Complex microbial agent having antibacterial effect, and use thereof in preparation of yoghourt

Abstract

A complex microbial agent having an antibacterial effect, and a use thereof in preparation of yoghourt. The complex microbial agent comprises Lacticaseibacillus rhamnosus FMBL L23004CNN and Lactiplantibacillus plantarum FMBL L23036 CNN, wherein the Lacticaseibacillus rhamnosus FMBL L23004CNN is preserved in the China Center for Type Culture Collection on 26 June 2023, and the preservation number is CCTCC NO: M 20231099; and the Lactiplantibacillus plantarum FMBL L23036 CNN is preserved in the China Center for Type Culture Collection on 26 June 2023, and the preservation number is CCTCC NO: M 20231101. The complex microbial agent has the effect of preventing and / or treating diarrhea, and further has antibiotic sensitivity, and yoghourt obtained by means of fermentation by the complex microbial agent as a yoghurt starter has high activity.

Description

A composite bacterial agent with antibacterial effect and its application in preparing yogurt Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composite bacterial agent with antibacterial effect and application thereof in preparing yogurt. Background Art

[0002] Fermented milk is made from raw cow's (or sheep's) milk or milk powder, sterilized, and then fermented by specific microorganisms, resulting in a lower pH. Functional yogurt is a type of yogurt that provides essential nutrients to the human body, meeting people's needs for healthy foods. It offers significant dietary advantages and has a broader application prospect. Dairy products are the best carriers for probiotics to enter the human body. Numerous strains of bacteria are used to ferment probiotic yogurt. Research has shown that strains of Lactobacillus, Streptococcus, Leuconostoc, and Bifidobacterium can all be used to ferment yogurt. Common examples include Lactobacillus delbrueckii, Lactobacillus acidophilus, Streptococcus lactis, Streptococcus lactis, Leuconostoc serovar Laevigata, Leuconostoc lactis, Leuconostoc mesenteroides and its subspecies cremoris, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis, and Pediococcus acidilactici.

[0003] In humans and animals suffering from diarrhea, the vast majority of cases are found to be caused by dysbiosis due to pathogens. On the one hand, invading pathogens inhibit the growth of normal bacteria, resulting in a decrease in the number of beneficial bacteria in the gastrointestinal tract. On the other hand, the toxic substances produced by pathogens further cause abnormal intestinal function and immune responses, leading to the occurrence of diarrhea. Bacterial diarrhea is a global health problem, especially in developing countries, where enteric pathogens are the main cause of infectious diarrhea. Currently, Escherichia coli, Shigella, Salmonella, Campylobacter, Clostridium difficile, and Aeromonas are the main pathogens of diarrhea.

[0004] Probiotics can promote the growth and reproduction of beneficial bacteria in the host's intestines, modulate the host immune system, promote the absorption of beneficial nutrients in the intestine, improve digestive system efficiency, reduce intestinal inflammation, and reduce the absorption of intestinal toxins, thereby improving intestinal health and preventing the occurrence of intestinal diseases. Probiotics can treat diarrhea caused by pathogens by maintaining or improving the balance of the intestinal microbiota. This mechanism may be related to inhibiting the colonization of harmful bacteria through competition for nutrients and production of antimicrobial compounds. In addition, Lactobacillus rhamnosus LGG can regulate the maturation and differentiation of dendritic cells and the secretion of inflammatory factors, thereby protecting against rotavirus-induced diarrhea. In summary, the beneficial effects of probiotics on diarrhea are related to the strain and dose, and the selection and use of optimal probiotics for treating diarrhea require further clinical trials.

[0005] The present invention screens out excellent strains with the ability to resist diarrheal pathogens, conducts multi-strain combination tests, screens out a pair of composite bacterial agents with good anti-diarrhea ability, and further applies them to yogurt production. By optimizing various yogurt indicators of traditional starter strains and analyzing the influence of probiotic strain combinations on yogurt quality, the foundation is laid for the research and development of anti-diarrhea functional fermented dairy products.

