Production and Evaluation of Probiotic Brown Yoghurt Made from Buffalo Milk as an Innovative Functional Dairy Product

doi:10.21203/rs.3.rs-4844222/v1

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Research Article

Production and Evaluation of Probiotic Brown Yoghurt Made from Buffalo Milk as an Innovative Functional Dairy Product

https://doi.org/10.21203/rs.3.rs-4844222/v1

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Purpose

Brown fermented milk has become more popular with consumers due to its high nutritional value, creamy texture, delicious caramel flavor, and brownish color. Brown yoghurt (BY), made from buffalo milk fortified with probiotic bacteria was evaluated as an innovative functional dairy product.

Methods

Standardized buffalo milk with a 1:1 protein/fat ratio was homogenized and browned at 97 ± 1°C for 4 h. At 42°C, it was inoculated with a 2.0% mixed starter culture and then divided into 4 portions. B. bifidum and L. rhamnosus, as probiotic bacteria, were added individually or in combination at a rate of 1.0% to create three treatments. The last portion without probiotics was served as a control BY.

Results

B. bifidum showed the highest viable counts when added alone or in combination with L. rhamnosus, particularly on days 7 and 15. However, the addition of B. bifidum did not improve the physical and sensory properties of the BY, which were similar to those of the control. Adding L. rhamnosus, either alone (T₃) or in combination with B. bifidum (T₄), greatly improved the viscosity, hardness, flavor compounds, and sensory scores of the BY. The antioxidant activity against DPPH and ABTS radicals was also significantly enhanced. T3 and T4 also had a thicker body, a smoother and creamier texture, and a light caramel taste combined with a pleasant sour taste. HMF concentration in By being affected slightly by bacteria strains and storage time.

Conclusions

standardized buffalo milk fortified with L. rhamnosus alone or in combination with B. bifidum can produce a higher-quality BY that is more acceptable as an innovative functional dairy product.

Brown yoghurt

Probiotic bacteria

Maillard reaction

Physiochemical properties

Sensory qualities

Fermented milk, also known as cultured milk, has been consumed for centuries and is popular for its nutritional value and appealing flavor. Around 400 different kinds of fermented milk products, such as yoghurt, Greek yoghurt, yammer, kefir, cultured buttermilk, filmjölk, cultured cream, and koumiss, are produced around the world and categorized based on the microorganisms (LAB, mold, or yeast) and fermentation processes ((Loveday et al., 2013; Turek and Wszołek, 2021). Depending on the type of microorganism utilized, lactic acid, acetic acid, carbon dioxide, diacetyl, acetaldehyde, alcohol, and many other compounds are produced throughout the fermentation process, giving the products their characteristic fresh taste and aroma (Zhenqiang, 2022). In addition to enhancing the product's flavor, the fermentation process also increases the product's shelf life and improves its digestibility (Savaiano and Hutkins, 2021).

Brown–fermented milk (BFM) or baked milk, which is produced by LAB fermentation and the Maillard reaction, is fermented milk that is particularly well-liked in Russia, Belarus and Ukraine. BFM is produced by holding milk at a high temperature for a long time (95–100°C, 3–5 h), causing the Maillard reaction between lactose and milk proteins, which causes the milk to turn brown (Ni et al., 2017; Han et al., 2019; Li et al., 2023). The bacterial starter culture is then added to initiate the fermentation process after the Maillard reaction is complete. Apart from the color difference, this manufacturing process also leads to variations in taste, flavor, and nutritional value compared with other fermented products (Martins et al., 2000). These functional and sensorial differences occur due to chemical changes during the heating and fermentation process, which result in milk metabolomics profiles that vary depending on the type of milk and the phase of fermentation. Typically, the fat content of BFM made in industrial settings ranges from 3.5 to 4%; however, it is generally permitted to range from 0.5–8.9%. Additionally, it has at least 3% protein while usually has 4–5% carbohydrates (ГОСТ 31455 − 2012). Ryazhenka and Varenets are the most popular names of BFM in Ukraine, Belarus, and Russia; Qatiq and Kaymak in Turkic countries. Recently, BFM became popular with consumers in China, Japan, Southeast Asia, Europe and other regions (Zhi-Yuan et al., 2010). Ma et al. (2016) reported that FBM has emerged as a new product in China since 2 year ago, with a variety of different forms; these include cooked fermented milk and Russian-style char-grilled fermented milk. Each country has its own standards for milk as a raw material, starter culture, production procedures, quality standards, and regulatory restrictions.

