A Lactobacillus helveticus strain for preparing a live lactic acid bacteria beverage with a cleaning formula and its application

Abstract

The present invention provides a Lactobacillus helveticus strain for preparing a live lactic acid bacteria beverage with a clean formula and its application. The Lactobacillus helveticus strain Zhegu LBH-VI provided by the present invention is deposited in the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: M 2023095. The Lactobacillus helveticus Zhegu LBH-VI provided by the present invention has the advantages of high extracellular polysaccharide production, high tolerance to gastrointestinal fluids, and strong intestinal colonization ability.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Lactobacillus helveticus and its applications. Background Technology

[0002] Probiotics are live microorganisms that produce beneficial effects on the host's health, primarily exerting their beneficial functions by colonizing the small intestine through adhesion. Lactic acid bacteria belong to the category of probiotics and are abundant in the human gut, serving as indispensable beneficial bacteria. They regulate the balance of intestinal flora, promote the absorption and utilization of nutrients, inhibit pathogens, enhance the body's immune regulation capabilities, and strengthen the intestinal barrier. Maintaining a balance of lactic acid bacteria in the gut is of great importance to overall health.

[0003] Extracellular polysaccharides (EPPs) of lactic acid bacteria are a class of natural high-molecular-weight sugar compounds secreted by lactic acid bacteria outside their cell walls to adapt to their environment. Based on their biological activity and physicochemical properties, such as immunomodulation, antioxidation, antibacterial, and antitumor effects, they have broad application prospects. Furthermore, EPPs can bind to casein and whey protein, forming a porous network structure that significantly influences the texture, flavor, and stability of fermented milk. It has been reported that EPPs can be used as thickeners and stabilizers in cheese to improve its textural properties and increase its water content. In dairy production, EPPs can replace food additives to improve the stability, gelling properties, emulsifying properties, viscosity, and water-holding capacity of fermented dairy products.

[0004] Lactic acid bacteria beverages, produced at low temperatures and containing live bacteria, are a popular product category in the market due to their refreshing taste and high concentration of live bacteria. These beverages are made by fermenting cow's milk or skim milk powder with lactic acid bacteria (such as Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus casei, and Lactobacillus rhamnosus) to produce acidic curds. When the acidity reaches above 130°T, this curd is used as a base for further formulation. The stability of lactic acid bacteria beverages is related to the particle size and viscosity of casein during fermentation. The denaturation and precipitation of milk proteins under acidic conditions has always been a key issue affecting the production and development of acidic dairy beverages. Currently, the main approach to improving the stability of formulated lactic acid bacteria beverages is by adding stabilizers, which contradicts the current pursuit of purely natural foods and simple formulations. Summary of the Invention

[0005] In view of the problems of easy separation, easy sedimentation, and instability that often occur in the preparation of lactic acid bacteria beverages in the prior art, the present invention provides a Lactobacillus helveticus, and the lactic acid bacteria beverage prepared using Lactobacillus helveticus is not easy to separate, not easy to settle, and is more stable.

[0006] In a first aspect, the present invention provides a strain of Lactobacillus helveticus Zhegu LBH-VI, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2023095.

[0007] Preferably, the 16S rDNA sequence of the Lactobacillus helveticus Zhegu LBH-VI strain is shown in SEQ ID NO.1.

[0008] Secondly, the present invention provides a fermentation preparation method for Lactobacillus helveticus inoculum, the method comprising the following steps: culturing the Lactobacillus helveticus strain.

[0009] Preferably, the preparation method includes the following steps:

[0010] (1) The Lactobacillus helveticus strain was cultured in a large-scale manner;

[0011] (2) Add the product obtained in step (1) to a liquid culture medium and ferment it at 35-39℃.

[0012] Thirdly, the present invention provides a microbial agent containing the aforementioned Lactobacillus helveticus strain.

[0013] Preferably, the microbial agent is obtained by the fermentation preparation method described above.

[0014] Fourthly, the present invention provides the application of the aforementioned Lactobacillus helveticus or the aforementioned bacterial agent in food, health products or food additives.

[0015] Preferably, the food is a fermented dairy product or a fermented fruit and vegetable product. More preferably, the fermented dairy product is one or a combination of two or more of fermented milk, cheese, milk powder, and lactic acid bacteria beverages.