[0006] Summary of the Invention

[0007] The primary purpose of the present invention is to provide a composite bacterial agent with an antibacterial effect, wherein the composite bacterial agent includes Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN, wherein the Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099; the Lactobacillus plantarum FMBL L23036 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231101.

[0008] The second object of the present invention is to provide the use of the composite bacterial agent in the preparation of drugs for inhibiting pathogenic bacteria.

[0009] Preferably, the pathogenic bacteria are one or more of enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Salmonella enterica subspecies Typhimurium, enterohemorrhagic Escherichia coli, Listeria monocytogenes and Salmonella enterica subspecies Typhimurium.

[0010] The third object of the present invention is to provide the use of the composite bacterial agent in the preparation of a drug for preventing and / or treating diarrhea.

[0011] The fourth object of the present invention is to provide the use of the composite bacterial agent in the preparation of food, food additives or health products.

[0012] The fifth object of the present invention is to provide the use of the composite bacteria in preparing yogurt or yogurt starter.

[0013] The sixth object of the present invention is to provide yogurt obtained by fermentation of the composite bacterial agent.

[0014] The beneficial effects of the present invention are as follows: the present invention provides a composite bacterial agent with an antibacterial effect, the composite bacterial agent comprises Lactobacillus rhamnosus FMBL L23004 CNN and Lactiplantibacillus plantarum FMBL L23036 CNN, the composite bacterial agent has the effect of preventing and / or treating diarrhea, the composite bacterial agent also has antibiotic sensitivity, the composite bacterial agent is used as a yogurt starter, and the yogurt fermented by the composite bacterial agent has suitable acidity and high water holding capacity, as well as good texture characteristics, has typical yogurt flavor characteristics, and the yogurt fermented by the composite bacterial agent has high activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Live bacteria count during yogurt storage

[0016] Figure 2 Standard curve and amplification curve of Lactobacillus plantarum

[0017] Figure 3. Number of viable Lactobacillus plantarum and Lactobacillus rhamnosus cells during yogurt storage DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0019] Titratable acidity represents the total amount of all acidic substances in the yogurt system, while the pH of yogurt reflects the H + The concentrations of the two are both related and different.

[0020] Acidity has a crucial impact on yogurt's processing cycle, production efficiency, and flavor. High acidity can negatively impact its water retention capacity and viscosity. Therefore, controlling acidity is essential for yogurt production and crucial for ensuring product quality.

[0021] Yogurt's water-holding capacity refers to the ability of proteins in yogurt to retain water, forming a gel-like network that maintains the yogurt's consistency. When this capacity is weak, the protein network becomes loose, whey precipitates, and the yogurt's texture deteriorates. Therefore, yogurt's water-holding capacity reflects the density of the gel network and the yogurt's texture, making it an important indicator of yogurt quality. Factors such as additives, temperature, and pH can affect yogurt's water-holding capacity, thereby affecting its texture and taste. Therefore, careful control of these factors during yogurt production is crucial to ensure optimal water-holding capacity and quality.

[0022] Many substances contribute to the flavor of yogurt. Most studies suggest that diacetyl and acetaldehyde are the primary flavor components of yogurt, making their determination essential and a key factor in evaluating yogurt flavor quality. Yogurt typically produces its most flavorful substances after fermentation. To maintain its flavor and texture, the yogurt must be ripened for approximately one day. Therefore, diacetyl and acetaldehyde levels are measured after one day of ripening.

[0023] Yogurt is popular among consumers for its unique firmness, dense texture, sweet dairy aroma, and moderate tartness. Therefore, viscosity is one of yogurt's most important qualities. Yogurts made with different bacterial strain combinations exhibit varying textural properties due to differences in the bacterial strains used and their specificity. This indicates that yogurts produced with different bacterial strain combinations have distinct textures. Yogurt firmness is a key characteristic of its gel structure; greater firmness improves storage and transport. Compared to the control yogurt, the sample yogurt exhibited significantly higher firmness and viscosity than the control. Higher viscosity indicates better rheological properties. Cohesion reflects the degree of aggregation within the yogurt, and the sample exhibited higher cohesion. The viscosity index reflects the degree to which yogurt is affected by temperature fluctuations; a higher viscosity index indicates a lower temperature dependency of viscosity.