Yoghurt is the most popular fermented milk product in Egypt. It comes in a variety of types, including plain or stirred yoghurt, partially skimmed or skimmed, as well as sweetened and flavored forms. Probiotics have also become more and more popular as beneficial ingredients around the world, particularly in dairy products. They can be used as a starter culture alone or in combination with traditional starters. Probiotics add several functional aspects to the food, including improved flavor, taste, and texture (Gao et al., 2021). Additionally, probiotics have several beneficial effects on health, such as enhanced immune responses, relief from lactose intolerance symptoms, treatment for diarrhea, decreased blood cholesterol, vitamin synthesis, and anticarcinogenic properties (Markowiak and Śliżewska, 2017). The probiotic strain Lactobacillus rhamnosus is one of the most extensively utilized and studied. It was identified as a potential probiotic due to its tolerance to bile and acid, strong growth characteristics, and capacity to adhere to the intestinal epithelial layer. Another kind of probiotic found in the intestines is called Bifidobacterium bifidum. It encourages the stomach to produce acetic and lactic acid (Hoffmann et al., 2021). B. Bifidum is one of the bacteria that can help in the digestion of food, the absorption of nutrients, and the defense against harmful organisms (Chen et al., 2021). However, BFM is not available in the Egyptian market, and buffalo milk represents approximately 50% of dairy production in Egypt. In addition, many consumers, especially Egyptian consumers, may prefer these products due to their unique burnt flavor and brown color (Yu et al., 2020). Probiotics can also be effectively transported by BFM, and their manufacture and consumption can help people's health. Probiotics must be viable during fermentation and storage, interact with starter cultures in the product, and have an impact on the organoleptic qualities of the final product. Therefore, the aim of the study was to produce brown yogurt (BY), as innovative dairy fermented milk made from buffalo milk fortified with probiotic bacteria, and evaluate its physiochemical, microbiological, and sensory qualities, as well as the effect of probiotic bacterial diversity on the product's technological and nutritional characteristics.

1. Materials

Fresh buffalo milk was obtained from the farm of the Faculty of Agriculture, Cairo University, Egypt. Both starter cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus) and probiotic bacteria (Bifidobacterium bifidum and Lactobacillus rhamnosus) were obtained from stock cultures at the Dairy Microbiology Lab, National Research Centre, Cairo, Egypt. Three successive transfers into modified MRS were used to individually activate each strain, followed by three further transfers into sterile 11% reconstituted skim milk powder. The 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazinyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), semicarbazide hydrochloride, 5-Hydroxymethylfurfural (5-HMF) standard and analytical-grade zinc sulfate and potassium hexacyanoferrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol was procured from Merck in Darmstadt, Germany. All chemicals and reagents were analytical grade and obtained from different sources.

2. Methods

2.1 Brown yoghurt making

Standardized fresh buffalo milk (4.1% fat, 4.09% protein, 4.91% lactose, and 0.87% ash) was preheated to 65°C, homogenized using a laboratory homogenizer (POLYTRON PT10-35-GT-D, KINEMATICA, Swaziland) at 21,000 rpm for 5 min, and then browned at 97 ± 1°C for 4 h in an electric oven (Li et al., 2023). The milk container was wrapped in foil to avoid evaporation. Browned buffalo milk was immediately cooled to 42°C with cold water, then divided into 4 equal portions after being inoculated with 2.0% mixed starter culture (L. bulgaricus and S. thermophiles, at a ratio of 1:1). The first portion served as the control, while the second, third, and fourth portions were additionally inoculated with 1% B. bifidum, 1% L. rhamnosus, or 1% of their mixture as probiotic bacteria, respectively. All treatments were poured into 120 mL plastic cups and incubated at 42°C until pH reached 4.6–4.7. The brown yoghurt (BY) samples were quickly cooled, stored at 5 ± 1°C and taken for testing on days 1, 7, 14, and 21 of the storage period.

2.2 Chemical analysis

2.2.1 Measurement of pH: The pH value of the BY samples was measured during the storage period using a laboratory pH meter with a glass electrode (HANNA, equipment, Portugal).

2.2.2 Determination of flavor compounds: The flavor compounds were determined by measuring the acetaldehyde and diacetyl content in the BY samples using the Conway microdiffusion-semicarbazide method of Less and Jago (1970; 1976). The carbonyl compounds react with the semicarbazide to form semicarbazone, which has an absorption peak at 224 nm for acetaldehyde and at 270 nm for diacetyl. A standard curve with a range of 5 to 50 µmol/100 g was used to calculate the acetaldehyde and diacetyl content in all BY samples.

