Lactobacillus salivarius, in-vitro probiotic characteristics and application of lactobacillus salivarius in yogurt fermentation

Abstract

The invention relates to the field of food, in particular to probiotic characteristics of lactobacillus salivarius JCF5 and a method for preparing Jersey yogurt through synergistic fermentation of the lactobacillus salivarius JCF5. The strain provided by the invention is named as Lactobacillus salivarius JCF5, and the Latin is Ligillactobacillussavarius. The invention further discloses a preparation method of the strain. The strain provided by the invention has the advantages that the strain is named as Lactobacillus salivarius JCF5; the strain is obtained by randomly selecting six newly-born Jersey and six Jersey-and-cattle hybrid calves with good health conditions from a pasture in Heilongjiang province, collecting fetal manure of the Jersey and six Jersey-and-cattle hybrid calves after the Jersey and cattle hybrid calves are born, and separating manure samples after the Jersey and cattle hybrid calves eat colostrum. The water-holding capacity and whey precipitation rate of the Jersey yoghourt can be remarkably improved, and the viscosity of the Jersey yoghourt can be increased. In addition, the strain has better acid resistance, cholate resistance and simulated gastrointestinal fluid resistance, does not show hemolysis capacity and gelatinase activity, has stronger antibacterial capacity and antibiotic sensitivity, and has the characteristics of probiotics. The application potential of the strain in fermenting the Jersey yogurt is fully explored, the quality of the Jersey yogurt is expected to be improved by changing the strain proportion of the leavening agent sold in the market, and a new leavening agent is provided for fermentation of the Jersey yogurt.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to a strain of Lactobacillus salivarius, its in vitro probiotic properties, and its application in yogurt fermentation. Background Technology

[0002] The concept of probiotics first appeared in 1974 and was defined in 2002 as: "live microorganisms that, when given in adequate amounts, confer health benefits upon the host." In recent years, probiotics have become increasingly popular among consumers worldwide, particularly in dairy products, ice cream, snacks, breakfast cereals, and functional beverages. Consumer interest in probiotics is partly due to their health benefits, such as preventing the overgrowth of pathogenic bacteria, enhancing the gut's resistance to pathogen invasion, and improving the epithelial barrier.

[0003] *Lactobacillus salivarius*, belonging to the genus *Lactobacillus*, is a probiotic strain widely used in human and animal foods and diets. It has received considerable attention due to its probiotic and functional properties, as well as various health-promoting effects. *Lactobacillus salivarius* possesses excellent antioxidant and antibacterial properties. Furthermore, many strains of *Lactobacillus salivarius* are capable of producing bacteriocins with antagonistic activity. These probiotic properties of *Lactobacillus salivarius* indicate its significant potential in promoting human health. In addition, previous studies have shown that most *Lactobacillus salivarius* strains have a positive impact on the composition of the gut microbiota, improving gut microbiota metabolism and promoting a healthy and balanced internal environment. On the other hand, research has also shown that *Lactobacillus salivarius* possesses several functional properties beneficial to the food industry. These properties include improved nutritional quality, enhanced flavor characteristics, and extended shelf life of food.

[0004] Yogurt is one of the most popular fermented products, based on milk and fermented with probiotics. The dairy matrix is ​​rich in nutrients, containing bioactive minerals, proteins, lipids, and carbohydrates. Besides providing nutrition, yogurt offers additional health benefits due to its unique bioactive compounds and active cultures. Therefore, yogurt is considered a healthy food and an excellent carrier of probiotics. Studies have found that regular consumption of yogurt can balance trace elements, combat aging, moisturize skin and hair, brighten eyes, supplement calcium, enhance immunity, regulate brain activity, and alleviate irritable bowel syndrome.

[0005] Jersey milk has a higher milk solids content than Holstein milk, and its protein and fat content are also significantly higher, with high-quality milk protein content exceeding 3.5%, making it a good raw material for fermented yogurt. However, current fermentation of Jersey milk using commercially available starter cultures has yielded unsatisfactory results. Therefore, this invention utilizes a combination of Lactobacillus saliva-associated with commercially available starter cultures to ferment Jersey milk, exploring its effectiveness compared to fermentation using commercially available starter cultures alone.

[0006] This invention uses six healthy newborn Jersey and six Jersey-Wabi crossbred calves randomly selected from a ranch in Heilongjiang Province. Samples of meconium, feces after consuming colostrum, colostrum, and regular milk were collected after birth to isolate the strain. Physiological and biochemical identification and sequence analysis confirmed the strain to be *Lactobacillus salivarius*, which was named *Lactobacillus salivarius*. The safety and in vitro probiotic properties of this strain were clarified. Further research revealed that this bacterium has antibacterial effects against pathogenic bacteria such as *Escherichia coli*, *Staphylococcus aureus*, and *Listeria monocytogenes*. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide an excellent strain of *Lactobacillus salivarius* that can improve the quality of Jersey yogurt.

[0008] This experiment used Lactobacillus salivarius JCF5, which was isolated and purified by the research group, as a co-fermenting agent. Jersey milk was fermented with Lactobacillus salivarius JCF5 alone and Jersey milk fermented with commercially available starter culture alone as controls. The effects of Lactobacillus salivarius fermentation alone and fermentation with commercially available starter culture alone on the quality changes of Jersey yogurt were evaluated.

