Introduction

Yoghurt is a widely enjoyed dairy product and has a number of health benefits such as increased bone mineralization [1] and gut-associated immune response and laxation [2]. It is an excellent vehicle for the production of functional foods, especially those containing probiotic bacteria [3].

The health effects of yoghurt can be enhanced by the release of bioactive peptides [4] as a result of proteolytic activities of the organisms triggering the fermentation [5].

These peptides are specific protein fragments that have a positive impact on body functions and influence on health [6]. They are released through one of the following ways: (1) hydrolysis by digestive enzymes, (2) enzymatic cleavage by proteases derived from microorganisms or plants, and (3) via food processing or manufacturing (e.g., by acids, alkali, heating).

Fermentation of milk by lactic acid bacteria releases a large number of peptides and amino acids with varying biological actions, including inhibition of angiotensin-converting enzymes (ACE) and antioxidant activities [7], immune modulation, and opioid effect [8].

The present work aims to study the effect of starter culture on the antioxidant and antibacterial activities of bioactive peptides during cold storage of buffalo yoghurt.

Materials and methods

Materials

Buffalo’s milk and buffalo’s skimmed milk were obtained from Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture, Dokki, Egypt. Yoghurt culture (Streptococcus salivarius subsp. thermophilus CH1 and Lactobacillus delbrueckii subsp. bulgaricus LB-12-DRI-VAC) and Lactobacillus helveticus CH 5 were obtained from Chr. Hansen laboratories, Hoersholm, Denmark, while Lactobacillus acidophilus 20552 ATCC was obtained from Cairo Microbiological Resources Centre (Cairo, MIRCEN), Faculty of Agriculture, Ain Shams University, Egypt. Bacillus cereus ATCC 6538, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 9027 and Escherichia coli ATCC 2592 were obtained from Food Technology Research Institute, ARC, Ministry of Agriculture, Egypt, while Pseudomonas aeruginosa ATCC 9027 was obtained from Cairo University Research Park (CURP), Faculty of Agriculture, Giza, Egypt. Nutrient agar was obtained from Difico, Sparks, Maryland (USA). All chemicals were purchased from Sigma Chemical Company (Saint Louis, MO, USA).

Experimental procedure

Buffalo’s milk was standardized to 3% fat. The chemical composition of the milk was 11.70% total solids (TS), 3.5% total protein (TP), 0.78% ash, and 0.17% titratable acidity (TA). The milk was heated at 90 °C for 10 min, cooled to 38 ± 1 °C, and then divided into three portions. The first one (T1) was inoculated with yoghurt culture (2%) and served as control, while the second one (T2) was inoculated with 2% of a mixture of Lb. acidophilus 20552 ATCC + yoghurt culture (1:1).The third portion (T3) was inoculated with 2% of a mixture of yoghurt culture + Lb. helveticus CH 5 (1:1). All starter cultures were activated in MRS broth before use. The inoculated milk was packed in sterilized plastic cups and incubated at the optimum temperature for the growth of starter culture (38 °C for T2 and 42 °C for T1 and T3) until pH4.6 (0.6–0.7% titratable acidity). Yoghurt samples were cooled, maintained refrigerated at 4 °C, and analyzed at intervals.

Analysis methods

Chemical composition of milk and yoghurt

The milk fat, TS, protein content, and titratable acidity (TA%) of milk and yoghurt were determined according to the Association of Official Analytical Chemists (AOAC) [9].

Extraction of water-soluble peptides

Water-soluble peptide extract (WSPE) was prepared from yoghurt samples according to Bhardwaj and Singh [10]. The protein content of WSPE was determined according to Lowry et al. [11].

Determination of degree of hydrolysis

The degree of hydrolysis (DH) was determined according to Hoyle and Merritt [12].

$$ {\text{DH}}\left( \% \right) = \left[ {{\text{Soluble}}\;{\text{protein}}\left( {\text{mg}} \right)/{\text{Total}}\;{\text{protein}}\left( {\text{mg}} \right)} \right] \times 100 $$

Measurement of antioxidant activity

The antioxidant activity was evaluated using the following two different methods:

