Yoghourt containing plant lactobacillus and preparation method thereof

Abstract

The invention relates to the technical field of dairy product processing, and particularly provides yoghourt containing lactobacillus plantarum and a preparation method of the yoghourt containing the lactobacillus plantarum and the preparation method of the yoghourt containing the lactobacillus plantarum and the preparation method of the yoghourt containing the lactobacillus plantarum and the preparation method of the yoghourt containing the lactobacillus plantarum and the preparation method of the yoghourt containing the lactobacillus plantarum. The yoghurt is elastic in taste, high in gel strength, excellent in texture, high in viable organism content, very high in oxidation resistance and wide in market prospect.

Description

Technical Field

[0001] The invention relates to the technical field of dairy product processing, and in particular to a yogurt containing Lactobacillus plantarum and a preparation method thereof. Background Art

[0002] Lactic acid bacteria (LAB) are Gram-positive bacteria widely distributed in the human intestine and are also key industrial microorganisms for industrial food fermentation worldwide. Lactic acid bacteria have probiotic properties. When ingested in sufficient amounts, they can improve host health, promote nutrient digestion and absorption, maintain the balance of intestinal flora structure, regulate sugar and lipid metabolism, and improve the body's antioxidant level and immunity.

[0003] Lactic acid bacteria can increase the activity of antioxidant enzymes or regulate and alleviate circulating oxidative stress to protect cells from oxidative stress-induced damage and reduce inflammation. The most important species, such as Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactoaseibacillus casei, Lactiplantibacillus rhamnosus, Lactococcus lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus, have been widely used in functional foods to improve the quality of food (including flavor and taste), promote the beneficial biological activity of food, and prevent and treat a variety of diseases. For example, Lactobacillus plantarum KSWY02 can increase the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) in alveolar epithelial cells and prevent cellular oxidative damage. Its metabolites may also enhance the activity of antioxidant enzymes and inhibit the increase of intracellular ROS in cell models (Wang et al., 2020).

[0004] Lactic acid bacteria fermented foods include yogurt, kimchi, cheese, etc. These foods produce lactic acid through lactic acid bacteria fermentation, which gives them a unique flavor. They can effectively improve intestinal flora imbalance, promote digestion, relieve constipation, and have the effects of moisturizing the intestines, replenishing the appetite, and enhancing immunity, which is beneficial to maintaining human health. Summary of the invention

[0005] The object of the present invention is to provide a yogurt containing plant lactobacillus and a preparation method thereof. The yogurt has a chewy texture, high gel strength, excellent texture, high content of live bacteria, strong antioxidant performance, and broad market prospects.

[0006] In one aspect, the present invention provides a yogurt, wherein the preparation method of the yogurt comprises the following steps:

[0007] Sodium carboxymethyl cellulose and sugar are added to fresh milk at a mass ratio of 0.1-0.5% and 6-12% respectively, homogenized, sterilized, and a milk fermentation base is obtained; the milk fermentation base is cooled to 42 DEG C, an activated lactobacillus plantarum bacterial liquid is inoculated into the milk fermentation base at a volume ratio of 3-5%, and fermented at 38-45 DEG C for 8-12 hours; after the fermentation is completed, the milk is refrigerated at 4 DEG C and ripened to obtain yogurt.

[0008] Further preferably, the plant lactobacillus is plant lactobacillus W8 (Lactiplantibacillus plantarum W8), which has been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on August 1, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 31521.

[0009] Further preferably, the amount of viable bacteria in the plant lactobacillus liquid is higher than 10 9 CFU / mL.

[0010] Further preferably, the method for preparing the yogurt comprises the following steps:

[0011] Sodium carboxymethyl cellulose and sucrose are added to fresh milk at a mass ratio of 0.5% and 12% respectively, homogenized, sterilized, and a milk fermentation base is obtained; cooled to 42°C, activated Lactobacillus plantarum W8 bacterial liquid is inoculated into the milk fermentation base at a volume ratio of 5%, and fermented at 45°C for 8h; after the fermentation is completed, refrigerated at 4°C and ripened to obtain yogurt.