[0009] Its significance lies in:

[0010] 1. The biosafety and in vitro probiotic properties of this strain were fully elucidated;

[0011] 2. Improve the quality of Jersey yogurt by changing the strain ratio of commercially available starter cultures, and provide a new starter culture for Jersey yogurt fermentation;

[0012] 3. Fully explore the application potential of this strain in fermented Jersey yogurt and enrich its application scope.

[0013] Based on the above objectives, this invention first prepares Jersey yogurt by co-fermentation of Lactobacillus salivae JCF5 and a commercially available starter culture. Two control groups were set up: control group 1 (Jersey milk fermented with Lactobacillus salivae JCF5 alone) and control group 2 (Jersey milk fermented with a commercially available starter culture alone). The effect of Lactobacillus salivae JCF5 on improving Jersey yogurt was evaluated by measuring the water-holding capacity, whey separation rate, texture, and nutrient content of the finished yogurt products in each group. Attached Figure Description

[0014] Figure 1 Acid resistance of Lactobacillus salivarius JCF5

[0015] Figure 2 Bile salt tolerance of Lactobacillus salivae JCF5

[0016] Figure 3Simulated gastrointestinal results of Lactobacillus saliva-associated with JCF5

[0017] Figure 4 Hemolysis results of Lactobacillus saliva JCF5

[0018] Figure 5 Results of antibacterial activity of Lactobacillus saliva JCF5: Note: Oxford cup diameter is 7.83±0.17mm.

[0019] Figure 6 Gelatinase activity results of Lactobacillus saliva-derived JCF5: a: Lactobacillus saliva-derived JCF5; Escherichia coli (negative control); c: Staphylococcus aureus (positive control)

[0020] Figure 7 Antibiotic susceptibility results of Lactobacillus saliva-associated with antibiotic JCF5

[0021] Figure 8 Antibiotic susceptibility test of Lactobacillus saliva-associated 5 (JCF5): Note: S, susceptible; R, resistant.

[0022] Figure 9 Growth curves and pH changes of *Lactobacillus salivarius* JCF5 mixed with commercially available starter culture (1:1), *Lactobacillus salivarius* JCF5, and commercially available starter culture.

[0023] Figure 10 Three sets of Jersey yogurt fermentation endpoint diagrams

[0024] Figure 11 Water-holding capacity and whey separation of three groups of Jersey yogurts

[0025] Figure 12 Textural characteristics of three groups of Jersey yogurt: Note: Different lowercase letters in the same row represent significant differences (P < 0.05).

[0026] Figure 13 Basic ingredient list of three Jersey yogurts: Note: Different lowercase letters in the same row indicate significant differences (P < 0.05). Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0028] The isolation of Lactobacillus JCF5 from salivary samples, evaluation of its probiotic properties, and fermentation of Jersey milk with a commercially available starter culture in conjunction with salivary samples were achieved according to the following steps:

[0029] Experimental Example 1: Screening and Identification of Lactobacillus saliva-associated with JCF5

[0030] 1. Isolation and purification of Lactobacillus saliva-associated with Salivary Lactobacillu

[0031] All samples were provided by a ranch in Heilongjiang Province. Six healthy newborn Jersey calves and six Jersey-Wabi crossbred calves were randomly selected from the ranch. Meconium and fecal samples from the calves after they ingested colostrum were collected, totaling 24 samples. Under aseptic conditions, 1g of fresh fecal matter from each Jersey and Jersey-Wabi crossbred calf was accurately weighed and added to a centrifuge tube containing 10mL of PBS; this was the original bacterial culture. The original bacterial culture was then subjected to 10 ppm PBS. -1 Up to 10 -10 Serial dilutions were performed, and the concentrations were labeled. 100 μL of each concentration of bacterial solution was spread onto MRS agar plates and incubated upside down in a constant temperature microbial incubator at 37°C for 48 h. After single colonies grew, colonies with good growth status and characteristic features (color, size, shape) were picked and streaked onto MRS solid medium plates and incubated at 37°C for 24 h. Single colonies were picked and cultured for three more generations for purification to finally obtain pure colonies grown on MRS agar plates.

[0032] 1. Biological identification of Lactobacillus salivarius JCF5

[0033] The bacterial strain was expanded and cultured, and the bacterial solution was centrifuged to obtain bacterial cells, which were then sequenced. Sequencing was performed by Nanjing Paisennong Gene Technology Co., Ltd. The 16S rDNA sequence of strain JCF5 was submitted after sequencing was completed.

[0034] A comparison with the NCBIBLAST database revealed that the gene sequence of strain JCF5 had 99.32% homology with *Ligilactobacilus salivarius*. Strain JCF5 was identified as *Ligilactobacilus salivarius*, which is deposited at the China Microbial Culture Collection Center, accession number: CGMCC No. 30923.

[0035] Experimental Example 2: Activation and Acid and Bile Salt Tolerance Experiments of Lactobacillus salivans JCF5

[0036] 1. Activation of Lactobacillus saliva-associated 5 (JCF5)

[0037] After thawing the Lactobacillus saliva-associated strain JCF5 preserved in glycerol at -20℃, it was inoculated into 5 mL of liquid MRS medium and incubated at 37℃ for 24 h. Then, the activated strain was inoculated into MRS liquid medium and incubated at 37℃ for 24 h. The above operation was repeated twice to ensure that the strain was fully activated.