2.2.3 Determination of hydroxymethylfurfural (HMF)

HMF was extracted from BY according the method of Shi et al. (2019) with some modifications. Briefly, 2 g of BY samples were weighed into 15 mL centrifuge tubes, added with 5 mL of methanol, and then vortex vigorously for 2 min. The extractives were centrifuged 6000 g for 20 min. The clear supernatant was poured into another centrifuge tube, and then 250 mL of Carrez I (15% potassium hexacyanoferrate solution) and 250 µL of Carrez II (30% zinc sulfate solution) were added. 2 mL of clear supernatant was put into a conical-bottom glass tube and dried at 40°C using a nitrogen-pressure blowing concentrator after centrifugation also at 6000 g for 20 min. The remaining residue was re-dissolved with 2 mL of distilled water for further purification. UPLC H Class Waters (Detector PDA, Column C18 phenomenex; 150 mm*4.6 mm*5 micron) were used to measure the HMF in purified samples. A 4:1 v/v acetonitrile-water combination was employed as the mobile phase for 20 minutes at a flow rate of 0.6 mL/min. The injection volume was 20 µL, and the UV detection wavelength was 284 nm. The concentration of HMF was determined by comparing the UV spectrogram and retention time value with those of the appropriate standards. The peak area values obtained from the various HMF standards were used to produce a standard curve with a concentration range of 2.5–15 µg/mL.

Antiradical activities

The antiradical activity of the BY samples was evaluated in filtrated whey using both stable DPPH (DPPH) and ABTS radicals (ABTS) assays, according to Brand-Williams et al. (1995) and Re et al. (1999), respectively. 200 µL of BY filtered supernatant was added to 3.8 mL of DPPH or ABTS working solution (25 mg DPPH/L methanol). A spectrophotometer (Shimadzu spectrophotometer, UV-Vis. 1201, Japan) was used to determine the degree of decolorization after an incubation period of 30 min in the dark at room temperature. The wavelengths used were 517 nm for the DPPH and 700 nm for the ABTS radical-scavenging assays. In the same way, control solutions—DPPH and ABTS without whey were prepared. Both ABTS and DPPH scavenging activities were calculated using the formula shown below

BY antiradical activity (%) = [(A₀ – A₁)/A₀] x 100

A₀ is the absorbance of the control (DPPH or ABTS solution), and A₁ is the absorbance of the sample

Viable counts of microorganisms

The BY sample was homogenized in 25 ml for 1 min, and buffered peptone water (BPW) was used to provide the dilutions sufficient for the microbiological examination. Ten-fold diluted homogenate samples were used as the inoculant for selective media plates. The L. bulgaricus strain was counted on MRS agar, and the S. thermophilus strain was counted on M17 agar (Oxoid) after aerobic incubation at 30°C for 48 h, as described by Abosereh et al. (2016). B. Bifidum was counted using modified MRS agar supplemented with 0.05% L-cysteine-HCl and lithium chloride, while L. rhamnosus was counted using MRS agar plates treated with 1.1 mM bromocresol purple (El-Shenawy et al. (2016). The plates were incubated at 37°C for 48 h under anaerobic conditions.

Physical properties

Apparent viscosity

The apparent viscosity of the BY samples was measured using a Brookfield digital viscometer (Model DV-II, Canada) with the measuring spindle 04 rotating at 12 rpm. The samples were completely stirred using a mixer with 2.6 cm blades (Heidolph No. 50 111, Type RZRI, Germany) at speed 5 for 20 sec and then poured into a 100-mL glass cylinder. The viscosity was then measured at 7 ± 1°C. Each result was recorded in triplicate in mPa.s after a 30-sec rotation.

Color attributes: The color attributes of BY samples (L*, a*, and b*) were measured using a spectrocolorimeter (Tristimulus Color Machine) outfitted with the CIE lab color scale (Hunter, Lab Scan XE-Reston, VA, USA) in the reflection mode. L* represents a range of darkness from black (0) to white (100), a* for range of redness (+) to greenness (-), and b* for a range of yellowness (+) to blueness (-). Each time, a white tile from the Hunter Lab Color Standard (LX No. 16379) was used to standardize the instrument. The results were as follows: X = 72.26, Y = 81.94 and Z = 88.14 (L* = 92.46; a* = -0.86; b* = -0.16).

Sensory evaluation

The yogurt samples were evaluated for their appearance, body & texture, and flavor by experts chosen from the dairy department staff at the National Research Centre (Egypt). The nine-point hedonic scale, as described by Shazly et al. (2022), ranges from extremely like (9 points) through like or dislike (5 points) to extremely dislike (1 point). BY samples were provided in three-digit-coded clear plastic containers, and they were tasted for the first time 15 min after being taken out of the refrigerator. In order to refresh their palates in between samples, water and unsalted crackers were provided.