[0016] Fifthly, the present invention provides the application of the aforementioned Lactobacillus helveticus or the aforementioned bacterial agent in products for improving intestinal function.

[0017] In a sixth aspect, the present invention provides the application of the aforementioned Lactobacillus helveticus or the aforementioned bacterial agent in the production of polysaccharides.

[0018] In a seventh aspect, the present invention provides a fermented dairy product, characterized in that the fermenting agent contains the aforementioned Lactobacillus helveticus strain or the aforementioned inoculum.

[0019] Preferably, the fermented dairy product contains acetaldehyde and / or dimethyl ethyl ketone; more preferably, the fermented dairy product contains 2.9-3.0 mg / L dimethyl ethyl ketone; or more preferably, the fermented dairy product contains 29-30 mg / L acetaldehyde.

[0020] Eighthly, the present invention provides a lactic acid bacteria beverage, characterized in that the fermenting agent contains the aforementioned Lactobacillus helveticus strain or the aforementioned bacterial agent.

[0021] Preferably, the lactic acid bacteria beverage is made from raw materials comprising the following parts by weight: 20-40 parts milk, 60-80 parts water, 3-6 parts sweetener, the Lactobacillus helveticus strain or the bacterial agent, wherein each milliliter of the lactic acid bacteria beverage contains ≥10 8 The Lactobacillus helveticus strain or the bacterial agent described in CFU;

[0022] Preferably, the sweetener is one or a combination of two or more of sucrose, xylitol, or sucralose.

[0023] Ninthly, the present invention provides a milk powder made from raw materials comprising the following parts by weight, on a dry matter basis: 5-50 parts of the Lactobacillus helveticus strain or the bacterial agent, 5-50 parts of skim milk powder, 5-50 parts of the protective agent oligosaccharide, and 2-10 parts of glycerol.

[0024] Preferably, the protective agent is selected from oligosaccharides;

[0025] Preferably, the oligosaccharide is selected from one or more combinations of maltodextrin, fructodextrin, galacto-oligosaccharide, xylooligosaccharide, soybean oligosaccharide, and inulin.

[0026] Preferably, the total number of active Lactobacillus helveticus LBH-VI in the milk powder is ≥10. 11 CFU / g.

[0027] The *Lactobacillus helveticus* Zhegu LBH-VI provided by this invention has the advantages of high extracellular polysaccharide production, high tolerance to gastrointestinal fluids, and strong intestinal colonization ability. Using *Lactobacillus helveticus* Zhegu LBH-VI provided by this invention to prepare lactic acid bacteria beverages can overcome the shortcomings and defects of existing lactic acid bacteria beverages, such as poor stability, easy stratification, and easy sedimentation. It can provide a clean and stable live lactic acid bacteria beverage; furthermore, this strain can colonize the intestines after entering the human body with the lactic acid bacteria beverage, truly playing a role in promoting intestinal health.

[0028] strain preservation

[0029] The *Lactobacillus helveticus* Zhegu LBH-VI strain provided by this invention was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO: M 2023095. The deposit address is: Wuhan University, No. 16 Luojia Mountain Road, Wuchang District, Wuhan, Hubei Province, China, Postcode: 430072; Telephone: 027-68754052. Attached Figure Description

[0030] Figure 1 The image shows the colony morphology of Lactobacillus helveticus Zhegu LBH-VI strain.

[0031] Figure 2 The image shown is a microscopic image of Lactobacillus helveticus Zhegu LBH-VI strain.

[0032] Figure 3 The figure shows the weight changes of a constipation model mouse during its growth period in this example. Detailed Implementation

[0033] The *Lactobacillus helveticus* strain provided by this invention was isolated from homemade yak milk residue from herders on the Zhegu Grassland at an altitude of 4600 meters in Tibet. It was identified as *Lactobacillus helveticus*, a naturally occurring wild-type microbial strain. It is included in the updated list of microbial strains that can be used in food, published by the National Health Commission on August 25, 2022, and can be widely used in various fermented dairy products. The *Lactobacillus helveticus* strain provided by this invention is named *Lactobacillus helveticus* Zhegu LBH-VI. This *Lactobacillus helveticus* strain possesses characteristics such as high production of extracellular polysaccharides, high tolerance to gastrointestinal fluids, and strong intestinal colonization ability. When applied to lactic acid bacteria beverages, it overcomes the shortcomings and defects of existing lactic acid bacteria beverages, such as poor stability, easy stratification, and easy sedimentation. It provides a clean-formulated, texture-stable live lactic acid bacteria beverage. Furthermore, this strain can colonize the intestines when it enters the human body with the lactic acid bacteria beverage, significantly improving intestinal health by relieving constipation.