[0038] In this embodiment, the MRS liquid culture medium was prepared as follows: 49.3g of MRS culture medium powder was dissolved in 1L of distilled water in an Erlenmeyer flask, and then autoclaved at 121°C for 20 minutes.

[0039] 2. Acid resistance test of Lactobacillus saliva-associated with JCF5

[0040] After two generations of activation in MRS medium at 37℃, the strain was inoculated at a 2% inoculum into MRS medium with pH adjusted to 4.0, 3.0, and 2.0 using 4.0 mol / L HCl, as well as into unadjusted MRS medium with pH 6.8. 100 μL of bacterial suspension was collected at 0 h and 3 h, and then serially diluted with physiological saline at a ratio of 1:9. Plates were then prepared, with each sample plated three times. After incubation at 37℃ for 24 h, colony counting was performed. Untreated MRS medium served as a control.

[0041] Probiotics must enter the human gastrointestinal tract to exert their beneficial functions. After being ingested, their activity is affected by the acid in the gastrointestinal tract, so acid tolerance is an important indicator of the quality of probiotics. In normal individuals, the pH of gastric juice on an empty stomach is 1.5-2.0. After food intake, gastric juice is diluted, causing the pH to change to approximately 3.5. Therefore, this experiment selected pH values ​​of 2, 3, and 4 to test the acid tolerance of the strains. Figure 1 It can be concluded that Lactobacillus salivarius JCF5 exhibits strong acid resistance at pH 4 and 3, while its acid resistance is weaker at pH 2 compared to pH 4 and 3. However, after 3 hours of culture, its survival rate can still reach 42.8%, indicating that the strain has good acid resistance.

[0042] 3. Bile salt tolerance test of Lactobacillus saliva-associated with Lactobacillus JCF5

[0043] After two generations of activation, *Lactobacillus salivarius* JCF5 strain was inoculated at a 2% inoculum into MRS medium containing no bile salts and 0.1% and 0.3% bile salts, respectively. 100 μL of bacterial suspension was collected at 0 h and 3 h, and then serially diluted with physiological saline at a ratio of 1:9. Plates were then prepared, with each sample plated three times. After incubation at 37°C for 24 h, colony counting was performed. MRS medium without bile salts served as a control.

[0044] Bile salts have antibacterial properties, and adapting to bile salt stress in the human small intestine is a crucial prerequisite for probiotics to exert their beneficial effects. Therefore, tolerance to bile salts is also an important indicator of the quality of probiotics. The concentration of bile salts in the small intestine of most people is around 0.3% (w / v). Therefore, this experiment selected two different mass fractions of bile salts (0.1% and 0.3%) and no bile salts as a control. Figure 2 It was found that at 0h and 3h, the viable count of Lactobacillus salivarius JCF5 gradually decreased with the increase of bile salt concentration, and after 3h of culture in a medium containing 0.3% bile salt, the viable count of Lactobacillus salivarius JCF5 was 3log (CFU / mL).

[0045] Experimental Example 3: Simulated Gastrointestinal Experiment Using Saliva-Associated Lactobacillus JCF5

[0046] After two generations of activation in MRS medium at 37℃, the strain was centrifuged at 8000g for 10 min, the supernatant was discarded, and the precipitate was added to 5 mL of artificial gastric fluid. After treatment at 37℃ for 3 h, the strain was centrifuged at 8000g for 10 min, the supernatant was discarded, and the bacterial sludge was retained. The sludge was then washed three times with sterile physiological saline. The precipitate was collected aseptically and mixed thoroughly with physiological saline at a ratio of 1:9. The mixture was serially diluted and plated, with each sample plated three times. After incubation at 37℃ for 24 h, colony counts were performed, and the average value was taken.

[0047] Take 1g of bacterial sludge after 3 hours of treatment with the gastric fluid, add 5mL of artificial intestinal fluid, treat at 37℃ for 3 hours, centrifuge and discard the supernatant, collect the precipitate, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge to physiological saline of 1:10, mix well, perform serial dilution, and count the colonies.

[0048] Survival rate of artificial gastrointestinal fluid = N t / N0

[0049] Where: N t N0 represents the number of viable bacteria after treatment with artificial gastric and intestinal fluids; N0 represents the number of viable bacteria before treatment.

[0050] Probiotic strains must survive adverse conditions encountered in the gastrointestinal tract to colonize the colon and positively impact consumer / host health. Food typically remains in the stomach for approximately 1-3 hours, retaining liquid foods. Once food is ingested and digestion begins, the pH gradually decreases; therefore, this experiment involved 3 hours of gastric juice treatment. After digestion in the stomach, food enters the intestines. The small intestine has an alkaline environment, and food typically resides there for 1-6 hours; therefore, this experiment involved 3 hours of intestinal juice treatment. Figure 3 It can be seen that after 3 hours of treatment with gastric juice, the viable count of Lactobacillus salivarius JCF5 reached 10.30±0.032 log(CFU / g), and the survival rate reached 90.67±0.407%. After 3 hours of treatment with intestinal juice, the viable count was 9.65±0.097 log(CFU / g), and the survival rate was 84.97±1.4%, indicating that Lactobacillus salivarius JCF5 has a good tolerance to artificial gastrointestinal juice.