[0024] Alcohols are important components of yogurt flavor, primarily produced through lactose fermentation, amino acid metabolism, and aldehyde conversion. They impart yogurt with its distinctive aroma and mellow taste. Lactose fermentation is the primary method for producing alcohols, and the alcohols produced after lactose fermentation can effectively enhance the flavor of yogurt, giving it a rich aroma and mellow taste. Amino acid metabolism is also a key method for producing alcohols, and the alcohols produced after amino acid metabolism can modulate the sourness of yogurt and give it a mellower taste. Furthermore, aldehyde conversion is another method for producing alcohols, and the alcohols produced after aldehyde conversion can add a subtle sweetness to yogurt, making it even more delicious.

[0025] Acids are primarily produced by lactic acid bacteria and lactose fermentation. These processes break down proteins, carbohydrates, and fats, thereby changing the taste and texture of food. Acids play an important role in human health, helping the digestive system better digest food and promoting its absorption, thereby improving nutritional intake.

[0026] Esters are primarily produced through esterification reactions. They have a pleasant sweet and fruity flavor and are a key component of yogurt, contributing to its mouthfeel. The ester content affects the flavor and texture of yogurt. Aldehydes, primarily produced through lactose fermentation and bacterial fermentation, can undergo aromatic substitution reactions, forming the primary aromatic compounds in yogurt. They are also important components of lactic acid beverages and yogurt. Acetaldehyde is a typical flavoring compound in yogurt, improving its mouthfeel and flavor, thereby enhancing its quality. Ketones are primarily derived from raw milk, bacterial fermentation, and citric acid fermentation. They contribute to its unique flavor and play a crucial role in yogurt. Ketones in raw milk are primarily derived from the action of lactic acid bacteria and lactosidase. During fermentation, lactic acid bacteria produce lactic acid, which in turn produces ethyl lactate, which is then converted into ketones. Citric acid fermentation is also a common method for producing ketones. During fermentation, citric acid is oxidized to form ketones such as acetolactate and ethyl acetolactate. Bacterial fermentation can also produce ketones.

[0027] In the following examples, the abbreviations and full names of the strains are shown in the table below.

[0028] Pathogenic bacteria and culture medium

[0029] Example 1: Anti-diarrhea effect of composite bacterial agent

[0030] 1. Strains

[0031] The composite bacterial agent includes Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN. The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a preservation number of CCTCC NO: M 20231099; the Lactobacillus plantarum FMBL L23036 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a preservation number of CCTCC NO: M 20231101.

[0032] 2. Experimental Methods

[0033] (1) Sugar metabolism experiment

[0034] The strains were subjected to lactose, glucose, fructose and galacto-oligosaccharide metabolism experiments. 1 mL of activated two-generation culture solution to the logarithmic phase was centrifuged at 10,000 rpm for 5 minutes to collect the bacteria, washed twice with sterilized saline and resuspended to prepare a bacterial suspension. Rhamnosus lactobacillus FMBL L23004 CNN and plant lactobacillus FMBL L23036 CNN were mixed at a ratio of 1:1 and inoculated at a 2% inoculum into MRS medium containing different sugars as the sole carbon source. Single strains were fermented as controls and cultured anaerobically at 37°C for 24 hours. The OD values ​​were measured. 600 ,The experiment was set up in triplicate.

[0035] (2) Antibacterial properties

[0036] The antibacterial ability of the strains was determined by the Oxford cup method, with enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Salmonella enterica subspecies Typhimurium, enterohemorrhagic Escherichia coli, Listeria monocytogenes and Salmonella enterica subspecies as indicator bacteria, and 100 μL (about 10 7 CFU / mL) was spread on the surface of the corresponding solid culture medium, and a sterile Oxford cup was evenly placed on the plate at equal intervals. 200 μL of mixed bacterial suspension (combination 1:1) was added to the Oxford cup. After pre-diffusion in a 4°C refrigerator for 6 h, the plate was placed in a 37°C constant temperature incubator and cultured for 24 h. The diameter of the inhibition zone was observed and measured, and the average value was obtained by three parallel measurements.