[0012] The yogurt provided by the present invention has high hardness, viscosity, elasticity and cohesion, a chewy texture, high gel strength and excellent texture. The pH of the yogurt is 4.25-4.32, the titrated acidity is 0.19%-0.23%, the water holding rate is 53.88-55.40%, and the number of viable bacteria is higher than 10 8 CFU / g, and the free amino acid content reached 0.42-0.48 mg / mL, with significant antioxidant effect; the scavenging rates of DPPH free radicals and ABTS free radicals of the yogurt prepared in Example 7 were as high as 82.03% and 88.41%, respectively, achieving unexpected technical effects.

[0013] The yogurt provided by the present invention exhibits a strong free radical scavenging ability during storage for 28 days; on the 28th day, the scavenging rates of DPPH and ABTS free radicals are still as high as 72.40% and 80.0%, respectively, with a significant antioxidant effect, achieving an unexpected technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology and Gram staining of Lactobacillus plantarum W8 on MRS plate;

[0015] Figure 2 The results of MTT cell toxicity assay;

[0016] Figure 3 The results of the determination of antioxidant enzymes and oxidative stress products in Caco2 cells;

[0017] Figure 4 The results of Caco2 cell permeability assay;

[0018] Figure 5 is the serum diamine oxidase (DAO) and D-lactic acid (D-LA) content of mice in each group;

[0019] Figure 6 is the cytokine level in colon tissue of mice in each group;

[0020] Figure 7 HE staining images of colon tissues of mice in each group;

[0021] Figure 8 Tight junction gene expression in colon tissue of mice in each group;

[0022] Fig. 9 The antimicrobial peptide gene expression in colon tissue of mice in each group;

[0023] Fig.10 The expression of LGR5 protein in the colon of mice in each group;

[0024] Fig.11 The microbial composition of intestinal contents at the genus level in each group of mice.

[0025] Fig.12 To determine the antioxidant capacity of yogurt during storage. DETAILED DESCRIPTION

[0026] The following examples are provided to better illustrate the present invention, and those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the protection and claims of the present invention are not limited to the examples provided.

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present invention, but the present invention is not limited thereto.

[0028] Example 1 Isolation, Identification and Performance Analysis of Lactobacillus plantarum W8

[0029] The present invention isolates a Lactobacillus W8 strain that has a strong scavenging ability for both DPPH and ABTS free radicals from naturally fermented soybean paste in Northeast China. Combining the colony morphology, physiological and biochemical characteristics and molecular biological identification results, the applicant determines that the screened W8 strain is a new type of plant lactobacillus, and names it as Lactiplantibacillus plantarum W8, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (referred to as CGMCC) on August 1, 2024, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 31521.

[0030] like Figure 1 As shown, the Gram stain of Lactobacillus plantarum W8 is blue-purple, and the morphology is single-rod, double-rod or chain-rod arrangement; the colonies on the MRS plate are milky white, round, smooth in surface, and have obvious frankincense characteristics.

[0031] The scavenging rates of the fermentation supernatant of Lactobacillus plantarum W8 on DPPH free radicals and ABTS free radicals reached 96.92% and 94.31% respectively, and the antioxidant effect was very significant.

[0032] Lactobacillus plantarum W8 has significant antibacterial ability against Salmonella and Enterobacter sakazakii, with antibacterial diameters of 23.47mm and 19.82mm respectively, which are more than twice that of standard Lactobacillus plantarum ATCC8014. It has a significant antibacterial effect and achieved unexpected technical results.

[0033] Lactobacillus plantarum W8 has a strong lipid-lowering function. Its cholesterol removal rate reaches 89.05% and its triglyceride degradation rate is 72.74%, achieving unexpected technical results.

[0034] Example 2 Analysis of acid and bile resistance of Lactobacillus plantarum W8

[0035] 2.1 Acid resistance determination

[0036] The culture medium was inoculated into MRS liquid culture medium and cultured at 37° C. Samples were taken every 2 hours, and the absorbance was measured at 600 nm to obtain the growth curve of Lactobacillus plantarum W8.

[0037] (1) Control group: The activated Lactobacillus plantarum W8 solution was inoculated at a ratio of 2% (V / V) into liquid MRS medium with a pH value of 6.7;

[0038] (2) Experimental group: The activated Lactobacillus plantarum W8 culture was inoculated into liquid MRS medium with pH values ​​of 2.0, 2.5, and 3.0 at a ratio of 2% (V / V); cultured at 37°C for 3 h, and the absorbance (OD) was measured at 600 nm. 600 ).

[0039] The survival rate of the strains was calculated according to the following formula.