[0051] Experimental Example 4: Biosafety Experiment of Saliment-Associated Lactobacillus JCF5

[0052] 1. Saliva-associated hemolysis test with Lactobacillus JCF5

[0053] Two generations of activated *Lactobacillus salivarius* JCF5 in MRS medium were spread onto blood agar plates using an inoculation loop and incubated at 37°C under anaerobic conditions for 48 hours. The presence of a clear, translucent halo around the colony was observed. A translucent halo indicated β-hemolysis, a green halo indicated α-hemolysis, and no halo indicated γ-hemolysis (i.e., no hemolytic activity). *Staphylococcus aureus* was used as a positive control.

[0054] observe Figure 4 It can be concluded that when Lactobacillus salivarius JCF5 was grown on blood agar medium, it did not exhibit hemolytic ability, while Staphylococcus aureus produced strong β-hemolysis.

[0055] 2. Saliva-based combined experiment with Lactobacillus JCF5 inhibition.

[0056] After activation, the indicator bacteria were inoculated into LB liquid medium and incubated at 37°C for 24 h. *Lactobacillus salivarius* JCF5 was also activated and inoculated into liquid MRS medium and incubated at 37°C for 24 h. Using the Oxford cup agar diffusion method, *Escherichia coli*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Bacillus subtilis*, and *Pseudomonas aeruginosa* were spread on plates, with blank MRS medium serving as a control. Two Oxford cups were evenly placed on each plate; one Oxford cup contained 200 μL of bacterial culture, and the other contained 200 μL of blank MRS medium. The plates were incubated at 37°C for 24 h. The presence of inhibition zones around the bottom of the Oxford cups was observed, and the diameter of the inhibition zones was measured and averaged.

[0057] In this embodiment, the LB liquid culture medium is prepared as follows: 10g peptone, 5g yeast extract, 10g sodium chloride, 1L distilled water are added and dissolved in an Erlenmeyer flask, and then autoclaved at 121℃ for 20min.

[0058] Figure 5 The table shows the antibacterial activity of Lactobacillus salivarius JCF5. It can be seen that inhibition zones were observed in all experiments with Lactobacillus salivarius JCF5, and the diameter of each inhibition zone was greater than 17.00 mm. This indicates that it inhibits Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, and Bacillus subtilis. The diameters of the inhibition zones were 17.77±0.40 mm, 22.00±1.00 mm, 18.33±0.5 mm, 20.00±2.00 mm, and 21.33±2.08 mm, respectively, demonstrating significant and strong antibacterial activity.

[0059] 2. Saliva-based assay for gelatinase activity in Lactobacillus JCF5

[0060] First, place the biochemistry tube in a 37°C incubator for 10 minutes. At this point, the culture medium in the tube will be liquid. Open the tube and add 100 μl of *Lactobacillus saliva-associated* JCF5 bacterial suspension. Seal the tube with sealing film and incubate at 37°C under anaerobic conditions for 48 hours. Then, place the tube in a refrigerator for 20 minutes to cool the culture medium to below 10°C. Tilt the tube and observe whether the culture medium solidifies. If the culture medium does not solidify and remains liquid, it indicates that the gelatin has been hydrolyzed, and the gelatinase test is positive. *Staphylococcus aureus* was used as a positive control, and *Escherichia coli* was used as a negative control.

[0061] observe Figure 6 The biochemical tubes containing *Lactobacillus saliva* JCF5 were found to be in a coagulated state, indicating the absence of gelatinase activity and a positive result for gelatinase. In contrast, the positive control, *Staphylococcus aureus*, showed a positive result for gelatinase, indicating that the gelatin was broken down into a liquid state.

[0062] 3. Saliva-based antibiotic susceptibility test with Lactobacillus JCF5

[0063] The susceptibility of the following six antibiotics was assessed using the disk diffusion method: ampicillin, tetracycline, erythromycin, clindamycin, vancomycin, and chloramphenicol. *Lactobacillus saliva-associated* JCF5 bacterial suspension was inoculated onto MRS agar at a 2% inoculum. Antibiotic disks were then added to the petri dishes, gently pressing them to ensure they adhered tightly to the culture medium surface. The petri dishes were incubated at 37°C for 48 hours. Antibiotic susceptibility was assessed by measuring the size of the inhibition zone.

[0064] Depend on Figure 7 and Figure 8 The results showed that Lactobacillus salivarius JCF5 was sensitive to ampicillin, tetracycline, erythromycin, clindamycin and chloramphenicol, but showed resistance to vancomycin.

[0065] Experimental Example 5: Growth curve and acid production capacity determination of Lactobacillus JCF5 in saliva

[0066] 1. Growth characteristics of Lactobacillus saliva-associated with JCF5 co-culture

[0067] To investigate the effects of *Lactobacillus salivarius* JCF5 under mixed culture conditions, *Lactobacillus salivarius* JCF5 and a commercially available starter culture were cultured in MRS medium at 37°C for 24 h. Under the same conditions, *Lactobacillus salivarius* JCF5 and the commercially available starter culture were then mixed at a 1:1 inoculation ratio. Under mixed culture conditions, the growth of the strains was measured at 600 nm every 2 h, and the pH of acid production was measured using a digital pH meter during the 24 h culture period.