[0034] The source information of the instruments and reagents used in the embodiments of the present invention is shown in Table 1 below.

[0035] Table 1

[0036]

[0037]

[0038] Example 1: Screening and Characterization of Lactobacillus helveticus

[0039] 1. Sample collection and bacterial enrichment

[0040] Take 25g of homemade yak milk residue from the Zhegu Grassland in Tibet and mix it with 225mL of sterile physiological saline to obtain a homogeneous sample solution. Then, perform serial dilutions on the sample solution, taking 10g of each solution separately. -3 10 -4 10 -5 10 -6 The diluted solution was spread on MRS agar plates and incubated at 37°C for 48 hours, after which colonies grew on the MRS agar.

[0041] 2. Initial screening of bacterial strains

[0042] Based on the standard colony characteristics of Lactobacillus helveticus, single colonies were selected for isolation, purification, and further cultured. The isolation and purification were repeated at least three times to obtain purified colonies.

[0043] Culture characteristics: The optimal growth temperature is 37℃. It is a facultative anaerobe and grows in MRS medium.

[0044] 3. Acid production experiment

[0045] The single colonies obtained from the initial screening of the bacterial strain were cultured on MRS medium plates containing 0.2% CaCO3. After culturing at 37°C for 48 hours, the presence or absence of a clear zone around the colony was observed. Single colonies with strong acid production capacity and large clear zones were selected for further isolation and purification.

[0046] Morphological characteristics: The growth state on MRS agar medium was as follows: The colonies were milky white, round, with smooth edges, raised cells, and a rough surface. The colony morphology of the obtained strain is shown in the figure below. Figure 1 As shown.

[0047] 4. Gram staining

[0048] Gram staining was performed on the strains obtained from the secondary screening. If the Gram staining was typically positive, the resulting strain was the target strain, and its microscopic image is shown below. Figure 2 As shown. Under a microscope, the cells appear as short rods, without flagella, do not produce spores, and are not motile.

[0049] 5. Identification of strains

[0050] The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA gene sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain is *Lactobacillus helveticus*. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.1:

[0051]

[0052] The strain was named Lactobacillus helveticus Zhegu LBH-VI and was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO:M 2023095.

[0053] 6. The strain's ability to produce extracellular polysaccharides

[0054] A. Lactobacillus helveticus Zhegu LBH-VI, Lactobacillus delbrueckii subsp. bulgaricus CICC6047 and Lactobacillus paracasei CICC6108 were activated and inoculated into MRS broth medium at a volume ratio of 6% to form a fermentation broth. The broth was cultured at 37°C for 48 hours under constant temperature and rate.

[0055] B. Place the fermentation broth obtained in step A in a 95°C water bath for 10 minutes. The purpose of the water bath is to inactivate any extracellular polysaccharide-degrading enzymes that may exist in the fermentation broth. Then, let it stand and cool to room temperature. Add 80% trichloroacetic acid to the fermentation broth to make the final concentration of the fermentation broth 5%. Stir the fermentation broth magnetically at 120 r / min for 2 hours at room temperature. Then, centrifuge at 4°C and 12000 r / min for 40 minutes. After centrifugation, retain the supernatant for removing bacteria and proteins.

[0056] C. The supernatant from step B was precipitated with 95% ethanol at a volume three times that of the supernatant in step B. The mixture was centrifuged at 12000 r / min and 4℃ for 40 min to obtain extracellular polysaccharide precipitate. The extracellular polysaccharide precipitate was washed three times and dissolved by sonication at 50℃ for 20 min in pure water. The solution was measured into a dialysis bag (molecular weight cutoff 14000 Da). The solution was dialyzed with pure water at 120 r / min magnetic stirring for 24 h at room temperature. The pure water was changed every 8 h. The solution after dialysis was brought to a final volume of 200 ml.

[0057] The content of extracellular polysaccharides in lactic acid bacteria producing extracellular polysaccharides was determined using the phenol-sulfuric acid method. The results are shown in Table 2.