Statistical analysis

Statistical analysis with SAS (2008) software was done using the GLM technique. Two-way analysis of variance (ANOVA) and Duncan's multiple comparison method were used to compare the means. A probability of P ≤ 0.05 was chosen for establishing statistical significance. Results were presented as means ± SE.

Viable counts of microorganisms

The viable counts of the starter cultures S. thermophilus and L. bulgaricus as well as the probiotic bacteria B. bifidum and L. rhamnosus for brown yoghurt (BY) made from buffalo milk during storage at 5 ± 1°C for 21 days are presented in Fig. 1. In general, viable counts of starter culture and probiotic above the recommended limit of 10⁶ cfu/g were the crucial quality parameters for the final product that could be stored for up to 21 days. The highest viable counts of all bacteria in all BY samples were observed on day 7 of storage; following that, the counts began to decrease. S. thermophilus counts were more stable during the 21-day storage period, with either B. bifidum (T2) or L. rhomnosus (T3) present. Similarly, Shori et al. (2022) showed that L. rhomnosus enhanced the viability of Lactobacillus spp. and S. thermophilus in yogurt. With the exception of the S. thermophiles counts, the reduction reached statistical significance (P ≤ 0.05) on day 21. Conversely, the highest counts of starters, S. thermophilus and L. bulgaricus, were found on the first day of storage in the symbiotic low-fat yoghurt (Ramchandran and Shah, 2010) and probiotic yoghurt made with aloe vera gel (Ahmed et al., 2023) and on the third day of storage in the probiotic yoghurt made from ewe's milk (Shazly et al., 2022). Although most strains of Bifidobacterium grow slowly in milk due to their poor proteolytic activity, the viable counts of B. bifidum were the highest in the T2 and T4. A possible explanation for the increase in B. bifidum counts is the metabolic activity of S. thermophiles and L. rhamnosus, which supplies nutrients in the form of di-, tri-, and oligo-peptides (Liu et al., 2018; Li et al., 2020).

Chemical properties

1. Changes in pH and flavor compounds

The pH, flavor compounds measured as acetaldehyde and diacetyl (µmol/100g sample), and antioxidant activity against DPPH radicals of BY fortified with probiotic bacteria during storage at 5 ± 1°C for 21 days are displayed in Table 1. Probiotic BY samples did not differ significantly from control BY samples in pH (P > 0.05), suggesting that neither L. rhamnosus nor B. bifidum contribute to increased acidity. Similarly, Jia et al. (2016) reported that L. rhamnosus produced modest acidification potential in goat milk. All BY samples showed a drop in pH during storage; the rate of drop was significant until day 15 (P < 0.05) and then non-significant. The decline in pH values is associated with the metabolic activity of bacteria, which have the capability of degrading lactose and producing organic acids. The pH values dropped considerably more during storage, reaching a range of 3.99 to 4.10, which was comparable to much research as well (Kang et al., 2019; Shazly et al., 2022; Zhuet al., 2023). As a result, neither the browning process nor the probiotic fortification had a significant effect (P > 0.05) on the fermentation process or the pH changes during storage.

The addition of L. rhamnosus alone (T3) or in combination with B. bifidum (T4) showed the highest concentrations of both acetaldehyde and diacetyl. The differences between T3 and control BY (T1) were significant (P ≤ 0.05) at days 1 and 15 for acetaldehyde content, whereas for daicetly, the differences were significant at days 1, 7, and 21 (P ≤ 0.05). However, all of the BY samples had an acetaldehyde concentration that ranged from 84.82 ± 4.45–99.15 ± 6.31µmol/100g on day 1 and decreased during storage to 40.40 ± 3.28–45.17 ± 3.31µmol/100g on day 21. Conversely, the diacetyl concentration of BY samples ranged from 15.11 ± 2.54–23.11 ± 1.44 µmol/100g on day 1 and significantly increased (P < 0.05) during storage to 79.41 ± 6.74–89.68 ± 6.56 µmol/100g on day 21. These values were higher than the acetaldehyde concentrations that could be detected in traditional yoghurt made from various starter cultures, which were found to range from 12.09 to 43.60 µmol/100g (Tamime and Robinson, 2000). According to previous studies, the ranges of acetaldehyde concentrations were 9.30–40.70 (Hernandez et al., 1995), 38.37–66.28 in non-fat yoghurt from a high milk protein powder (Mistry and Hassan, 1992), and 7.50–21.66 µmol/100g in probiotic concentrated yoghurt fortified with CLA (Abd El-Salam et al., 2011). Han et al. (2019) reported that the distinctive flavor of a product is formed by flavor components such as carboxylic acids, aldehydes, alcohols, and ketones, which are produced during the browning stage and then increased during fermentation (Han et al., 2019). Similar, the concentrations of diacetyle found in BY samples were higher than those found in studies by Abd El-Salam et al. (2011) and Hassan et al. (2015), which were 2.54–7.35 and 0.92–3.22 µmol/100g, respectively. Thus, BY fermented with starter culture alone or in combination with probiotics is characterized by a high concentration of flavor compounds.