[0068] according to Figure 9It can be seen that when Lactobacillus salivarius JCF5 is added under mixed culture conditions, the activity of the mixed strain is higher than that of the commercially available starter culture alone; compared with Lactobacillus salivarius JCF5 culture alone, the mixed strain has vigorous metabolism in the logarithmic growth phase and the highest number of viable cells at 16h; and the pH value of the culture medium of the three can also be seen from the pH value of the mixed culture strain, which also shows a sharp decrease.

[0069] Experimental Example 6: Preparation of Jersey Yogurt

[0070] Jersey yogurt was made by mixing whole-fat sterilized Jersey milk and white sugar, sterilizing and homogenizing, adding starter culture, and fermenting. The control was made by fermenting fresh milk alone with Lactobacillus salivarius JCF5 and fermenting fresh milk alone with commercial starter culture. Fermentation was carried out at 42℃. After fermentation, the yogurt was placed in a refrigerator at 4℃ for post-fermentation and storage.

[0071] In this embodiment, the fermentation of Lactobacillus saliva-associated with JCF5 in combination with a commercially available starter culture, the fermentation of Lactobacillus saliva-associated with JCF5 alone, and the fermentation of a commercially available starter culture alone are referred to as (SJCF5 group, JCF5 group, and S group), respectively.

[0072] The yogurts fermented with Lactobacillus salivarius JCF5 in combination with a commercially available starter culture, as well as those fermented with Lactobacillus salivarius JCF5 alone, showed no significant difference in appearance compared to the commercially available starter culture. All yogurts were glossy, milky white, and showed little or no whey separation. This indicates that Lactobacillus salivarius JCF5, as a starter culture, does not adversely affect the sensory properties of the yogurt.

[0073] Experimental Example 7: Determination of the fermentation endpoint of Jersey yogurt

[0074] According to national standards, the acidity of yogurt must reach 70°T or higher. Therefore, the fermentation endpoint of yogurt in this experiment was set at an acidity of 70°T.

[0075] Acidity determination: Samples were taken every 2 hours, with 10g of yogurt sample weighed each time and the mass M recorded. The sample was diluted with 20.00mL of distilled water, and 2-3 drops of phenolphthalein were added as an indicator and shaken well. Titration was performed with 0.1mol / L standard NaOH solution until the solution turned slightly pink and remained unchanged for 30 seconds. The volume V of standard NaOH solution consumed was recorded, and the acidity of the sample was calculated. The acidity value in the sample was calculated using the following formula, expressed in Gilnell degrees (°T), and denoted as X. Three parallel samples were taken from each group for determination.

[0076]

[0077] In the formula, X represents the acidity of the sample, expressed in degrees (°T).

[0078] C – Molar concentration (mol / L) of sodium hydroxide standard solution;

[0079] V — The volume (mL) of sodium hydroxide standard solution consumed during titration;

[0080] M—mass of the sample (g);

[0081] 0.1 — Molar concentration of sodium hydroxide (mol / L)

[0082] Depend on Figure 10 It can be seen that the saliva-based fermentation group combined with Lactobacillus and commercially available starter culture (SJCF5 group) and the commercially available starter culture group alone (S group) reached the fermentation endpoint simultaneously after 4 hours of fermentation, and the acidity of the two groups was 77.38±0.89, respectively. a °T, 77.4±2.11 a °T; while the saliva-associated lactobacillus-only fermentation group (JCF5 group) reached the fermentation endpoint after 9 hours, with an acidity of 77.53±9.91. a °T. The above results indicate that fermentation with Lactobacillus salivarius JCF5 in combination with a commercially available starter culture does not delay the fermentation time of the commercial starter culture, and there is no significant difference in acidity between the two at the fermentation endpoint. However, the group fermented solely with Lactobacillus salivarius JCF5 as the starter culture reached the fermentation endpoint after 9 hours of fermentation, which is slower than the other two groups. This may be because the optimal growth temperature of Lactobacillus salivarius JCF5 is 37°C, while the fermentation temperature of yogurt is 42°C, which leads to the slow growth of Lactobacillus salivarius, further resulting in slow acid production, thus causing a longer yogurt fermentation time.

[0083] Experimental Example 8: Quality Determination of Jersey Yogurt

[0084] Water-holding capacity is an important indicator of yogurt quality. The higher the water-holding capacity, the better the texture and stability of the yogurt. Higher water-holding capacity also makes the yogurt taste better.

[0085] Water-holding capacity determination: Take 10g of yogurt samples from different groups (W1), centrifuge at 8000×g for 10min at 4℃, discard the supernatant, and record the yogurt mass at this point (W2). Three parallel samples are taken from each group for determination. Water-holding capacity (WHC) is calculated according to the following formula:

[0086]

[0087] Whey separation is a key quality defect in both stirred and set yogurts, leading to the loss of water-soluble nutrients and unpleasant sensory characteristics.