2,2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic (ABTS) assay

The antioxidant activity of WSPE of yoghurt samples, either fresh or during cold storage, was determined spectrophotometrically according to De Gobba et al. [13] at 405 nm. The addition of antioxidant compounds reduces the ABTS acid cations, thus causing a reagent decolorization, which is measurable spectrophotometrically, depending on the antioxidant type and concentration as well as the reaction time. The ABTS radical scavenging activity (%) was calculated using the following:

$$ {\text{Radical}}\;{\text{scavenging}}\;(\%)= 100 - \left[ {100 \, \times \, \left( {{\text{A}}_{\text{sample}} } \right)/\left( {{\text{A}}_{\text{ctrl}} } \right)} \right], $$

where Actrl is the absorbance of the control sample with water instead of hydrolysate and A sample is the absorbance of the hydrolysate sample.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay

The DPPH radical scavenging activity of the WSPE of fresh and cold stored yoghurt samples was spectrophotometrically determined at 517 nm according to Ahmed et al. [14].

$$ {\text{Radical}}\;{\text{scavenging}}\;{\text{activity}}\left( \% \right) = \left( {{\text{A}}0 - {\text{A}}1/{\text{A}}0} \right) \, \times \, 100, $$

where A0 is the absorbance of the control at 517 nm and A1 is the absorbance of the WSPE at 517 nm.

SDS-PAGE analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of WSPE of either fresh or cold stored yoghurt samples was performed according to Dei Piu et al. [15] by using handcast 16% (v/v) polyacrylamide gels. Gel preparation and vertical electrophoresis were carried out using Bio-Rad (Mini-PROTEANW system) equipment. To evaluate the protein molecular weights, 5 µL of standard protein and 15 µL of each sample containing around 7 µg/mL of protein were loaded. Gels were stained in a water solution containing 0.05% (w/v) Coomassie Blue, 50% (v/v) methanol, and 10% (v/v) acetic acid at 50 °C for a few minutes. They were then destained by washing with water a few times at 50 °C.

Peptide profile by ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS)

Peptides were characterized by LC–MS/MS analysis using an UHPLC + Ultimate 3000 (Thermo Fisher Scientific, 4000 Roskilde, Denmark) coupled with a Q Exactive Biotech mass spectrometer (Thermo Fisher Scientific, 4000 Roskilde, Denmark) as described by De Gobba et al. [16], with some modifications. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. Ten microliters of the samples were injected. The flow rate was 0.25 mL/min and the gradient consisted of 100% buffer A for 5 min, followed by a linear increase from 0 to 60% buffer B in 70 min. UV spectra were recorded at 214 and 280 nm. On-line MS/MS spectra were recorded in the positive mode using first the Full MS method from 200 to 2000 m/z with a resolution of 7000. Mass spectra of peptides were exported and compared to the buffalo’s proteome from the Swissprot database using Proteome Discoverer (v 1.4, Thermo Fisher Scientific, 4000 Roskilde, Denmark).

Antibacterial activity

The antibacterial activity of the WSPE of either fresh or cold stored yoghurt samples was examined according to Correa et al. [17] by using the disc diffusion assay. Indicator microorganisms were activated in the nutrient broth and the final concentration was 108 CFU mL−1. Aliquots of 25 µL of sample (50 mg mL−1) were spotted on the freshly prepared indicator strain, and plates were incubated at the optimal temperature for each test microorganism (37 °C for E. coli, S. Aureus, and S. typhimurium; 40 °C for B. cereus and 5–6 °C for Ps. aeruginosa). Subsequently, zone diameters of growth inhibition (represented by clear zone) were measured and presented as inhibition zones (mm). Streptomycin 500 mg was used as a positive control and sterilized water was used as a negative control.

Statistical analysis

All experiments were carried out in triplicates and the mean values were tabulated. Differences between samples with respect to bioactivity were tested using one-way analysis of variance (GLM procedure), and means of samples were compared using Duncan’s Multiple Range test (SPSS 23 static analysis, IBM).

Results and discussion

Chemical composition

Table 1 shows the chemical composition of yoghurt samples under cold storage. TS content ranged 11.71–11.75% in fresh samples and significantly increased (p < 0.05) during storage, up to 12.64–2.94%. The same trend was observed for both protein and fat contents. The TA increased significantly (p < 0.05) in all treatments during storage.

Table 1 Chemical composition (%) of yoghurt during storage at 4 °C
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DH represents the percentage of peptide bonds cleaved. The degrees of protein hydrolysis during cold storage under the different treatments, as shown in Fig. 1, were 56.88, 70.14, and 79.45% for T1, T2, and T3, respectively; they increased in all treatments at the end of cold storage to reach 71.71, 80.62, and 81.62%, with significant difference (p < 0.05) between control samples (T1) and both T2 and T3. The highest values were recorded in T3, in which Lb. helveticus CH 5 was used owing to its higher proteolytic activity, followed by T2 with non-significant differences, and lastly T1. Lb. helveticus has higher proteolytic activity than most other lactobacilli and hydrolyses more casein in culture media than other species [18].