[0058] Table 2 Extracellular polysaccharide content

[0059] strain number Extracellular polysaccharides (mg / L) Lactobacillus helveticus Zhegu LBH-VI 451.8 Lactobacillus delbrueckii subsp. bulgaricus CICC6047 402.7 Lactobacillus paracasei CICC6108 327.3

[0060] As shown in Table 2 above, the extracellular polysaccharide content produced by Lactobacillus helveticus Zhegu LBH-VI provided by the present invention is high, reaching 451.8 mg / L.

[0061] 7. Test on resistance to gastrointestinal fluids

[0062] Preparation of artificial gastric fluid: Adjust the pH of 0.2% NaCl solution to 2.0 with concentrated hydrochloric acid, then add 0.05g of pepsin (specific activity 1:10000), and filter through a 0.22μm sterile filter membrane for sterilization.

[0063] Preparation of artificial intestinal fluid: 1.2g NaH2PO4, 0.1g trypsin, 0.45g bile salts, 100mL distilled water, adjust pH to 8.0 with sodium hydroxide, and filter sterilize using a 0.22μm sterile filter membrane.

[0064] Lactobacillus helveticus LBH-VI, Lactobacillus delbrueckii subsp. bulgaricus CICC6047, and Lactobacillus paracasei CICC6108 were inoculated into MRS medium and cultured at 37°C for 24 h, followed by two subcultures. 1 mL of this medium was then added to simulated gastric and intestinal fluids, respectively, and incubated at 37°C. Samples were taken at 0 h and 3 h to determine the total bacterial count. The results are shown in Table 3.

[0065] Table 3 Gastrointestinal Fluid Tolerance

[0066]

[0067]

[0068] The results in Table 3 above indicate that Lactobacillus helveticus Zhegu LBH-VI has good acid and bile salt resistance.

[0069] 8. Intestinal colonization capacity experiment

[0070] (1) Preparation of strains

[0071] Lactobacillus helveticus Zhegu LBH-VI was inoculated into MRS liquid medium at a 3% inoculum. After two generations of activation culture at 37°C for 24 h, the bacterial culture that had reached the end of the logarithmic growth phase was centrifuged at 5000 r / min for 10 min, the supernatant was discarded and the bacterial cells were collected. The cells were washed twice with sterile PBS solution (pH=7.2) and then resuspended in sterile PBS solution.

[0072] (2) Cell hydrophobicity assay

[0073] Mix 3 mL of bacterial suspension with 1 mL of xylene (a hydrophobic agent) until homogeneous, let stand for 10 min to separate the organic and aqueous phases, and collect the aqueous phase. Measure the OD values ​​At and A0 of the aqueous phase and bacterial suspension at 600 nm. Repeat the experiment three times and calculate the values.

[0074] Hydrophobicity (%) = [(1-At) / A0] × 100%

[0075] (3) Cell self-agglutination ability assay

[0076] The absorbance (B0) of the bacterial suspension at 600 nm was measured. 4 mL of the bacterial suspension was taken, shaken to mix, and incubated at room temperature for 5 h. The OD value (Bt) of the bacterial supernatant was measured at 600 nm. The experiment was repeated three times and the results were calculated.

[0077] Self-agglomeration ability (%) = [(1-Bt) / B0] × 100%

[0078] Table 4 Hydrophobicity and Self-aggregation Ability

[0079] strain number Hydrophobicity (%) Self-agglomeration ability (%) Lactobacillus helveticus Zhegu LBH-VI 94.6 72.5 Lactobacillus delbrueckii subsp. bulgaricus CICC 6047 84.2 54.6 Lactobacillus paracasei CICC 6108 93.4 64.9

[0080] Cellular hydrophobicity and self-agglutination ability are prerequisites for probiotics to colonize the gastrointestinal tract by clearing intestinal pathogens and adhering to intestinal epithelial cells. As shown in Table 4 above, among the three lactic acid bacteria strains, *Lactobacillus helveticus* Zhegu LBH-VI exhibited higher hydrophobicity and self-agglutination abilities, at 94.6% and 72.5%, respectively.