[0088] Whey separation rate determination: 25g of yogurt was placed in a funnel with filter paper, and the filtered whey was collected in a beaker for 2 hours. The mass of the beaker (m) and the mass of the whey (m1) were recorded. Three replicates were set up for each sample.

[0089]

[0090] Figure 11 The water-holding capacity and whey separation of the three groups of Jersey yogurt were compared. It can be seen that the group fermented with the commercially available starter culture alone (S) had the highest water-holding capacity, at 93.81 ± 0.44. a The water-holding capacity of the salicylic acid-based Lactobacillus JCF5 fermentation group (SJCF5) was 86.71 ± 11.51%. a The percentage was %, while the water-holding capacity of the Lactobacillus salivarius JCF5 fermentation group alone (JCF5) was the lowest, at 68.44 ± 1.75%. b The percentage was %, but there was no significant difference between the group fermented with commercially available starter culture alone and the group fermented with Lactobacillus salivarius JCF5 combined with commercially available starter culture, indicating that the participation of Lactobacillus salivarius JCF5 in the fermentation of commercially available starter culture does not affect the water-holding capacity of Jersey yogurt; regarding whey separation, it can be seen that the whey separation rate of the group fermented with Lactobacillus salivarius JCF5 combined with commercially available starter culture (SJCF5) was the lowest, at 17.06±0.28%. c The percentage was followed by fermentation alone using commercially available starter cultures (S), at 26.12 ± 1.11%. b The highest percentage was observed in the Lactobacillus saliva-associated fermentation group (JCF5), at 34.11 ± 3.55%. a The percentage was %, and there were significant differences among the three groups. These results indicate that fermentation using saliva combined with Lactobacillus JCF5 and a commercially available starter culture can improve the quality of Jersey yogurt.

[0091] Experimental Example 9: Texture Determination of Jersey Yogurt

[0092] Texture is an important indicator for evaluating the quality of yogurt. This experiment used the TA-XT EXPRESS physical property tester to determine the textural properties of yogurt, including hardness, adhesiveness, elasticity, cohesiveness, chewiness, and resilience. Specific parameters are as follows: P / 36R probe was selected; the test distance was set to 15.0 mm; the probe speeds before, during, and after the test were 5.0, 2.5, and 2.0 mm / s, respectively; the test time was 5.00 s; and the sensing force was 5.0 g. Three parallel samples were taken from each group of yogurt samples for testing.

[0093] Figure 12The table shows the textural properties of three groups of Jersey yogurt. As can be seen from the table, the adhesiveness, elasticity, cohesiveness, and chewiness of the group fermented with Lactobacillus salivarius JCF5 and commercial starter culture (SJCF5) are higher than those of the group fermented with commercial starter culture alone (S). This indicates that the participation of Lactobacillus salivarius JCF5 in the fermentation of commercial starter culture can improve the adhesiveness, elasticity, cohesiveness, and chewiness of Jersey yogurt.

[0094] Experimental Example 7: Determination of the Basic Components of Jersey Yogurt

[0095] Ash content: determined according to GB 5009.4-2016.

[0096] Protein: Determined by the Kjeldahl method according to GB 5009.5-2016.

[0097] Fat: Determined by alkaline hydrolysis method according to GB 5009.6-2016.

[0098] Total solids: Add 20g of quartz sand to a petri dish and dry in a drying oven at 100℃±2℃ for 2h. Cool in a desiccator for 0.5h, weigh, and repeat drying until constant weight. Record the mass as m1. Weigh 5.0g of yogurt sample onto a petri dish with constant weight, record the mass as m2, dry in a drying oven at 100℃±2℃ for 3h, remove and cool in a desiccator for 0.5h, weigh, and then dry in a drying oven at 100℃±2℃ for 1h. Remove, cool, and weigh until the difference between the two masses does not exceed 1.0mg. Record the mass as m3.

[0099]

[0100] Depend on Figure 13 It can be seen that the solid content of the commercially available starter culture alone (S), the fermentation group of *Lactobacillus salivarius* JCF5 combined with the commercially available starter culture (SJCF5), and the fermentation group of *Lactobacillus salivarius* JCF5 alone (JCF5) were 21.2±0.53 g / 100g, 21.67±1.10 g / 100g, and 20.67±0.64 g / 100g, respectively, and the ash content was 0.6±0.09 g / 100g, 0... The protein content was 3.65±0.11g / 100g, 3.69±0.02g / 100g, and 3.71±0.04g / 100g, respectively, and the fat content was 4.66±0.05g / 100g, 4.78±0.07g / 100g, and 4.63±0.03g / 100g, respectively, all of which meet the national standards.