Table 1

pH values and flavor compounds of probiotic yoghurt made from buffalo milk fortified with probiotic bacteria during storage at 5 ± 1°C for 21 days
Brown yoghurt treatments	Storage periods (days)
Brown yoghurt treatments	1	7	15	21
pH
T1	4.56^Aa ± 0.06	4.34^Ab ± 0.07	4.09^Ac ± 0.08	4.03^Ac ± 0.04
T2	4.50^Aa ± 0.06	4.33^Ab ± 0.05	4.03^Ac ± 0.07	4.01^Ac ± 0.05
T3	4.55A^a ± 0.04	4.29^Ab ± 0.06	4.12^Ab ± 0.08	4.10^Ac ± 0.07
T4	4.52A^a ± 0.06	4.28^Ab ± 0.05	4.14^Ac ± 0.06	4.07^Ac ± 0.05
Acetaldehyde (µmol/100 g)
T1	86.52^Ba ± 5.71	78.93^Aa ± 3.97	44.13^Bb ± 3.01	40.40^Ab ± 3.28
T2	84.82^Ca ± 4.45	86.22^Aa ± 4.37	43.66^Bb ± 4.45	42.39^Ab ± 5.19
T3	99.15^Aa ± 6.31	90.59^Ab ± 3.42	53.27^Ac ± 2.82	45.17^Ac ± 3.31
T4	97.78^ABa ± 7.71	92.70^Aa ± 4.56	48.38^ABb ± 3.81	42.96^Ac ± 4.02
Diacetyl (µmol/100 g)
T1	15.11^Bd ± 2.54	40.89^Bc ± 2.31	62.55^Ab ± 2.89	79.41^Ba ± 6.74
T2	20.01^ABd ± 2.51	37.78^Bc ± 4.11	60.39^Ab ± 5.05	81.70^Ba ± 2.66
T3	22.78^Ad ± 1.92	47.75^{A c}±3.27	63.83^Ab ± 2.71	89.68^Aa ± 6.56
T4	23.11^Ad ± 3.44	46.33^Ac ± 3.51	64.56^Ab ± 3.31	86.08^ABa ± 3.41

^ABCMeans (n = 3 ± SE) with different alphabets are significantly different between each type of yoghurt for a particular day of storage; ^abcdMeans with different alphabets are significantly different within each type of yoghurt; T1, brown yoghurt fermented with starter culture; brown yoghurt fermented with starter culture and L. rhamnosus; brown yoghurt fermented with starter culture and B. bifidum; brown yoghurt fermented with starter culture, L. rhamnosus and B. bifidum.

2. Changes in HMF

After fermentation (1 day) and on day 15 of storage at 5 ± 1°C, the HMF concentrations of BY as influenced by probiotic bacteria were determined. On day 1, T2, which fermented with B. bifidum, had the highest HMF concentration, followed by T4, which fermented with L. rhamnosus and B. bifidum. These findings might be explained by B. bifidum fermentation, which accelerates the breakdown of lactose into glucose and galactose and stimulates Maillard reactions, which rapidly produce HMF. Conversely, On the other hand, T4, which contains L. rhamnosus, had the lowest HMF content; nevertheless, the differences were not statistically significant (P > 0.05), as illustrated in Fig. 2. In contrast to predictions, all BY treatments showed a small drop in HMF concentrations during cold storage. Hang et al. (2019) found a similar finding after storage brown fermented milk at C for 21 days Albalá-Hurtado et al. (1998) reported that no variations in the HMF levels have been observed, which aligned with the values found in the baby milk kept at 20°C but not in the milk kept at 37°C, where an increase in HMF levels was detected.

T1, brown yoghurt fermented with starter culture; brown yoghurt fermented with starter culture and L. rhamnosus; brown yoghurt fermented with starter culture and B. bifidum; brown yoghurt fermented with starter culture, L. rhamnosus and B. bifidum.