[0037] 3. Experimental Results

[0038] (1) Sugar metabolism experiment

[0039] As shown in Table 1, compared with single bacteria, the composite bacteria agent has better utilization ability of glucose, oligofructose and oligogalactose, and its utilization of lactose is higher than that of single bacteria Lactobacillus rhamnosus FMBL L23004 CNN, and slightly lower than that of Lactobacillus plantarum FMBL L23036CNN.

[0040] Table 1 Utilization of lactose, glucose, fructose and galactose by composite bacterial agents

[0041] Note: Different letters in the same row represent significant differences (P<0.05)

[0042] (2) Antibacterial experiment

[0043] As shown in Table 2, compared with the single strains, the composite strain showed enhanced inhibitory activity against hemorrhagic Escherichia coli, diarrheagenic Escherichia coli, Salmonella typhimurium, and serovar Enteritidis. Compared with the single strain Lactobacillus rhamnosus FMBL L23004 CNN, the composite strain showed enhanced antibacterial activity against all but enterotoxigenic Escherichia coli. Compared with the single strain Lactobacillus plantarum FMBL L23036 CNN, the composite strain showed enhanced antibacterial activity against all five indicator bacteria, except Listeria monocytogenes.

[0044] Table 2 Antibacterial activity of different strains

[0045] Note: Different letters in the same column represent significant differences (P<0.05)

[0046] Example 2: Antibiotic Sensitivity

[0047] 1. Experimental Methods

[0048] The two strains were cultured anaerobically at 37°C for 24 h with a 2% inoculum volume, and 100 μL (about 1×10 7 cfu / mL) was spread on the surface of MRS solid culture medium. Then, drug-sensitive paper discs (purchased from Oxoid, UK) were gently pressed against the center of the culture medium. The discs were incubated anaerobically at 37°C for 24 hours. The antibacterial activity of the drug-sensitive paper discs was observed, and the diameter of the inhibition zone was measured. Information about the drug-sensitive paper discs is shown in Table 3.

[0049] Table 3 Drug susceptibility paper information

[0050] 2. Experimental Results

[0051] The results showed that the two strains were resistant to gentamicin, acamycin, vancomycin, teikanin, norfloxacin, polymyxin, kanamycin and ciprofloxacin, and were sensitive to penicillin, ampicillin, cephalosporin, clindamycin, tetracycline, minocycline, chloramphenicol, rifampicin and amoxicillin.

[0052] Table 4 Antibiotic susceptibility of the two strains

[0053] Note: R: resistant; I: intermediately sensitive; S: sensitive

[0054] Example 3: Preparation of yogurt by fermentation with composite bacterial agent

[0055] 1. Experimental Methods

[0056] (1) Preparation of yogurt

[0057] Through aseptic operation, the strain combination was inoculated into 100 mL of sterilized whole milk at a 2% inoculation rate and stirred thoroughly to ensure uniform mixing. It was then cultured at 42°C until the curd state was reached and finally refrigerated in a 4°C refrigerator for 24 hours.

[0058] (2) Sensory evaluation of yogurt

[0059] The sensory evaluation of yogurt is shown in Table 5.

[0060] Table 5 Sensory evaluation scoring criteria

[0061] (3) Acidity determination

[0062] Titration is used to determine the acidity of fermented yogurt after ripening. First, weigh 10g of yogurt into a beaker and add 20mL of distilled water. Mix thoroughly. Then, add 2-3 drops of 0.5% phenolphthalein indicator and mix thoroughly. Titrate with 0.1mol / L NaOH standard solution, mixing constantly until the solution turns light pink and does not fade within 30 seconds. The acidity (°T) is calculated by multiplying the number of milliliters of NaOH standard solution consumed by 10.

[0063] (4) pH measurement

[0064] The pH of the yogurt samples was measured using a pH meter. A glass rod was used to stir and mix the samples to ensure accurate measurements.