[0081] Example 2: Preparation of probiotic powder containing Lactobacillus helveticus Zhegu LBH-VI

[0082] 1. Cell culture: Two generations of activated Lactobacillus helveticus Zhegu LBH-VI seed culture medium was inoculated into the fermentation medium at a 3% inoculum rate. Under suitable culture conditions, the viable cell concentration was increased to 10⁻⁶. 10 Centrifuge at CFU / mL for 5000 rpm for 10 min, discard the supernatant, wash twice with sterile water, and collect the bacterial cells.

[0083] 2. Composition of freeze-drying protectant: 20 parts of Lactobacillus helveticus Zhegu LBH-VI, 15 parts of skim milk powder, 15 parts of soybean oligosaccharides, 2 parts of glycerol, and 45 parts of deionized water, totaling 1000g. After mixing evenly, place in a -80℃ freezer for 2 hours for pre-freezing.

[0084] 3. Freeze-drying treatment: The pre-frozen probiotic composition was freeze-dried at -80℃ and 15Pa to obtain probiotic powder. The total number of active Lactobacillus helveticus Zhegu LBH-VI in the prepared probiotic powder was ≥10. 11 CFU / g.

[0085] Example 3: Preparation of a dairy beverage containing Lactobacillus helveticus Zhegu LBH-VI

[0086] After browning pure milk at 95-97℃ for 2.5 hours, it was rapidly cooled to 37℃ to prepare a fermentation medium. Three portions were prepared according to the above method, and then 10... 6CFU / mL of *Lactobacillus helveticus* Zhegu LBH-VI, *Lactobacillus delbrueckii* subsp. bulgaricus CICC6047, and *Lactobacillus paracasei* CICC6108 were inoculated into the obtained fermentation medium and mixed thoroughly. Fermentation was carried out at a constant temperature of 37°C until the fermentation acidity remained constant at pH 130°T. Fermentation was then terminated to obtain the milk beverage base. In a mixing tank, the ingredients (water, 10% granulated sugar, and 30% fermentation base) were thoroughly mixed. Acidity was adjusted at 10-20°C by spraying the acid solution (prepared with room temperature water to a concentration of less than 10% citric acid, lactic acid, and sodium citrate) evenly into the mixture, adjusting the acidity to 70°T. Any remaining acid was supplemented with citric acid. The mixture was then homogenized (pressure 20 MPa), pumped into cans, cooled, filled, and refrigerated. The obtained probiotic beverages containing *Lactobacillus helveticus* Zhegu LBH-VI, *Lactobacillus delbrueckii* subsp. bulgaricus CICC6047, and *Lactobacillus paracasei* CICC6108 all had a viable count ≥10⁻⁶ for each of these bacteria. 8 CFU / mL.

[0087] The three probiotic beverages obtained above were subjected to sensory evaluation by 50 professional tasters. The sensory evaluation criteria are shown in Table 5. The evaluation results are shown in Table 6.

[0088] Table 5 Sensory Evaluation Standards for Dairy Beverages

[0089]

[0090] The different strains of bacteria mentioned in the above-mentioned dairy beverages are shown in Table 6, and their preparation steps and processes are exactly the same.

[0091] Table 6 Evaluation of fermentation characteristics of strains

[0092]

[0093] As shown in Table 6 above, the lactic acid bacteria beverage fermented with the present invention, *Lactobacillus helveticus* Zhegu LBH-VI, had the shortest coagulation time, and its viscosity and viable cell count were higher than those of the control strain. Sensory evaluation of the milk beverage revealed that the milk beverage prepared with *Lactobacillus helveticus* Zhegu LBH-VI was milky white, uniform, and smooth, without layering or sedimentation, and possessed a refreshing and delicious flavor characteristic of milk beverages, resulting in the highest overall score.

[0094] The prepared lactic acid bacteria beverage was refrigerated at 4°C and observed at 0, 7, 14, and 21 days to evaluate the effect of different bacterial strains on stability.

[0095] Table 7 Evaluation of the storage stability of lactic acid bacteria beverages

[0096]

[0097] The stability of the Lactobacillus helveticus LBH-VI fermented lactic acid bacteria samples stored under refrigeration was observed regularly. As shown in Table 7 above, the Lactobacillus helveticus LBH-VI fermented lactic acid bacteria samples were homogeneous and stable, with no stratification or precipitation in the liquid.