[0040] Survival rate (%) = (test OD 600 / Control OD 600 )×100%.

[0041] The results showed that the Lactobacillus plantarum W8 provided by the present invention has a strong acid resistance. After being treated at pH 3.0 for 3 hours, its survival rate can still reach 92.54%, indicating that the strain can enter the intestine through gastric juice and has the prerequisite for entering the intestine to play a probiotic role.

[0042] 2.2 Bile salt tolerance test

[0043] The inoculum was inoculated with 2% of the culture medium containing 0.10%, 0.20%, 0.30%, and 0.40% of sodium taurocholate, respectively, and cultured at 37°C for 4 h. The absorbance (OD) was measured at 600 nm. 600 ).

[0044] The results showed that the survival rates of Lactobacillus plantarum W8 provided by the present invention reached 85.76% and 66.11% respectively after being treated for 4 hours in the presence of 0.2% and 0.3% bile salts, indicating that the strain has a strong bile salt tolerance and is capable of entering the intestine to exert a probiotic effect.

[0045] 2.3 Simulated gastrointestinal tolerance test

[0046] The plant lactobacillus W8 bacterial suspension was mixed with simulated gastric fluid at a volume ratio of 1:9 and cultured at 37°C for 2 hours. The cultured bacterial liquid was then washed and mixed with simulated intestinal fluid at the same ratio and cultured at 37°C for 24 hours. Samples were taken at 3h, 6h, 12h, and 24h, and the number of viable bacteria was determined by the plate count method to calculate the survival rate of plant lactobacillus W8. The calculation formula is as follows:

[0047] Survival rate (%) = (number of live bacteria after passing through intestinal juice / number of live bacteria before adding gastric juice) × 100%.

[0048] Table 1 Survival rate of Lactobacillus plantarum W8 after digestion in simulated gastrointestinal fluid

[0049]

[0050] From the results in Table 1, it can be seen that after being treated with simulated gastric juice for 2 hours and simulated intestinal juice for 3 hours, the survival rate of Lactobacillus plantarum W8 reached 90.35%, and after being treated with simulated intestinal juice for 24 hours, the survival rate was still as high as 41.18%. This shows that the Lactobacillus plantarum W8 provided by the present invention has strong tolerance to the digestive tract environment and can achieve effective colonization in the intestine, which is conducive to exerting its probiotic effect.

[0051] Example 3 Regulatory effect of Lactobacillus plantarum W8 on oxidative stress in Caco2 cells

[0052] 3.1 Strain activation

[0053] The glycerol-preserved Lactobacillus plantarum W8 was activated in liquid MRS medium, cultured at 37°C for 18 h, and obtained the experimental strain after three generations.

[0054] 3.2 Recovery, culture and subculture of Caco2 cells

[0055] Human colorectal cancer cell line Caco-2 was taken out of liquid nitrogen, thawed in a 37°C water bath within 3 minutes, then centrifuged quickly, the culture medium was discarded, 1 mL of DMEM complete culture medium was added and dispersed, and then transferred to a culture flask for culture. When the cells grew to about 80% of the surface of the culture flask, the cells were digested with trypsin-EDTA for passage, and the cells were used for subsequent experiments when they were cultured for 5 generations.

[0056] 3.3 Cytotoxicity assay

[0057] Caco-2 cells were cultured at 2 × 10 per ml. 5 The cells were inoculated into 96-well plates at a concentration of 10 cells / mL. The cells were washed with phosphate buffered saline (PBS). Then, 100 μL of bacterial suspension (at a concentration of 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL), and the cells were incubated for 24 hours, with dimethyl sulfoxide (DMSO) without antibiotics as a control. Then, 20 microliters of 0.5 mg / mL thiazolyl blue (MTT, diluted with PBS buffer) were incubated at 37°C for 4 hours in the dark. The supernatant was removed, 150 microliters of dimethyl sulfoxide were added, and the mixture was shaken for 10 minutes. The absorbance was read at a wavelength of 490nm using an ELISA reader. The results are expressed as the percentage of live cells compared to the control. The absorbance of the control is considered to represent 100% cell viability.