3. Antiradical activities

As shown in Table 1, probiotic BY samples (T2, T3 and T4) exhibited higher antioxidant activity against DPPH and ABTS radicals compared to control BY. The proteolysis degree by probiotic strains and the type of peptides released are considered other factors that participated in the observed increment in the antioxidant activity (Taha et al., 2017). The antioxidant activity against DPPH radicals was more pronounced in T3, followed by T4 (P ≤ 0.05). Similarly, Liu et al. (2018) found that L. rhamnosus significantly enhanced the DPPH radical scavenging behavior of cheddar cheese during the ripening period compared to the control group. L. rhamnosus may stimulate the production of smaller-molecule polypeptides through proteolysis. Moreover, L. rhamnosus has the ability to produce exopolysaccharides, which have antioxidant properties (Faraki and Rahmani, 2020). In general, Hoffmann et al. (2021) reported that lactobacilli cell-free supernatants like L. rhamnosus exhibit strong antioxidant activities against DPPH radical scavenging, inhibition of linoleic acid peroxidation, hydroxyl radical scavenging and reducing power (RP) assays. As the time of storage increased, the antioxidant activity against DPPH radicals increased, the increase was significant (P ≤ 0.05) at day 7 for T1 and T2, whereas for T3 and T4 the increase (P ≤ 0.05) was significant at day 21. Increased antioxidant activity during storage could have been related to protein hydrolysis (Shazly et al., 2022). Similarly, Liu et al. (2018) found that L. rhamnosus significantly enhanced the DPPH radical scavenging behavior of cheddar cheese during the ripening period compared to the control group.

Table 2

Antiradical activities of brown yoghurt made from buffalo milk fortified with probiotic bacteria during storage at 5 ± 1°C for 21 days
Brown yoghurt treatments	Storage periods (days)
Brown yoghurt treatments	1	7	15	21
DPPH scavenging activity (%)
T1	8.84^Bc ± 0.71	11.52^Bbc ± 0.21	12.95^Bab ± 1.11	15.71^Ba ± 0.37
T2	10.75^Bb ± 0.57	15.65^ABa ± 1.31	14.73^ABa ± 0.53	17.22^ABa ± 0.73
T3	14.45^Ab ± 0.93	17.72^Aab ± 1.81	16.50^Aab ± 0.66	19.99^Aa ± 1.65
T4	13.19^Ab ± 1.35	14.88^Bab ± 1.45	17.79^Aa ± 0.93	17.87^ABa ± 1.21
ABTS scavenging activity (%)
T1	33.30^Bb ± 1.97	36.65^Bab ± 2.36	38.55^Ba ± 2.61	39.61^Ba ± 2.28
T2	38.05^Ab ± 1.24	45.34^Aab ± 1.54	47.72^Aa ± 1.47	47.56^Aa ± 2.54
T3	40.92^Ab ± 2.31	44.67^Aab ± 1.25	45.56^Aa ± 2.11	47.73^Aa ± 2.24
T4	39.97^Ab ± 0.97	44.48^Aab ± 1.65	45.73^Aa ± 2.15	49.02^Aa ± 1.24

Physical properties

1. Apparent viscosity and firmness

According to a previous study (Akalın et al., 2012; Ichimura et al., 2023), the extended heating of the milk at high temperatures reduced the texture properties of the resultant fermented milk: viscosity and firmness. Viscosity plays a crucial role in consumer acceptance of yoghurt. Viscosity reflects the consistency and hardness of yoghurt samples; the higher, the better (Hasani et al. 2016). As shown Fig. 3, T3 and T4 exhibited higher viscosity and hardness (P ≤ 0.05) in comparison to T1 and T2, indicating that L. rhamnosus fortification improved BY viscosity and hardness regardless of whether B. bifidum was present or not. For samples fortified with a combination of L. rhamnosus and B. bifidum (T4), the improvement in BY hardness was more noticeable. Yang et al. (2010) reported that capsular polysaccharide produced by L. rhamnosus (composed mainly of galactose and N-acetylglucosamine in a ratio of 5:1) was identified during fermentation using both optical and transmission electron microscopy. L. rhamnosus can be used in the dairy industry to improve the rheological properties of fermented milk products by increasing their viscosity and water-holding capacity. Similarly, Jia et al. (2016) found that adding L. rhamnosus GG can improve the quality of goat milk yoghurt. L. rhamnosus grew and acidified milk, as well as being able to increase the viscosity of and confer a desirable texture to the fermented product (Salazar et al., 2009). However, the viscosity and hardness of BY samples were unaffected significantly (P > 0.05) by B. bifidum fortification alone. This result is in accordance with the findings of Prasanna et al. (2014), who reported that, like many other bacteria, bifidobacteria produce EPS, although it is a very rare phenomenon compared to other genera. Over the storage period of 21 days, BY samples exhibited a continuous increase in viscosity until day 15 (P ≤ 0.05) and then a slight decrease, whereas hardness continued to increase until day 21. Viscosity is increased because the particles are more swollen and attached over a greater area, and gel particles have stronger connections (Walstra et al., 1999). Also, Jia et al. (2016) reported that when the acidity value is too high, the protein gel becomes dehydrated. Thus, it reduced the yoghurt's capacity to hold water and caused the whey to dissolve, increasing the yoghurt's hardness. Such an effect has been confirmed by other researchers (Doleyres et al., 2005; Shazly et al., 2022).