[0065] (5) Determination of water holding capacity

[0066] Select a 50mL centrifuge tube and weigh it, recording its mass as m1; take about 10g of yogurt into the centrifuge tube and record the mass of the centrifuge tube and yogurt as m2; centrifuge the centrifuge tube at 5000r / min at room temperature for 30 minutes, pour out the supernatant, invert the centrifuge tube for 10 minutes and weigh it, recording its mass as m3. The water holding capacity (WHC) of yogurt is calculated as follows: WHC = (m3 - m 1) / (m2-m1)

[0067] (6) Determination of acetaldehyde content in yogurt

[0068] The sample was stirred with 16% TCA and other substances in a certain proportion, centrifuged at 3500 r / min for 10 min, 25 mL of the supernatant was placed in an iodine volumetric flask, 5 mL of 1% NaHSO3 was added, shaken, and placed in the dark for 1 hour, then 1 mL of 1% starch was added, and it was titrated with 0.1 mol / L iodine solution until there was almost no color, and then 0.01 mol / L iodine solution was added until it showed light blue, and then 20 mL of 1 mol / L sodium bicarbonate solution was added, shaken, and then titrated with 0.01 mol / L iodine standard solution until it showed light blue again, and the volume of iodine solution used was adjusted, and three parallel experiments were performed.

[0069] Acetaldehyde content calculation formula: Acetaldehyde (g / mL) = (V1-V2)C × 0.022) / 25

[0070] V 2－ The volume of I2 standard solution consumed in the blank control titration (mL); V 1－ The volume of I2 standard solution consumed in the sample titration (mL); C-the concentration of I2 standard solution (mol / L); 25-the weight of the acetaldehyde sample (mL); 0.022-the basic unit of acetaldehyde chemical reaction (g).

[0071] (7) Determination of diacetyl content in yogurt

[0072] Get the fermented sour milk that 10g afterripening is finished and put into 50mL centrifuge tube, add 10mL 16%TCA solution to mix, 3500r / min centrifugal 10min under normal temperature condition, draw the supernatant of 5mL, add the o-phenylenediamine solution 0.25mL of 1g / 100mL respectively, vibrate and be evenly placed in dark place 30min, the HCl solution 1.0mL purpose that adds 4.0mol / L afterwards is termination reaction, measures the absorbance under wavelength 335nm with quartz cuvette.Each sample is done 3 times in parallel.Then contrast diacetyl standard curve figure (standard curve is with reference to) can obtain diacetyl mass concentration in testing sample.

[0073] (8) Yogurt texture determination and principal component analysis

[0074] Texture analysis of ripened yogurt samples was performed using a method slightly modified from that described by Changkun Li et al. A 35-mm-diameter A / BE probe was used in a 200-ml glass container (64-mm diameter, 70-mm height). Pre-, mid-, and post-test speeds were 1.0 mm / s, 1.0 mm / s, and 5.0 mm / s, respectively. The test parameters included a penetration distance of 20 mm and a surface trigger force of 10 g.

[0075] (9) Determination of viable bacteria count during yogurt storage

[0076] The number of thermophilic Streptococcus and the total number of lactic acid bacteria in yogurt were determined by plate counting method; the number of Lactobacillus plantarum and Lactobacillus rhamnosus in yogurt were determined by real-time fluorescence quantitative method.

[0077] A. Lactic acid bacteria plate count

[0078] According to GB 4789.35-2016, about 1 g of the sample was taken out and diluted 10 times with 0.85% sterile saline. 0.1 mL of the diluent was added to 0.9 mL of sterile saline with a concentration of 8.5 g / L, and then diluted to 10. -7 , select two continuous appropriate dilutions, take 100 μL of dilution liquid for each dilution, and spread them on M17 plates (for thermophilic Streptococcus) and MRS plates (for the total number of lactic acid bacteria), respectively. Each dilution is repeated in two parallels, and cultured under anaerobic conditions at 37°C for 48 hours. Count the number of colonies on the plate, and combine the dilution and sampling volume to obtain the number of lactic acid bacteria. The data results are expressed as CFU / g.