[0058] 3.4 Caco2 cell grouping and modeling

[0059] Caco2 cells were treated as follows:

[0060] (1) Blank group: no intervention;

[0061] (2) Model group: Caco2 cells were treated with 1 μg / mL LPS (lipopolysaccharide) for 6 h;

[0062] (3) Experimental group: Caco2 cells were treated with 1 μg / mL LPS for 6 h, and then treated with the cell-free supernatant of Lactobacillus plantarum W8 for 24 h.

[0063] 3.5 Detection of oxidative stress indicators

[0064] After the intervention, the cells were washed by centrifugation and ultrasonicated in cold PBS. The malondialdehyde (MDA) content and the activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and catalase (CAT) in Caco2 cells were detected using commercial kits produced by Nanjing Jiancheng.

[0065] 3.6 Results Analysis

[0066] Depend on Figure 2 It can be seen that 10 7 -10 10 CFU / ml bacterial concentrations were non-toxic to Caco2 cells, so the middle value of bacterial concentration was 10 8 CFU / mL for further experiments. Figure 3 As shown, compared with the normal group, LPS can induce Caco2 cell oxidative stress, thereby causing Caco2 cell MDA content to significantly increase, and the activity of GSH-PX, SOD and CAT to significantly reduce. After the intervention of plant lactobacillus W8 cell-free supernatant, the levels of MDA, GSH, SOD and CAT induced by LPS were reversed, and the effect was very significant, close to the normal group level. Thus illustrating, plant lactobacillus W8 provided by the invention can effectively alleviate the oxidative stress induced by LPS.

[0067] Example 4 Repair effect of Lactobacillus plantarum W8 on Caco-2 cell barrier damage

[0068] 4.1 Activation of strains, cell culture, grouping and modeling as in Example 3

[0069] 4.2 Determination of epithelial resistance

[0070] Logarithmically growing Caco-2 cells were plated at 1×10 9The cells were inoculated into a 6-well Transwell plate at a concentration of 1.5 mL / mL. 1.5 mL and 2.5 mL of culture medium were added to the apical side (AP) and the basal side (BL), respectively. The medium was changed every other day in the first week, and the medium was changed every day from the second week onwards. The cells were cultured for about 21 days until the cells were completely differentiated. The cell grouping and modeling methods were the same as in Example 3. After the modeling was completed, the epithelial resistance value was measured using an epithelial resistance meter.

[0071] 4.3 Fluorescein isothiocyanate-dextran (FITC-Dextran) permeability assay

[0072] After modeling, wash the monolayer membrane three times with HBSS, then add 500 μL of DMEM medium containing 1 mg / mL FITC-Dextran to the AP side, and add 1.5 mL of DMEM medium to the BL side. After 6 hours of treatment, aspirate 0.1 mL of culture medium from the BL side and add it to a light-proof black 96-well plate. Use an ELISA reader to measure the fluorescence intensity at 490-520 nm, and repeat each test three times. Calculate the concentration of FITC-Dextran on the outside and the apparent permeability coefficient Papp based on the standard curve.

[0073] 4.4 Results Analysis

[0074] Depend on Figure 4 It can be seen that compared with the normal group, the epithelial resistance of Caco2 cells induced by LPS was significantly reduced, and intervention with the supernatant of Lactobacillus plantarum W8 fermentation significantly increased the epithelial resistance of Caco2 cells. This shows that the supernatant of Lactobacillus plantarum W8 fermentation has the potential to increase the tightness of intestinal epithelial cells and improve the intestinal barrier.

[0075] Compared with the normal group, the FITC permeability of Caco2 cells in the LPS group increased significantly, which was 1.5 times that of the normal group; the Lactobacillus plantarum supernatant group significantly improved the increase in permeability caused by LPS, approaching the level of the normal group. The results once again show that the supernatant of Lactobacillus plantarum W8 has a significant ability to improve the intestinal barrier.

[0076] Example 5 Application of Lactobacillus plantarum W8 in reducing intestinal permeability and improving intestinal inflammation in mice

[0077] 5.1 Preparation of Lactobacillus plantarum W8 bacterial suspension

[0078] Lactobacillus plantarum W8 was incubated in MRS liquid medium at 37°C for 18 h and subcultured three times. After centrifugation, the bacterial cells were collected and washed twice with saline. Then, the bacterial cells were resuspended in saline to a bacterial concentration of 1 × 10 9 CFU / mL.