Claims

1. A method for isolating and evaluating the probiotic properties of *Lactobacillus salivarius* JCF5 (accession number: CGMCC NO.30923) and preparing Jersey milk by combining it with a commercially available starter culture, characterized in that... It is implemented in the following steps: All samples were provided by a ranch in Heilongjiang Province. Six healthy newborn Jersey calves and six Jersey-Wabi crossbred calves were randomly selected from the ranch. Meconium and fecal samples from the calves after they ingested colostrum were collected, totaling 24 samples. Under aseptic conditions, 1 g of fresh fecal matter from each Jersey and Jersey-Wabi crossbred calf was accurately weighed and added to a centrifuge tube containing 10 mL of PBS; this was the original bacterial solution. The original bacterial solution was then subjected to 10 mL of PBS. -1 Up to 10 -10 Serial dilutions were performed, and concentrations were clearly labeled. 100 μL of each concentration of bacterial culture was spread onto MRS agar plates and incubated upside down in a constant temperature microbial incubator at 37°C for 48 h. After single colonies emerged, colonies with good growth and characteristic color, size, and shape were picked and streaked onto MRS solid medium plates and incubated at 37°C for 24 h. Single colonies were then cultured for three generations for purification, ultimately obtaining pure colonies grown on MRS agar plates. The obtained single bacteria were molecularly identified and named *Lactobacillus salivarius* JCF5. The isolated strain was cryopreserved in glycerol. The cryopreserved *Lactobacillus salivarius* JCF5 was activated and aseptically inoculated into sterile MRS liquid medium in a clean bench and incubated at 37°C for 24 h. This process was repeated three times to ensure full activation of *Lactobacillus salivarius*. The fully activated and expanded bacterial culture was then inoculated into MRS medium at different pH values ​​and incubated at 37°C for 24 h. For example, the following experiments were conducted: * **Acid tolerance test of Lactobacillus salivae JCF5:** * **Bile salt tolerance test of Lactobacillus salivae JCF5:** * **Bacterial suspension test of Lactobacillus salivae JCF5:** * **Gastrointestinal simulation test of Lactobacillus salivae JCF5:** * **Hemolysis test of Lactobacillus salivae JCF5:** * **Inoculation test of indicator bacteria:** * **Inoculation test of indicator bacteria:** * **Inoculation test of Lactobacillus salivae JCF5:** * * **Inoculation test of indicator bacteria ... h, gelatinase activity experiment of Lactobacillus salivarius JCF5 was performed; the fully activated and expanded bacterial culture was inoculated onto MRS agar, and then antibiotic paper discs were added to the petri dishes to perform antibiotic sensitivity experiment of Lactobacillus salivarius JCF5; the activated Lactobacillus salivarius JCF5 starter culture was cultured in MRS medium at 37 ℃ for 24 h, and under the same conditions, Lactobacillus salivarius JCF5 and commercial starter culture were mixed and cultured at an inoculation ratio of 1:1, and the growth curve and pH value were measured under mixed culture conditions; the activated Lactobacillus salivarius JCF5 bacterial culture was expanded twice in 40 mL and 800 mL of MRS liquid medium, respectively; whole milk sterilized Jersey milk and white sugar were mixed, sterilized, homogenized, and Lactobacillus salivarius JCF5 and starter culture were added for fermentation to obtain yogurt, with commercial starter culture alone fermenting Jersey milk as a control; fermentation was carried out at 42 ℃, and after fermentation, the yogurt was placed in a refrigerator at 4 ℃ for post-ripening and storage.

2. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The Lactobacillus saliva-associated 5 used was isolated in our laboratory from fresh feces of Jersey and Jersey-Wabi crossbred calves.

3. The method for isolating, evaluating the probiotic properties of *Lactobacillus salivarius* JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... In the acid tolerance test of Lactobacillus salivarius JCF5, the pH of MRS liquid medium with a concentration of 4.0 mol / L HCl was adjusted to 4.0, 3.0, and 2.0 from a pH of 6.

8. The strain was inoculated into the medium at different pH values ​​and cultured. Plates were prepared at 0 h and 3 h, respectively, and then incubated at 37℃ for 24 h. The survival rate was calculated using MRS liquid medium with a pH of 6.8 as a control, and the acid tolerance of Lactobacillus salivarius JCF5 was evaluated.

4. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that: The bile salt tolerance test of Lactobacillus salivarius JCF5 was conducted by inoculating the strain with 2% inoculum into MRS medium containing 0.1% and 0.3% bile salt concentrations and culturing it. Plates were prepared at 0 h and 3 h, respectively, and then incubated at 37℃ for 24 h. MRS medium without bile salt was used as a control. The survival rate was calculated to evaluate the effect of bile salt on Lactobacillus salivarius JCF5.

5. The method for isolating, evaluating the probiotic properties of *Lactobacillus salivarius* JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The method for preparing the simulated gastrointestinal tract experiment of *Lactobacillus salivarius* JCF5 was as follows: *Lactobacillus salivarius* JCF5 was activated twice in MRS medium, centrifuged, and the resulting bacterial slurry was added to 5 mL of simulated gastric fluid. After treatment at 37°C for 3 h, the precipitate was collected under aseptic conditions. Sterile physiological saline was added at a ratio of 1 g bacterial slurry: 10 mL physiological saline, mixed thoroughly, diluted, and spread. The mixture was then incubated at 37°C for 24 h, and colony counting was performed. Subsequently, 1 g of the bacterial slurry treated with simulated gastric fluid was centrifuged and added to 5 mL of simulated intestinal fluid. After treatment at 37°C for 3 h, the bacterial slurry was collected, diluted, spread, and incubated at 37°C for 24 h, and colony counting was performed. The colony count in the untreated MRS medium was used as a control, and the survival rate was calculated.