[0079] Extraction of DNA from yogurt

[0080] The DNA of yogurt samples was extracted according to the operating procedures of the kit. The yogurt samples should be diluted before DNA extraction and the DNA products should be stored at -20°C to prevent degradation.

[0081] B. Real-time fluorescence quantitative PCR method for lactic acid bacteria counting

[0082] First, a standard curve was prepared by diluting the extracted DNA 10-fold until 10 5 times, then 10 1 -10 5 Using a 1000-fold nucleic acid dilution as a template, standard curves for lactic acid bacteria counts were generated, representing the relationship between the logarithmic values ​​and Ct values ​​of lactic acid bacteria. The reaction system and conditions for real-time fluorescence quantitative PCR are shown in Table 6. The specific primer sequences for Lactobacillus plantarum and Lactobacillus rhamnosus and the lengths of the amplified products are shown in Table 7.

[0083] After the yogurt sample DNA was extracted using the kit, real-time fluorescence quantitative PCR was performed according to the reaction system and reaction conditions of the standard curve. The obtained Ct value was substituted into the corresponding standard curve. The corresponding number of Lactobacilli in the yogurt sample was calculated based on the sampling volume and the dilution factor of the yogurt sample DNA. The result was expressed as Log CFU / mL.

[0084] Table 6 Real-time fluorescence quantitative PCR

[0085] Table 7 Specific primers for different lactic acid bacteria

[0086] (10) Determination of yogurt flavor substances

[0087] A. Sample Pretreatment and SPME Extraction Method

[0088] The volatile components of fermented milk were analyzed using solid-phase microextraction (SPME). The extraction tip was first placed in a gas chromatograph (GC) at 245-255°C for two hours. The tip was conditioned for an additional 10 minutes before extraction and between each sample. This eliminated any residual material in the tip, ensuring that it would not affect the determination of volatile components in the subsequent sample. Take 5g of sample, add it to a 20mL injection bottle, and add 1g NaCL and 1μL 2-methyl-3-heptanone, stabilize at 50℃ for 20min, and when equilibrium is reached, insert the 50 / 30μm DVB / CAR / PDMS extraction tip into the injection port and age it for 10min. Immediately insert the 50 / 30μm DVB / CAR / PDMS extraction tip into the injection port and age it for 10min. Immediately place the extraction tip into the injection bottle and adsorb at 50℃ for 30min. Immediately after collection, insert the extraction tip into the injection end and desorb at 250℃ for 5min. Release the collected volatile substances at the beginning of the column and carry out GC-MS analysis.

[0089] B. GC-MS Detection Conditions

[0090] GC conditions: HP-INNOWAX column, He gas, flow rate 1.0 mL / min, linear velocity 40 cm / s, split ratio 1:10. The sample injection temperature was set at 240°C. A temperature program was used: the initial temperature was set at 40°C for 10 minutes, then increased to 140°C at a rate of 4-5°C / min for 5 minutes, and finally, increased to 250°C at a rate of 10°C / min for 10 minutes. The transmission line temperature was set at 250°C.

[0091] MS conditions: ion source operating at 150°C in electron impact mode, voltage of 70 eV, ion source temperature of 230°C, scan range 40–400 m / z, mass spectra of all yogurt samples recorded with five scans and no solvent delay. Identification was performed using the NISI library.

[0092] 2. Experimental Results

[0093] (1) Determination of physicochemical properties of yogurt

[0094] The pH value of the combined yogurt was lower than that of the control group, and the acidity was higher than that of the control group. The sensory score of the combined yogurt was 88.77 points, the acidity was 96°T, the pH value was 4.46, and the water holding capacity was 67%.