[0079] 5.2 Animal grouping, modeling and drug administration

[0080] Experimental animals: 5-week-old healthy male C57BL / 6J mice (20 g ± 2 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SVXK (Liao) 2020-0001, and were kept in the SPF-level laboratory of the Experimental Animal Center of Shenyang Agricultural University. The animal experiment was approved by the Ethics Committee of the Experimental Animal Center of Shenyang Agricultural University, ethics approval number: NO.2023102702. 24 mice were randomly divided into 5 groups: control group (Con), model group (DSS), negative control group (NC, Lactobacillus plantarum standard bacteria ATCC8014), positive control group (PC, mesalazine), Lactobacillus plantarum intervention group (W8);

[0081] Acute colitis was induced using a 2.5% (w / v) dose of dextran sulfate sodium (DSS). During the 7-day experiment, mice in the control group received only water. Mice in the DSS group were gavaged with 2.5% DSS for 5 consecutive days. Mice in the normal group were orally administered with a standard bacterial suspension (10 mg / kg) once a day, mice in the W8 group were orally administered with a Lactobacillus plantarum W8 bacterial suspension (10 mg / kg) once a day, and mice in the PC group were gavaged with 200 mg / kg mesalazine / day for 7 consecutive days, and supplemented with DSS for 5 days. Body weight was recorded once a day. Mice were killed on the 7th day. The colon was removed and the length was measured. The colon specimens were fixed in 10% formalin and stained with hematoxylin-eosin (HE).

[0082] 5.3 Enzyme-linked immunosorbent assay

[0083] 100 μL of serum and 100 mg of colon tissue were collected from each group of mice and colon homogenate (100 mg) was prepared with phosphate buffered saline (PBS) (w / v 1 / 9). In colon homogenate and cell samples, IL-10, IL-1β and TNF-α cytokines and serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels were detected according to the manufacturer's instructions (Nanjing Jiancheng, Nanjing, China).

[0084] 5.4 H&E staining

[0085] Fresh colon tissue was rapidly fixed in 4% paraformaldehyde for 48 h. Subsequently, tissue samples were dehydrated, embedded in paraffin, and sectioned at 4 μm. After dewaxing, tissue samples were stained with H&E and analyzed using a light microscope.

[0086] 5.5 Determination of intestinal barrier-related gene expression in colon tissue

[0087] Total RNA was extracted from colon tissue using Trizol reagent, and 1 μg of RNA was reverse transcribed to synthesize cDNA using M5-Super-qPCR RT Kit with gDNA removal reagent. mRNA expression was measured using a 96-well instrument. The primer sequences are listed in Table 2. The levels of the target genes were quantified relative to the level of the Gapdh gene, and the results obtained were analyzed using the 2-ΔΔCt analysis method.

[0088] Table 2 Primer sequences

[0089]

[0090] 5.6 Expression of intestinal stem cell marker LGR5 in colon tissue

[0091] (1) Mouse colon tissue was obtained, quickly frozen in liquid nitrogen, and ground to extract total protein.

[0092] (2) Add an appropriate volume of cell tissue lysis buffer to the tissue to be lysed, lyse on ice for 10 min, centrifuge at 12,000 rpm for 10 min, and then collect the supernatant.

[0093] (3) Measure protein concentration using BCA kit.

[0094] (4) Protein denaturation: Add 5× SDS-PAGE loading buffer and cook at 100°C for 10 min.

[0095] (5) Gel preparation and electrophoresis: Take 10 μg of protein solution and perform SDS-PAGE gel electrophoresis. Adjust the voltage to 150 V and continue for 1 hour.

[0096] (6) Transfer: Pour enough transfer solution. Perform quantitative electrophoresis on the sample solution, transfer the protein to a 0.2 μm PVDF membrane at a constant voltage of 80 V for 2 h, and set the current to 200 mA to fix the protein on the membrane. The transfer time is determined according to the specific molecular weight of the protein, usually 40-90 min.

[0097] (7) Blocking: Place the membrane in 5% skim milk and shake for blocking at 37°C for 1 h.

[0098] (9) Incubation with primary antibody: dilute LGR5 antibody at a dilution ratio of 1:1000 and incubate overnight at 4°C on a shaker. After the primary antibody incubation, rinse the strips three times with PBST buffer at room temperature, each time for 10 min.