Fig. 1
figure 1

Degree of hydrolysis (DH) of yoghurt samples during cold storage at 4 °C. T1 yoghurt with yoghurt starter, T2 yoghurt made with a mixture of Lb. acidophilus 20552 ATCC + yoghurt culture (1:1), T3 yoghurt made with a mixture of yoghurt culture + Lb. helveticus CH5 (1:1). Different superscripts (a, b, c…) indicate significant difference between treatments

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SDS-PAGE patterns of the WSPE of the yoghurt samples are shown in Fig. 2. The results indicate that all caseins were completely hydrolyzed by all strains, whereas the whey protein fractions were present even after 15 days of cold storage, suggesting the limited capacity to hydrolyze whey proteins. These results are in agreement with Gonzalez-Olivares et al. [19], who found that Lactic acid bacteria (LAB) degrade casein more than whey proteins. The intensity of β-LG band in T3, which contained Lb. helveticus, is light compared with that in T1 and T2, reflecting the ability of Lb. helveticus to hydrolyse β-LG. A similar result was reported by Bu et al. [20], who observed high hydrolysis of β-LG and α-LA with combined strains of Lb. helveticus and S. thermophilus. During cold storage, low-molecular-weight peptides appeared and their intensity increased with the storage period.

Fig. 2
figure 2

SDS-PAGE patterns of the WSPE of yoghurt samples during cold storage at 4 °C. MW molecular weight (KDa), Mk internal marker, BM buffalo milk protein marker, 1 T1 control yoghurt (fresh), 2 (7 days), 3 (15 days), 4 T2 acidophilus yoghurt (fresh), 5 (7 days), 6 (15 days), 7 T3 helveticus yoghurt (fresh), 8 (7 days), 9 (15 days)

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Peptide profiles of the WSPE of the fresh or cold stored yoghurt samples shown in Table 2 indicate that the molecular weight of the bioactive peptides in fresh control yoghurt (T1) ranged 0.68–1.99 kDa. During cold storage, lower-molecular-weight peptides were released and ranged 0.68–1.08 kDa at the end of storage. The molecular weight of the bioactive peptides in fresh acidophilus yoghurt (T2) ranged 0.78–2.33 kDa. As the storage period increased, new peptides were released with molecular weights 0.78–2.22 kDa. In fresh helveticus yoghurt (T3), smaller peptides were released compared to those in control and acidophilus yoghurt, with molecular weight ranging 0.68–1.09 kDa. At the end of storage, the molecular weight of the obtained peptides ranged 0.77–1.15 kDa. These results suggest that Lb. helveticus followed by Lb. acidophilus and yoghurt starter have the ability to produce low-molecular-weight bioactive peptides during fermentation. Most of the bioactive peptides originated from β-casein and fewer came from K-and αs1 casein. However, no peptides originated from whey proteins; this result agrees with the previous reports on other types of fermented milk [21, 22].

Table 2 Peptide profiles of the WSPE of yoghurt samples during cold storage
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Antioxidant peptides present in food play a vital role in the maintenance of antioxidant defense systems by preventing the formation of free radicals or by scavenging free radicals and active oxygen species, which induce oxidative damage to biomolecules and possibly cause many diseases [23]. Antioxidants from natural sources are more desirable than synthetic ones due to their carcinogenic effects [24]. Fermented milk products are an excellent source of bioactive peptides because of the proteolytic activities of the organisms involved in the fermentation [25, 26]. These peptides have an antioxidative effect [5, 27]. Figure 3(A, B) reveal a significant increment in the antioxidant activity of the WSPE of the fresh or cold stored yoghurt samples. In addition, significant differences were recorded between all treatments by the two methods. The rate of increment of the antioxidant activity (%) as determined by the ABTS assay was the highest in T1 (control 13.7%), followed by T2 (13.2%) and lastly T3 (10.5%), in which Lb. helveticus was used in combination with yoghurt starter.