[0095] Table 8 pH value, acidity, water holding capacity and sensory evaluation results of different yogurt combinations

[0096] (2) Determination of acetaldehyde and diacetyl content

[0097] The peak period for yogurt to produce aromatic substances is generally after the fermentation is completed. In order for the yogurt to have a good taste and flavor, it must be ripened for about 1 day. Therefore, the diacetyl and acetaldehyde contents of the yogurt were measured after 1 day of ripening. The acetaldehyde content and diacetyl content of the yogurt prepared with the composite bacterial agent were higher than those of the control group, at 17.60 and 5.65 mg / L, respectively. When the acetaldehyde concentration is higher than 10 mg / L, the yogurt has a typical aroma, or when the ratio of acetaldehyde to diacetyl content is greater than 3:1, the yogurt will have a characteristic flavor, and the higher the acetaldehyde content, the more obvious the characteristic flavor. The yogurt prepared with the composite bacterial agent described in the present invention has a typical aroma and characteristic flavor.

[0098] Table 9 Determination results of acetaldehyde and diacetyl in different combinations of yogurt

[0099] (3) Determination of yogurt texture

[0100] Compared with the control group, the yogurt prepared with the composite inoculum of the present invention had higher hardness and viscosity, and better rheological properties. The yogurt prepared with the composite inoculum also had higher cohesion and better internal aggregation. The yogurt prepared with the composite inoculum had a higher viscosity index and was less affected by temperature. The yogurt prepared with the composite inoculum had better textural properties than the control group.

[0101] Table 10 Determination results of different combinations of yogurt texture characteristics

[0102] (4) Determination of the number of lactic acid bacteria in yogurt

[0103] A. Determination of the number of lactic acid bacteria in yogurt

[0104] In this study, M17 medium (for thermophilic Streptococcus) and MRS medium (for lactic acid bacteria) were used to count the total amount of thermophilic Streptococcus and lactic acid bacteria in yogurt.

[0105] The results are shown in Figure 1. The number of viable thermophilic streptococci and lactic acid bacteria showed a trend of first increasing and then decreasing with the increase of storage time. However, after 14 days of storage, the number of viable thermophilic streptococci and lactic acid bacteria was still greater than 10 7 CFU / mL, in line with national standards.

[0106] B. Fluorescent real-time quantitative PCR

[0107] Real-time fluorescence quantitative PCR was used to establish standard curves for the determination of bacterial counts of Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN.

[0108] As shown in Figure 2(A) and Figure 2(B). The results of fluorescent quantitative PCR amplification of gradient diluted DNA liquid showed obvious differentiation, as shown in Figure 2(C) and Figure 2(D), indicating that the test stability was good. The software automatically plotted the bacterial count and Ct value to form the standard curves of Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN. The calculation showed that the R value of the standard curve of Lactobacillus plantarum FMBL L23036 CNN was 0. 2 greater than 0.99, and the amplification efficiency of the standard curve was 102.9%; the R 2 greater than 0.99, the standard curve amplification efficiency was 104%, and R 2 The closer the value is to 1, the better the linear correlation. The ideal standard curve amplification efficiency range is 90%-110%. The amplification efficiency of this test is within this range and can well meet the basic experimental requirements of fluorescent quantitative PCR in this study.

[0109] According to the standard curves of Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN, the viable counts of Lactobacillus plantarum FMBL L23036 CNN and Lactobacillus rhamnosus FMBL L23004 CNN in yogurt DNA samples at different storage times were calculated. The results are shown in Figure 3. It can be seen that the viable counts of Lactobacillus plantarum FMBL L23036 CNN and Lactobacillus rhamnosus FMBL L23004 CNN gradually decreased with the increase of storage time after 5 days of storage, but were still greater than 10 at 14 days. 7 CFU / mL, indicating that Lactobacillus plantarum FMBL L23036 CNN and Lactobacillus rhamnosus FMBL L23004 CNN can survive well in yogurt and play a better role, which is in line with national standards.

[0110] (5) Determination of yogurt flavor substances

[0111] The determination of volatile flavor compounds in the two samples revealed a total of 40 volatile flavor compounds, including alcohols, acids, aldehydes, ketones, esters, and others. In the control group, 26 volatile flavor compounds were detected, including 2 alcohols, 8 acids, 1 ester, 1 aldehyde, 8 ketones, and 6 others. In the test samples, 28 volatile flavor compounds were detected, including 6 alcohols, 5 acids, 3 esters, 2 aldehydes, 7 ketones, and 5 others.

