mL in a special class and put on the equipment, then L*, a* and b* values were recorded.

Statistical Analyses
A one-way ANOVA (Analysis of Variance) was used to determine significant differences at α=0.05 level in the number of probiotic bacteria, pH, colors, and viscosity as a function of time and among products, using the SPSS version 19.0 (SPSS Inc., Chicago, USA). For the identification of the homogeneous groups of time and samples' variables, Duncan multiple comparison test was performed. Statistical analyses were applied to find how samples of ayran and storage time affect the probiotic bacteria and other parameters. observed by the addition of 1% WPH at the end of the storage among the only WPH containing samples ( Figure 1a). This increment could be due to WPH containing the branched-chain amino acid, which could play an important role in the growth of probiotic bacteria (Güler-Akın and Akın, 2007). Also, whey protein contains milk proteins about 20% (Hoffman and Falvo, 2004). High amounts of amino acids, minerals, and beneficial compounds could stimulate the growth of this bacteria, in agreement with the report of Fox (1986). Akalin et al. (2007) reported that the counts of S. thermophilus in low-fat yoghurt containing 1.5% WPC [(8.56 log CFU/mL) was higher than in the low-fat yoghurt without the addition of WPC (8.08 log CFU/mL)] after the 28 days of storage time.
Addition of probiotics had no significant effect (p>0.05) on the growth of S. thermophilus compared to the control till the end of the storage ( Figure 1b).
However, the highest growth of S. thermophilus (8.93 log CFU/mL) was determined by the addition of both WPH and probiotics ( Figure 1c) at 0.25% into ayran (p<0.05). Probiotics are sensible to environmental conditions as well as had nutritional requirements more than the other kind of bacteria. That the growth of probiotics may effect an increase/decrease by the other addition on a media (Gardiner et al., 2002).

Growth of Lactobacillus delbrueckii ssp. bulgaricus
Statistical analyses indicated that the increase in the concentrations of WPH caused to increase (p<0.05) the growth of L. delbrueckii ssp. bulgaricus . The highest growth of L. bulgaricus was found in the samples containing 1% WPH at 21 st days of storage ( Figure 2a). The addition of WPH worked as a buffering agent to prevent the changes in the acidity and to avoid the lethal pH value that may destroy the growth of L. delbrueckii ssp. bulgaricus (Dave and Shah, 1998). Changes in the growth of L. bulgaricus in samples prepared the addition of 0.25, 0.5 and 1% WPH with probiotics ( Figure 2c) into ayran were not significant (p>0.05). Vargas et al. (2015) keynoted that the addition of 3% whey protein isolate (WPI) significantly (p<0.05) increased the counts of L. delbrueckii ssp. bulgaricus than those of 1 and 2% WPI after the end of storage time.
Growth of Lactobacillus acidophilus Changes in the growth of L. acidophilus in the samples were recorded before and after the fermentation and during the storage time ( Figures  3a-c). The addition of WPH had a significant effect (p<0.05) on the growth of L. acidophilus after the fermentation, and at 21 st days of storage compared to the control (Figure 3a). Use of 0.5 and 1% WPH caused significantly (p<0.05) increase in the growth of L. acidophilus compared to the control samples. This could be due to that WPH could increase viability of lactic acid bacteria because of promoting the buffering ability of culture (Kailasapathy and Supriadi, 1996) as a unique source of nutrients and amino acid in WPH. The addition of lactic acid bacteria into milk may improve the production of free fatty acids by lipolysis of milk fat that caused to support the growth of L. acidophilus (Öndül, 2004(Öndül, , Yadav et al., 2007.
The nutrients supplementation of ayran by WPH had positive impacts on the promoting probiotics and lactic acid bacteria growth.