[0098] Example 4: Improvement of intestinal constipation in mice by gavage administration of lactic acid bacteria beverage

[0099] After consuming a lactic acid bacteria beverage containing Lactobacillus helveticus LBH-VI for 14 consecutive days, the study investigated whether this strain could improve constipation in mice and assessed its effect on the secretion of intestinal motility-related hormones.

[0100] Six- to seven-week-old mice were randomly divided into five groups: a control group (administered via gavage with sterile saline), a model group (administered only lopidine, i.e., the chlorpheniramine group), a low-dose group (1 x 10⁻⁶ g / L), and a low-dose group (administered via gavage with sterile saline). 8 CFU / kg.bw), medium (1x10) 9 CFU / kg.bw), high dose (1x10) 10 Mice in each group were administered probiotics (CFU / kg.bw) plus a constipation-related group (Lopaminergic amine + lactic acid bacteria beverage containing Lactobacillus helveticus Zhegu LBH-VI) via gavage at a dose of 0.1 mL / 10 g body weight. The animals were housed at an ambient temperature of 21±2℃ and humidity of 30-70%, with 12-hour light-dark cycles, and free access to water and feed. Bedding was changed every three days, and water and feed were replenished as needed. All animals were gavaged for three consecutive weeks. Body weight was measured weekly, and the gavage dosage was adjusted accordingly. Body weight changes during the growth period for each group of mice are shown below. Figure 3 As shown.

[0101] like Figure 3 As shown, the results indicate that no mice died during the observation period, and all mice in each treatment group gained weight. No mice were observed to lose weight or gain weight abnormally.

[0102] The mice were divided into groups as follows: Each group underwent intervention with the corresponding gavage for 2 weeks. On the evening of day 15, the mice were fasted overnight. The following morning at 8:00 AM, they were given a lactic acid bacteria beverage, and after a 30-minute wait, they were given ink via gavage. Half of the mice in each group were randomly selected and sacrificed 30 minutes after ink administration. Intestinal segments were harvested, and their length and "ink propulsion length" were measured to calculate the small intestinal propulsion rate. The other half of the mice were timed after ink administration, and the time of excretion of the first black stool was recorded. The results are shown in Table 8. Small intestinal and blood samples were collected from the sacrificed mice for analysis.

[0103] Table 8 Time of excretion of primordia in constipated mice

[0104] Group Time to expel the first black particle (min) Blank group 121.4 Model group 189.6 low-dose group 49.6 medium dose group 43.2 High-dose group 42.5

[0105] As shown in Table 8, oral administration of a lactic acid bacteria beverage containing Lactobacillus helveticus Zhegu LBH-VI significantly advanced the time of first black grain appearance.

[0106] The effect of a lactic acid bacteria beverage containing Lactobacillus helveticus (Zhegu LBH-VI) on constipation was assessed by observing the small intestinal propulsion rate 30 minutes after consuming foods containing coloring agents. The intestinal length was measured as "total small intestine length," and the distance from the pylorus to the ink front was defined as "ink propulsion length." The formula for calculating the ink propulsion rate is as follows:

[0107] Ink propulsion rate = Ink propulsion length (cm) / Total small intestine length (cm) × 100%

[0108] Table 9. Small intestinal propulsion rate in mice 30 minutes after feeding.

[0109] Group Ink propulsion rate (%) in 30 minutes Blank group 32.5 constipation group 24.6 low-dose group 28.8 medium dose group 32.9 High-dose group 36.6

[0110] As shown in Table 9 above, compared with the constipation group, the small intestinal propulsion rate of mice in the medium-dose and high-dose groups was significantly increased 2 hours after administration of ink. In conclusion, Lactobacillus helveticus fermented lactic acid bacteria beverage can significantly improve the intestinal propulsion speed in constipated mice.

[0111] The levels of somatostatin and gastrin in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA) according to the ELISA kit instructions. The results are shown in Tables 10 and 11.

[0112] Table 10. Somatostatin levels in mouse serum

[0113] Group Serum somatostatin levels (pg / mL) Blank group 523.6 constipation group 573.1 low-dose group 429.4 medium dose group 429.6 High-dose group 413.5

[0114] Table 11 Serum gastrin levels in mice

[0115] Group Gastrin (pg / mL) Blank group 53.3 constipation group 44.7 low-dose group 46.8 medium dose group 48.3 High-dose group 54.6

[0116] The results in Tables 10 and 11 indicate that long-term intake of Lactobacillus helveticus Zhegu LBH-VI lactic acid bacteria beverage has a certain effect on the secretion of digestive peptides (somatostatin and gastrin), and can be used to relieve constipation.