Color attributes

On day 1, there are no differences in the L*, a*, and b* colors of BY, indicating that the fortification with probiotic bacteria had no effect on the color attributes during fermentation (Table 3). The lightness, redness, and yellowness degrees were within the range found by Han et al. (2019) and were between 82.56 ± 0.95–82.77 ± 1.08, 2.28 ± 0.29–2.47 ± 0.16, and 18.06 ± 1.08–18.86 ± 1.05, respectively. Additionally, both T1 and T2 did not exhibit any discernible changes in their color attributes during storage, which suggests that colored compounds were not formed. Han et al. (2019) found a similar finding with brown fermented milk stored at 4–7°C. L. rhamnosus appears to have the capacity to bind or absorb some Maillard reaction products during storage, as indicated by the observation that T3 and T4 showed a slight rise in lightness and a decrease in yellowness. These results may be related to the ability of L. rhamnosus to produce capsular polysaccharides (Yang et al., 2010), which adsorb or reduce Millard reaction compounds. The color attributes—a slight increase in the degree of whiteness (L*) and a slight decrease in the degree of yellowness (b*)—are confirmed by a slight drop in the HMF concentration after storage at 5 ± 1°C.

Table 3

Color parameters of brown yoghurt made from buffalo milk fortified with probiotic bacteria during storage at 5 ± 1°C for 15 days
Brown yoghurt treatments	Storage periods (days)
Brown yoghurt treatments	1	15
L*
T1	82.77 ± 1.08^ns	82.02 ± 0.55
T2	82.72 ± 1.23	82.96 ± 1.08
T3	82.67 ± 1.06	83.61 ± 0.66
T4	82.56 ± 0.85	83.11 ± 0.98
a*
T1	2.47 ± 0.16^ns	2.53 ± 0.13
T2	2.28 ± 0.29	2.21 ± 0.43
T3	2.38 ± 0.24	2.41 ± 0.17
T4	2.42 ± 0.22	2.33 ± 0.42
b*
T1	18.63 ± 0.38^ns	18.79 ± 0.41
T2	18.64 ± 0.37	18.06 ± 1.08
T3	18.66 ± 0.41	17.76 ± 0.39
T4	18.86 ± 1.05	18.17 ± 0.54

Means (n = 3 ± SE) with the same letters are not significantly different at P ≤ 0.5; ns, non-significant; T1, brown yoghurt fermented with starter culture; brown yoghurt fermented with starter culture and L. rhamnosus; brown yoghurt fermented with starter culture and B. bifidum; brown yoghurt fermented with starter culture, L. rhamnosus and B. bifidum.

Sensory evaluation

Table 4 shows the scores for probiotic BY made from buffalo milk during storage for 21 days at 5 ± 1°C in terms of appearance, body & texture, and flavor. In general, all of the BY samples were characterized by a pleasant flavor (caramel flavor), a smooth texture, and a soft body. According to Li et al. (2020), the brown fermented milk has significantly more di- and tri-peptides, which contribute to a unique taste. With the exception of small whey droplets that appeared on the surface of T1 and T2 after two weeks of storage, there were no appreciable differences in the appearance among all of the BY samples. Additionally, no discernible change in flavor or texture attributes was found between T1 and T2 (P > 0.05), indicating that B. longum had no positive effect on sensory attributes of BY. Similarly, Tian et al. (2022) found that the flavor and taste of the yoghurt grown just with B. longum did not differ significantly from the yoghurt fermented with the starter culture. However, T3 and T4 were superior in terms of flavor—a light caramel taste combined with a desirable sour taste, a smoother and creamy texture, and a thicker body (P < 0.05). The quality of goat milk yoghurt was found to be improved by the inclusion of L. rhamnosus in the appropriate proportion (Jia et al., 2016). This suggests that adding L. rhamnosus, either with or without B. bifidum, can improve the sensory properties of BY. The sensory attributes of all BY samples changed little during storage, however on day 21, the taste appeared somewhat sour. During storage, similar and acceptable sensory qualities were observed, with a tendency for the quality to reduce with an extended storage period for day 21. Such an effect was found in fermented goat milk with B. animalis ssp. lactis or B. longum by Mituniewicz-Małek et al. (2017).