6. The method for isolating, evaluating the probiotic properties of *Lactobacillus salivarius* JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The hemolysis test of Lactobacillus salivarius JCF5 involved spreading Lactobacillus salivarius JCF5, which had been activated for two generations in MRS medium, onto blood agar plates using an inoculation loop. The plates were then incubated at 37°C under anaerobic conditions for 48 h, and the presence of a clear, translucent halo around the colonies was observed.

7. The method for isolating, evaluating the probiotic properties of *Lactobacillus salivarius* JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The antibacterial assay of Lactobacillus JCF5 in saliva was performed using the Oxford cup agar diffusion method. Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Bacillus subtilis, and Pseudomonas aeruginosa were used as indicator bacteria for plate spreading. Blank MRS medium was used as a blank control. Two Oxford cups were evenly placed on each plate. 200 μL of bacterial culture was added to one Oxford cup, and MRS liquid medium was added to the other Oxford cup. After incubation at 37°C for 24 h, the inhibition zone was observed.

8. The method for isolating, evaluating the probiotic properties of Lactobacillus salivarius JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... To evaluate the gelatinase activity of Lactobacillus salivarius JCF5, 100 μL of Lactobacillus salivarius JCF5 activated in MRS medium for two generations was added to a gelatin biochemical tube. The tube was sealed with sealing film and incubated at 37°C under anaerobic conditions for 48 h. Subsequently, the biochemical tube was placed in a refrigerator for 20 min to cool the medium to below 10°C. The tube was tilted to observe whether the medium could solidify, thus evaluating the gelatinase activity of Lactobacillus salivarius JCF5. Staphylococcus aureus was used as a positive control, and Escherichia coli was used as a negative control.

9. The method for isolating, evaluating the probiotic properties of Lactobacillus salivarius JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... In the antibiotic susceptibility assay for Lactobacillus salivarius JCF5, Lactobacillus salivarius JCF5 activated for two generations in MRS medium was inoculated onto MRS solid medium at an inoculum of 2%. Antibiotic discs were then added to the culture dishes, and the discs were gently pressed to ensure they adhered tightly to the surface of the medium. The culture dishes were incubated at 37°C for 48 h, and the antibiotic susceptibility of Lactobacillus salivarius JCF5 was assessed by measuring the size of the inhibition zone.

10. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) according to claim 1, and preparing Jersey milk by fermentation with a commercially available starter culture, characterized in that... The growth curve and pH preparation method of Lactobacillus salivarius JCF5 and commercially available starter culture in a 1:1 ratio were determined. Lactobacillus salivarius JCF5 and commercially available starter culture were cultured in MRS medium at 37 °C for 24 h. Under the same conditions, Lactobacillus salivarius JCF5 and commercially available starter culture were mixed at an inoculation ratio of 1:

1. Under the mixed culture conditions, the growth of the strain was measured at 600 nm every 2 h, and the pH value of acid production was measured using a digital pH meter during the 24 h culture period.

11. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The method for activating Lactobacillus salivarius JCF5 is as follows: after melting the Lactobacillus salivarius JCF5 strain preserved in glycerol at -20℃, inoculate it into 5 mL of liquid MRS medium and incubate it in a constant temperature incubator at 37℃ for 24 h. Then, take the activated strain and inoculate it into MRS liquid medium at 37℃ for 24 h. Repeat the above operation twice to ensure that the strain is fully activated.

12. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) according to claim 1, and preparing Jersey milk by fermentation with a commercially available starter culture, characterized in that... The Lactobacillus salivae used was a strain independently isolated by our research group, which has broad-spectrum antibacterial properties.

13. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The preparation method for Jersey yogurt by co-fermentation of Lactobacillus saliva and Lactobacillus JCF5 involves expanding the activated bacterial culture to ensure sufficient bacterial sludge for subsequent experiments. The specific operation is as follows: 2 mL of the activated bacterial culture is inoculated into 40 mL of MRS liquid medium and cultured in a constant temperature incubator at 37℃ for 24 h for the first expansion. Then, 40 mL of the bacterial culture after the first expansion is poured into 800 mL of MRS liquid medium and cultured at 37℃ for 24 h for the second expansion.

14. The method for isolating, evaluating the probiotic properties of Lactobacillus saliva-associated (JCF5) and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... The viable count of the activated and centrifuged bacterial sludge was obtained by plate counting. Lactobacillus salivarius JCF5 was mixed with a commercially available starter culture at a viable count ratio of 1:

1. Lactobacillus salivarius JCF5 was fermented alone and the commercially available starter culture was fermented alone as controls.

15. The method for isolating, evaluating the probiotic properties of Lactobacillus salivarius JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... Whole milk sterilized Jersey milk and 7% (m / v) white sugar were mixed and homogenized, sterilized at 85°C for 15 min, cooled to about 45°C and then fermented with starter culture. Jersey milk was fermented alone with Lactobacillus salivarius JCF5 and with commercially available starter culture alone as controls.

16. The method for isolating, evaluating the probiotic properties of *Lactobacillus salivarius* JCF5, and preparing Jersey milk by combining it with a commercially available starter culture, as described in claim 1, is characterized in that... Jersey yogurt, after fermentation at 42℃, was placed in a refrigerator at 4℃ for 12 hours for post-fermentation, and then various indicators were measured to evaluate the quality of the yogurt.

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