[0099] (10) Incubation with HRP secondary antibody: Dilute the HRP goat anti-mouse secondary antibody at a concentration of 1:5000 with 5% blocking solution, place the strips in the secondary antibody, and incubate on a shaker at room temperature for 1 hour. After the secondary antibody incubation, rinse the strips three times with PBST buffer solution at room temperature, each rinse for 10 minutes.

[0100] (11) Development: The signal was further visualized using HRP color imaging using the Amersham Imager 600 imaging system.

[0101] (12) Quantification: Western blot was performed using ImageJ software to quantitatively analyze LGR5 and the internal reference (β-actin), and the grayscale ratio of the target protein to the internal reference was calculated.

[0102] 5.7 Determination of intestinal flora composition

[0103] After the mice were killed, the colon contents were quickly collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing.

[0104] 5.8 Result analysis:

[0105] like Figure 5 As shown in the figure, compared with the normal group, the levels of D-LA and DAO in the colitis model group (DSS) and the negative control group (NC) were significantly increased, indicating that the intestinal mucosal barrier of mice was damaged; compared with the DSS group, the levels of D-LA and DAO in the Lactobacillus plantarum W8 intervention group were significantly reduced, and the effect was close to that of the positive drug group (PC). This shows that Lactobacillus plantarum W8 can significantly reduce intestinal permeability and alleviate DSS-induced intestinal damage.

[0106] like Figure 6 As shown in the figure, compared with the DSS group, the inflammatory factor content of mice in the W8 intervention group was significantly reduced, the IL-10 level was extremely significantly increased, and there was no significant difference between the W8 intervention effect and the PC group. This shows that Lactobacillus plantarum W8 intervention in mice can reduce the secretion of pro-inflammatory factors to varying degrees, thereby alleviating intestinal inflammation in mice and improving the intestinal barrier defense ability of mice.

[0107] like Figure 7 As shown in the figure, the colon mucosa of the normal group mice was intact and continuous, the glands were neatly arranged, the crypts were normal, and there was no inflammatory cell infiltration. The colon damage of the DSS group mice was serious, with a large number of inflammatory cells infiltrating the mucosa and submucosa. The NC group had lymph node hyperplasia in the colon tissue, accompanied by a small amount of inflammatory infiltration, while the W8 intervention group and the PC group had neatly arranged colon glands and normal crypts. It is further illustrated that the plant lactobacillus W8 provided by the present invention has the effect of relieving intestinal inflammation, reducing intestinal damage and repairing intestinal mucosa.

[0108] like Figure 8 The results showed that after intervention with Lactobacillus plantarum W8, the expression of intestinal tight junction genes ZO-1, Occludin, Claudin-1, and Claudin-2 in colitis mice increased significantly, reaching the level of the normal group. This is consistent with the results of in vitro experiments, indicating that Lactobacillus plantarum W8 can significantly enhance intestinal tight junctions and maintain intestinal barrier function.

[0109] like Fig. 9 As shown in the results, antimicrobial peptides (AMPs) play an important role in the establishment and maintenance of mucosal barriers. Therefore, the expression of AMPs genes Reg3a, Reg3g and Defa5 was detected. Compared with the normal group, the expression of AMPs genes in the DSS group and the NC group was significantly reduced, and the intervention of Lactobacillus plantarum W8 significantly inhibited the downregulation of AMPs gene expression, and the effect was slightly better than that of the PC group. This indicates that Lactobacillus plantarum W8 can help the intestine resist pathogenic bacteria invasion by regulating the expression of antimicrobial peptides.

[0110] like Fig.10 As shown, Lactobacillus plantarum W8 intervention significantly promoted the expression and distribution of LGR5, a marker of intestinal stem cells in colitis mice. The LGR5 protein level was twice that of the colitis model group and was equivalent to the expression level of the normal group. This shows that Lactobacillus plantarum W8 can effectively promote the recovery and renewal of intestinal stem cells.

[0111] like Fig.11 As shown in the figure, the changes in the intestinal flora of mice were analyzed, and the changes at the genus level were as follows Figure 7 As shown in the figure. Compared with the normal group, the abundance of short-chain fatty acid-producing bacteria Allobaculum, Muribaculaceae, and Bifidobacterium in DSS-induced colitis mice was significantly reduced, while the abundance of bacteria associated with colon inflammation, such as Faecalibaculum and Candidatus Saccharimonas, increased. However, Lactobacillus plantarum intervention significantly reversed this trend, and the abundance and composition of intestinal flora were closer to those of the normal group. The above results indicate that Lactobacillus plantarum W8 can regulate the composition of intestinal flora in colitis hosts, promote the growth of beneficial bacteria, and inhibit the proliferation of harmful bacteria, thereby balancing the homeostasis of intestinal flora.