[0112] Table 11 Volatile flavor substance determination results

[0113] The test results, shown in Table 11, show that a total of seven alcohol flavor compounds were detected. Of these, 2-butyl-1-octanol was only found in the control group, while six alcohols were detected in the samples: 1-hexanol, 3,5-octadien-2-ol, 1-octen-3-ol, 1-heptanol, 1-nonanol, and octaethylene glycol monododecyl ether. These alcohols contribute to the yogurt's pleasant flavor and also serve as nutrients for lactic acid bacteria, promoting their growth and thus improving the taste and quality of the yogurt.

[0114] Table 11 shows that a total of eight acidic flavor compounds were detected. The control group had eight acids but no unique acidic compounds, while the samples detected five acids but no unique compounds. Acetic acid, butyric acid, caproic acid, caprylic acid, and palmitic acid were common acids in both yogurt samples and play a significant role in yogurt flavor. Acetic acid and caproic acid are the primary contributors to yogurt's sourness, while caproic acid also enhances its odor and contributes to its floral aroma. Lactic acid contributes significantly to the refreshing sourness of yogurt, but due to its low volatility, it was not detected in either yogurt sample. These acids significantly influence both the flavor and aroma of yogurt.

[0115] Table 11 shows that three ester flavor compounds were detected in the sample, with ethyl acetate and octyl formate being unique to the test samples. The control group had one ester compound and no unique ester compounds. Only two aldehyde compounds were detected in the yogurt prepared with the composite inoculum, with 2-decenal being the unique aldehyde compound. This improves the taste and flavor of yogurt.

[0116] As shown in Table 11, a total of 10 ketone flavor compounds were detected, of which 7 were unique to the test samples, with 2,3-pentanedione and 2H-pyran-2-one tetrahydro-6-pentyl being the only compounds. Eight were unique to the control group, with 2-butanone, 2,3-butanedione, and 2-methyl-3-pentanone being the only compounds. Acetoin has a mild, creamy, slightly sweet, buttery flavor. It is a common flavor compound in dairy products and has a significant impact on flavor. Diacetyl is an important aromatic compound that imparts a buttery flavor. Small amounts of diacetyl contribute to the unique flavor and aroma of yogurt. Furthermore, diacetyl and acetoin combine to create a mild, pleasant, buttery flavor, which is crucial to the mouthfeel of yogurt.

[0117] In summary, the present invention provides a composite bacterial agent with an antibacterial effect. The composite bacterial agent includes Lactobacillus rhamnosus FMBL L23004 CNN and Lactiplantibacillus plantarum FMBL L23036 CNN. The composite bacterial agent has the effect of preventing and / or treating diarrhea, and the composite bacterial agent also has antibiotic sensitivity. The composite bacterial agent is used as a yogurt starter, and the yogurt fermented with the composite bacterial agent has suitable acidity and high water holding capacity, as well as good texture characteristics and typical yogurt flavor characteristics. The yogurt fermented with the composite bacterial agent has high activity.

[0119]

[0118]

Claims

1. A composite bacterial agent with antibacterial effect, characterized in that: The composite bacterial agent includes Lactobacillus rhamnosus FMBL L23004 CNN and Lactobacillus plantarum FMBL L23036 CNN, the Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099; the Lactobacillus plantarum FMBL L23036 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231101.

2. Use of the composite bacterial agent as claimed in claim 1 in the preparation of drugs for inhibiting pathogenic bacteria.

3. The use according to claim 2, characterized in that The pathogenic bacteria are one or more of enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Salmonella enterica subspecies typhimurium, enterohemorrhagic Escherichia coli, Listeria monocytogenes and Salmonella enterica subspecies.

4. Use of the composite bacterial agent according to claim 1 in the preparation of a drug for preventing and / or treating diarrhea.

5. Use of the composite bacterial agent according to claim 1 in the preparation of food, food additives or health products.

6. Use of the composite bacteria as claimed in claim 1 in preparing yogurt or yogurt starter.

7. Yogurt obtained by fermentation with the composite bacterial agent according to claim 1.

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