Claims

1. A Lactobacillus helveticus ( Lactobacillus helveticus ) Zhegu LBH-VI strain, characterized in that It is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M 2023095.

2. The Lactobacillus helveticus Zhegu LBH-VI strain according to claim 1, wherein The 16S rDNA sequence of the Lactobacillus helveticus Zhegu LBH-VI strain is shown in SEQ ID NO.

1.

3. A fermentation method for preparing a Lactobacillus helveticus agent, characterized in that: The method comprises the following steps: culturing the Lactobacillus helveticus strain according to claim 1 or 2.

4. The preparation method according to claim 3, characterized in that The preparation method comprises the following steps: (1) amplifying and culturing the Lactobacillus helveticus strain according to claim 1 or 2; (2) Add the product obtained in step (1) to liquid culture medium and ferment and culture at 35-39°C.

5. A bacterial agent, characterized in that The bacterial agent contains the Lactobacillus helveticus strain according to claim 1 or 2.

6. The microbial agent according to claim 5, characterized in that The bacterial agent is obtained by the fermentation preparation method according to claim 3 or 4.

7. Use of the Lactobacillus helveticus according to claim 1 or 2 or the bacterial agent according to claim 5 or 6 in food, health products or food additives.

8. The use according to claim 7, characterized in that The food is a fermented dairy product or a fermented fruit and vegetable product.

9. The use according to claim 8, characterized in that The fermented dairy product is one or a combination of two or more of fermented milk, cheese, milk powder and lactic acid bacteria beverage.

10. Use of the Lactobacillus helveticus according to claim 1 or 2 or the bacterial agent according to claim 5 or 6 in the preparation of a product for improving intestinal constipation.

11. Use of the Lactobacillus helveticus according to claim 1 or 2 or the bacterial agent according to claim 5 or 6 in producing polysaccharides.

12. A fermented dairy product, characterized in that The starter contains the Lactobacillus helveticus strain according to claim 1 or 2 or the bacterial agent according to claim 5 or 6.

13. The fermented dairy product according to claim 12, characterized in that The fermented dairy product contains acetaldehyde and / or diacetyl.

14. The fermented dairy product according to claim 13, characterized in that The fermented dairy product contains 2.9-3.0 mg / L diacetyl.

15. The fermented dairy product according to claim 13 or 14, characterized in that The fermented dairy product contains 29-30 mg / L acetaldehyde.

16. A lactic acid bacteria beverage, characterized in that The starter contains the Lactobacillus helveticus strain according to claim 1 or 2 or the bacterial agent according to claim 5 or 6.

17. The lactic acid bacteria beverage according to claim 16, characterized in that The invention is prepared by using the following raw materials in parts by weight: 20-40 parts of milk, 60-80 parts of water and 3-6 parts of sweetener, the Lactobacillus helveticus strain according to claim 1 or 2 or the bacterial agent according to claim 5 or 6, wherein each milliliter of the lactic acid bacteria beverage contains ≥10 8 CFU / mL The Lactobacillus helveticus strain according to claim 1 or 2 or the bacterial agent according to claim 5 or 6.

18. The lactic acid bacteria beverage according to claim 17, characterized in that The sweetener is one or a combination of two or more of sucrose, xylitol or sucralose.

19. A milk powder, characterized in that: The milk powder is prepared by using raw materials containing the following parts by weight, calculated on a dry matter basis: 5-50 parts of the Lactobacillus helveticus strain according to claim 1 or 2 or the bacterial agent according to claim 5 or 6, 5-50 parts of skim milk powder, 5-50 parts of oligosaccharides as a protective agent, and 2-10 parts of glycerol.

20. The milk powder according to claim 19, characterized in that The oligosaccharide is selected from one or a combination of two or more of malto-oligosaccharide, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, soybean oligosaccharide and inulin.

21. The milk powder according to claim 19 or 20, characterized in that The total number of active Lactobacillus helveticus ZheguLBH-VI in the milk powder is ≥10 11 CFU / g.

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