Table 4

Sensory evaluation of brown yoghurt made from buffalo milk fortified with probiotic bacteria during storage at 5 ± 1°C for 21 days
Brown yoghurt treatments	Period of storage (day)
Brown yoghurt treatments	1	7	15	21
Appearance
T1	8.18 ± 0.18^ns	8.09 ± 0.20	7.90 ± 0.19	7.81 ± 0.19
T2	8.00 ± 0.19	8.09 ± 0.21	7.81 ± 0.22	7.81 ± 0.23
T3	8.36 ± 0.20	8.36 ± 0.25	8.27 ± 0.19	8.27 ± 0.18
T4	8.27 ± 0.14	8.27 ± 0.14	8.27 ± 0.16	8.10 ± 0.12
Body & texture
T1	7.81 ± 0.22^Ba	7.81 ± 0.21^Ba	7.63 ± 0.21^Ba	7.63 ± 0.23^Ba
T2	7.71 ± 0.26^Ba	7.81 ± 0.21^Ba	7.72 ± 0.20^Ba	7.63 ± 0.26^Ba
T3	8.45 ± 0.31^Aa	8.63 ± 0.28^Aa	8.63 ± 0.28^Aa	8.45 ± 0.15^Aa
T4	8.36 ± 0.18^Aa	8.63 ± 0.15^Aa	8.54 ± 0.17^Aa	8.45 ± 0.23^Aa
Flavor
T1	7.17 ± 0.21^Ba	7.36 ± 0.20^Ba	7.36 ± 0.23^Ba	7.09 ± 0.24^Ba
T2	7.27 ± 0.22^Ba	7.36 ± 0.24^Ba	7.27 ± 0.19^Ba	7.18 ± 0.18^Ba
T3	8.36 ± 0.15^Aa	8.63 ± 0.24^Aa	8.63 ± 0.16^Aa	8.27 ± 0.22^Aa
T4	8.27 ± 0.19^Aa	8.45 ± 0.16^Aa	8.45 ± 0.14^Aa	8.27 ± 0.15^Aa

^ABMeans (n = 3 ± SE) with different alphabets are significantly different between each type of yoghurt for a particular day of storage; ^abMeans with different alphabets are significantly different within each type of yoghurt; T1, brown yoghurt fermented with starter culture; brown yoghurt fermented with starter culture and L. rhamnosus; brown yoghurt fermented with starter culture and B. bifidum; brown yoghurt fermented with starter culture, L. rhamnosus and B. bifidum.

Standardized buffalo milk (~ 4.0% fat, ~ 4.0% protein) heated to 97 ± 1°C for 4 h can be used to produce high-quality BY as an innovative dairy product with a more acceptable flavor and color. B. bifidum is not recommended to be added alone for making BY, but it can be added to mixed cultures as an adjunct to L. rhamnosus because of its health benefits. L. rhamnosus has probiotic properties, but it can also be technologically utilized to enhance the viscosity, hardness, flavor compounds, antioxidant activity and sensory properties of resultant BY. Brown fermented milks can be widely used in other dairy products, like frozen yogurt, because they are well-liked by customers. Furthermore, the production of these products depends on Maillard reaction products, which have the potential to be harmful. As a result, the whole product must be biologically evaluated, not just individual components, to decide whether there will be positive or negative effects on health and thus limit or expand its production.

Authors’ contributions

All authors contributed substantially towards the paper. The author(s) read and approved the final manuscript.

Consent for publication

All of the authors consent to the publication of this manuscript in Annals of Microbiology.

Competing interests

The authors declare no competing interests in publishing this manuscript.

Author details

1 Dairy Department, Food Industries and Nutrition Research Institute, National Research Centre, Giza, Egypt. 2 Regional Centre for Food and Feed, Agriculture Research Centre, Giza, Egypt

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

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Production and Evaluation of Probiotic Brown Yoghurt Made from Buffalo Milk as an Innovative Functional Dairy Product

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Abstract

Purpose

Methods

Results

Conclusions

Figures

INTRODUCTION

MATERIALS AND METHODS

1. Materials

2. Methods

2.1 Brown yoghurt making

2.2 Chemical analysis

2.2.3 Determination of hydroxymethylfurfural (HMF)

Viable counts of microorganisms

Physical properties

Statistical analysis

RESULTS AND DISCUSSION

Viable counts of microorganisms

1. Changes in pH and flavor compounds

2. Changes in HMF

3. Antiradical activities

1. Apparent viscosity and firmness

Color attributes

Sensory evaluation

Conclusion

Declarations

Authors’ contributions

Author details

Availability of data and materials

References

Status:

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