Fig. 3
figure 3

Antioxidant activity (%) of the WSPE of yoghurt samples during cold storage at 4 °C. (A) ABTS assay, (B) DPPH assay. T1 yoghurt made with yoghurt starter, T2 yoghurt made with a mixture of Lb. acidophilus 20552 ATCC + yoghurt culture (1:1), T3 yoghurt made with a mixture of yoghurt culture + Lb. helveticus CH5 (1:1). The superscripts a, b, c… indicate significant difference between treatments. The superscripts A, B, C… indicate significant difference between storage periods

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The DPPH method is based on the ability of a sample to scavenge free stable DPPH radicals by hydrogen donation [28]. The DPPH scavenging activity of the WSPE of the yoghurt samples was 56.88–71.71% in T1, 70.14–80.62% in T2, and 79.45–81.62% in T3, in which Lb. helveticus was used. Lb. helveticus has been shown to exhibit strong proteolytic activity. The DPPH radical scavenging activity of crude yoghurt (0.2 mg/mL) was 94.47% of different fractions of yoghurt [29]. The authors [29] suggested that the oxidative stability of yoghurt depends on the antioxidant peptides released during the fermentation of milk by lactic acid bacteria. These peptides act as electron donors and can react with free radicals to convert them to more stable products. Milk fermented with Lb. acidophilus recorded the highest antioxidant activity, 63.99%, after the completion of fermentation. The lowest antioxidant activity of the same culture, which was recorded on the 10th day of storage at 4 °C, was 48.60%. The lowest antioxidant activity was seen in the milk fermented with the symbiotic cultures Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, which was 39.43%, as measured on the third day of storage [30].

Table 2 lists several peptides identified in the different samples. Among them, only LQDKIHP, LVYPFPGPIPKSLPQN, VYPFPGPIPK, LYQEPVLGPVRGP, LVYPFPGPIPK, VYPFPGPIPK, VLPVPQK, VAPFPE, KVLPVPQ, YQEPVLGPVRGPFPII, and VLPVPQ are already known for their antioxidant activity [13, 14, 31,32,33,34]. The lower-molecular-weight protein fractions were found to possess greater antioxidative capacity than higher-molecular-weight protein fractions. These peptides therefore can be considered as majorly responsible for the increased antioxidant activity of the samples. It is indeed known that peptides rich in Y, P, and F can contribute to the antioxidant activity [13].

The antibacterial activities of the WSPE of the fresh and cold stored yoghurt samples are shown in Table 3. Clearly, all the treatments displayed antibacterial activity against E. coli and this effect significantly increased at the end of cold storage, which is in accordance with Sah et al. [35]. In addition, T3 containing Lb. helveticus had the highest inhibition activity, followed by T1 and T2.

Table 3 Antibacterial activity of the WSPE of yoghurt during cold storage (inhibition zone in mm) at a concentration of 50 mg/mL
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Concerning the antibacterial activity against S. aureus and B. cereus, both T1 and T3 had positive effect, which effect increased toward the end of the storage period, while T2 had an opposite trend.

The antibacterial activity of the WSPE of the yoghurt samples against S. typhimurium of all treatments decreased significantly as the storage period increased. The highest effect was observed for T1 (control yoghurt), followed by T3 and then T2.

The antibacterial activity of the control yoghurt against P. aeruginosa had a positive effect, which significantly increased with the storage period, while the antibacterial activity of both T2 and T3 significantly decreased toward the end of storage.

As shown in Table 2, only the peptides IKHQGLPQ, YQEPVLGPVRGPFPIIV, YPFPGPIPK, LVYPFPGPIPK, and LVYPFPGPIPKSLPQN were already known for their antibacterial activity [31, 33, 34]. The different behaviors of the samples against different strains allow us to speculate that the peptides can act against only some of the bacteria and that they can also have some synergistic effect, since there is no clear pattern for the samples with the presence of a particular peptide and antimicrobial activity. Most antimicrobial bioactive peptides can act either by penetrating and disrupting microbial membrane integrity or by translocating across the membrane and acting on internal targets [35]. Lb. helveticus has higher proteolytic activity (because of the secretion of proteolytic enzymes) than most other lactobacilli, and it hydrolyses more casein in culture media than other species [18].

In conclusion, fermentation of the milk by lactic acid bacteria releases a large number of peptides and amino acids with varying biological actions. In this study, the yoghurt starters, Lb. acidophilus 20552 ATCC and Lb. helveticus CH 5, had variable proteolytic activity. The WSPE of yoghurt fermented with Lb. helveticus CH 5 displayed the highest antioxidant and antibacterial activities as compared to both yoghurt culture and Lb. acidophilus 20552.