Value of pH
Addition of 0.25, 0.5 and 1% of WPH had significant (p<0.05) effect on pH value after fermentation and 7 th , 14 th and 21 st days of storage compared to control ( Table 1). The WPH containing samples at 0.25, 0.5 and 1% had significantly (p<0.05) lower pH levels compared to the control after fermentation at 7 th and14 th of storage. However, the samples containing 0.25, 0.5 and 1% of WPH had significantly (p<0.05) higher pH levels compared to the control after 21 st of storage (Table 1). Nadal et al., (2010) reported that the addition of WP decreased the acidification value at the end of storage of product. Moreover, Almeida et al., (2009)   Different capital letters; A, B, C, D, F indicate statistical difference between the times at α= 0.05 level among products at each time obtained from One-Way ANOVA. Different small letters; a, b, c, d, f indicate statistical difference between the concentrations at α= 0.05 level among products at each time obtained from One-Way ANOVA.
Dave and Shah (1998) reported that addition of WPH can increase lactic acid, that causes to reduce pH level. They recorded that WPH could be used to control the pH levels. The increase in buffering capacity led to decrease pH slowly and promoted acidification level by starter bacteria (Shafiee et al., 2010).

Total solid and protein measurements
Total solid and protein contents of control and WPH containing samples (0.25, 0.50, and 1%) were measured after the end of the storage time (Table 2). At 0.5% of WPH containing samples had higher total solid level than that of the control, 0.25% and 1% of WPH. WPH contains about 45-50% of milk solids (Hoffman and Falvo, 2004). Use of probiotics at 0.5 and 1% concentrations showed a higher value of total solid compared to control. Besides, samples containing WPH and probiotics had higher total solid content compared to the control.
When the use of WPH increased from 0.25 % to 1%, protein content of ayran increased from 16.49 to 19.51 mg/L. Incorporation of 0.5 and 1 % WPH into ayran significantly increased protein content compared to the control. The highest protein content of ayran was measured with the addition of 1% probiotics, followed by 1% of WPH+probiotics. The increase of protein content in the sample could come from the rich amount of protein in WPH (Vargas Lopez, 2013).

The viscosity
Addition of WPH, probiotics, and WPH+probiotics showed a significant (p<0.05) effect on the viscosity compared to the control samples after fermentation and 7 th , 14 th and 21 st days of storage time (Table 3).  Vargas et al. (2015) reported that the addition of 1% WPI increased the apparent viscosity compared to the control after the first day of storage. The increase of the viscosity could be due to the ability of the new style globules of whey protein to bind a higher amount of water, while the hydrophilic section of amino acids is put on the outer section of protein globule (Ipsen et al., 2000).

Hunter color parameters
Hunter color parameters; a* redness-greenness, b* yellowness-blueness, L* lightness-darkness have been previously recorded to describe visual colors (Garza et al., 1999). Different capital letters; A, B, C, D, F indicate statistical difference between the times at α= 0.05 level among products at each time obtained from One-Way ANOVA. Different small letters; a, b, c, d, f indicate statistical difference between the concentrations at α= 0.05 level among products at each time obtained from One-Way ANOVA.
It can be stated that the color of fermented products is an important issue for consumer preferences as well as for the shelf life of products. Its measurement can supply reliable information about food quality characteristics.
Samples containing 0.25, 0.50, and 1% of WPH showed a significant effect on the lightness value compared to the control after the fermentation, 14 th and 21 st of the storage (Table 4). In WPH added samples at 0.25, 0.5 and 1%, Hunter`s a* values changed significantly during the storage.
WPH having samples at 0.25, 0.5 and 1% recorded (p<0.05) increase in b* level after the fermentation and 14 th days of storage compared to the control. The samples having 0.25, 0.5 and 1% of WPH had lower (p<0.05) in the b* level at 21 st days of fermentation.

CONCLUSIONS
In this study, addition of WPH and probiotics at different concentrations (0, 0.25, 0.5 and 1%) into the ayran effected growth of S. thermophilus, L. delbrueckii spp. bulgaricus, L. acidophilus, and B. lactis. Besides, addition of these supplements could affect pH, total solid value, protein value, viscosity, and Hunter color values of ayran through the fermentation and the storage time. WPH and probiotics had great potentials for enhancing the growth of probiotic bacteria and nutritional of ayran.