[0112] In summary, the Lactobacillus plantarum W8 provided by the present invention has significant antioxidant, antibacterial and anti-inflammatory effects, can repair intestinal barrier damage in colitis mice, promote intestinal stem cell renewal, promote the growth of beneficial intestinal bacteria, and inhibit the proliferation of harmful bacteria, and can be widely used in the prevention and treatment of enteritis.

[0113] Example 6 A yogurt containing Lactobacillus plantarum and a preparation method thereof

[0114] 1. Add sodium carboxymethyl cellulose and white sugar to fresh milk at a mass ratio of 0.1% and 6% respectively, homogenize, sterilize, and obtain milk fermentation base;

[0115] 2. Cool to 42°C and inoculate the milk fermentation base with activated Lactobacillus plantarum W8 solution (with a live bacterial count of more than 10 9CFU / mL), fermented at 38°C for 12h;

[0116] 3. After fermentation is completed, refrigerate at 4℃ and ripen to obtain yogurt.

[0117] Example 7 A yogurt containing Lactobacillus plantarum and a preparation method thereof

[0118] 1. Add sodium carboxymethyl cellulose and sucrose to fresh milk at a mass ratio of 0.5% and 12% respectively, homogenize, sterilize, and obtain milk fermentation base;

[0119] 2. Cool to 42°C and inoculate the milk fermentation base with activated Lactobacillus plantarum W8 solution (with a live bacterial count of more than 10 9 CFU / mL), fermented at 45°C for 8h;

[0120] 3. After fermentation is completed, refrigerate at 4℃ and ripen to obtain yogurt.

[0121] Example 8 Yogurt Performance Evaluation

[0122] 8.1 Detection Method

[0123] (1) Determination of pH:

[0124] Use a handheld pH meter to measure the pH of the yogurt.

[0125] (2) Determination of titrable acidity:

[0126] The titratable acidity was determined from the amount (%) of lactic acid in each sample.

[0127] The formula is as follows: Lactic acid (%) = V × f × 0.9 / m.

[0128] Wherein, V is the volume of 0.1 mol / L sodium hydroxide solution used in titration, in mL; f is the correction factor of 0.1 mol / L sodium hydroxide solution; 0.9 is the lactic acid conversion factor; and m is the sample mass.

[0129] (3) Determination of water holding capacity:

[0130] Weigh 50g of yogurt and centrifuge it at 10℃ and 4000rpm for 20min. Calculate the water holding capacity by the following formula:

[0131]

[0132] Where Wt is the weight of the supernatant (g), Wi is the weight of the sample (g).

[0133] (4) Determination of viable bacterial count:

[0134] The determination was carried out using the plate count method.

[0135] (5) Determination of DPPH free radical scavenging rate

[0136] Mix equal amounts of yogurt and 0.1 mol / L DPPH solution (dissolved in 95% ethanol) by oscillation for 1 min, and then place at room temperature in the dark for 30 min. Centrifuge and take the supernatant to measure the absorbance at a wavelength of 517 nm. Use an equal volume of ethanol as a blank control. The calculation formula is as follows:

[0137]

[0138] (6) Determination of ABTS free radical scavenging rate

[0139] Mix yogurt and ABTS working solution in a ratio of 1:7, incubate at 25°C for 10 min, measure the absorbance at 734 nm, and use an equal volume of anhydrous ethanol as a blank control. The calculation formula is as follows:

[0140]

[0141] (7) Determination of total free amino acids

[0142] The total free amino acids in yogurt were determined by ninhydrin colorimetric method.

[0143] A standard curve was drawn using glycine (0-0.5 mg / mL) as the standard, with glycine content (mg / mL) as the abscissa and absorbance value as the ordinate. The glycine standard curve was y=2.763x-0.2689, R 2 =0.9923, and the total free amino acids in the yogurt samples were calculated based on the standard curve.

[0144] 8.2 Test results

[0145] The physical and chemical indicators of the yogurts prepared in Examples 6 and 7 of the present invention are shown in Table 1.

[0146] Table 3 Physical and chemical indicators of yogurt

[0147]

[0148] As shown in Table 3, the pH of the yogurt prepared in Examples 6 and 7 of the present invention is 4.25-4.32, the titrated acidity is 0.19%-0.23%, the water holding rate is 53.88%-55.40%, and the number of viable bacteria is higher than 10 8 CFU / g, the free amino acid content reached 0.42-0.48 mg / mL, and the antioxidant effect was significant. The scavenging rates of DPPH and ABTS free radicals of the yogurt prepared in Example 7 reached 82.03% and 88.41% respectively, achieving unexpected technical effects.

[0149] Example 9 Determination of texture index of yogurt

[0150] The texture index of yogurt was measured using a CT3 texture analyzer with a TA10 probe.

[0151] The test conditions for the texture are as follows: the target distance is 20 mm, the trigger point load is 5 g, the test speed is 2.00 mm / s, and there are 2 cycles. Each set of experiments is performed in parallel 3 times. The test results are shown in Table 4.

[0152] Table 4 Texture characteristics of yogurt

[0153]

[0154] From the results in Table 4, it can be seen that the yogurts prepared in Examples 6 and 7 of the present invention have high hardness, viscosity, elasticity and cohesion, and the yogurts have a chewy texture, high gel strength and excellent texture.

[0155] Example 10 Changes in the antioxidant capacity of yogurt during storage

[0156] In the present invention, the yogurt prepared in Examples 6 and 7 was refrigerated at 4° C. for 28 days, during which samples were taken on the 0th day, the 7th day, the 14th day, the 21st day, and the 28th day to detect the scavenging rates of the yogurt for DPPH and ABTS free radicals.

[0157] The detection method is as follows:

[0158] (1) Mix equal amounts of yogurt and 0.1 mol / L DPPH solution (dissolved in 95% ethanol) by oscillation for 1 min, and then stand at room temperature in the dark for 30 min. Centrifuge and take the supernatant to measure the absorbance at a wavelength of 517 nm. Use an equal volume of ethanol as a blank control. The calculation formula is as follows:

[0159]

[0160] (2) Mix yogurt and ABTS working solution in a ratio of 1:7, incubate at 25°C for 10 min, and measure the absorbance at 734 nm. Use an equal volume of anhydrous ethanol as a blank control. The calculation formula is as follows:

[0161]

[0162] Test results such as Fig.12 As shown, during the storage period, the yogurts prepared in Examples 6 and 7 of the present invention showed strong free radical scavenging ability; on the 28th day, the scavenging rates of DPPH and ABTS free radicals were still as high as 72.40% and 80.0%, respectively, with significant antioxidant effects, achieving unexpected technical results.

Claims

1. A yogurt, characterized in that: The yogurt is made from milk and then treated with Lactobacillus plantarum ( Lactiplantibacillus plantarum ) is made by fermentation.

2. The yogurt according to claim 1, characterized in that The deposit number of the plant lactobacillus is CGMCC No.31521.

3. The yogurt according to claim 2, characterized in that The preparation method of the yogurt comprises the following steps: Sodium carboxymethyl cellulose and sugar are added to fresh milk at a mass ratio of 0.1-0.5% and 6-12% respectively, homogenized, sterilized, and a milk fermentation base is obtained; the milk fermentation base is cooled; an activated lactobacillus plantarum bacterial liquid is inoculated into the milk fermentation base at a volume ratio of 3-5%, and the milk fermentation base is fermented at 38-45°C for 8-12h; after the fermentation is completed, the milk fermentation base is refrigerated at 4°C and then ripened to obtain yogurt.

4. The yogurt according to claim 3, characterized in that The amount of live bacteria in the plant lactobacillus liquid is higher than 10 9 CFU / mL.

5. The yogurt according to claim 3, characterized in that The preparation method of the yogurt comprises the following steps: Sodium carboxymethyl cellulose and sucrose are added to fresh milk at a mass ratio of 0.5% and 12% respectively, the mixture is homogenized and sterilized to obtain a milk fermentation base; the mixture is cooled to 42°C, an activated Lactobacillus plantarum bacterial liquid is inoculated into the milk fermentation base at a volume ratio of 5%, and the mixture is fermented at 45°C for 8 hours; after the fermentation is completed, the mixture is refrigerated at 4°C and ripened to obtain yogurt.

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