Lactobacillus plantarum ZENIYOUTH-01 and application thereof

Abstract

The invention provides a strain of lactobacillus plantarum ZENIYOUTH-01 and application thereof, and belongs to the technical field of probiotics and application thereof. The fermented whole grain prepared by adopting the lactobacillus plantarum disclosed by the invention effectively retains nutritional ingredients such as vitamin B, vitamin E, nicotinic acid, mineral substances and the like in the whole grain; abundant enzyme systems secreted by lactobacillus plantarum are utilized to degrade macromolecular proteins, dietary fibers and the like in the raw materials to generate bioactive peptides, short-chain fatty acids, exopolysaccharides and flavor compounds, release of functional components such as polyphenols, flavonoid compounds and the like is promoted, the contents of protein peptides, lactic acid, acetic acid, citric acid and beta-glucan are increased, and the nutritional value of the beverage is improved. The traditional Chinese medicine composition can effectively proliferate intestinal effective microbial communities and promote intestinal peristalsis, and has a remarkable effect in the aspect of improving constipation.

Description

Technical Field

[0001] This invention belongs to the field of probiotics and their application technology, and in particular relates to a strain of Lactobacillus plantarum ZENIYOUTH-01 and its application. Background Technology

[0002] Currently, products for chronic constipation mainly include laxatives, dietary fiber supplements, and probiotics. Among them, laxatives may cause dependence in some patients, and the risk of constipation recurrence is high after discontinuation. Dietary fiber supplements have a poor taste and require a lot of water intake, which may cause bloating and increased flatulence. Probiotics are safe, but slow to take effect and have limited effect on stubborn constipation, with significant individual differences.

[0003] Whole grains are the complete grains (or false grains) after removing only the inedible parts such as the husk and outer shell. They contain the complete endosperm, embryo, and seed coat, maximizing the retention of natural dietary fiber, B vitamins, and other micronutrients and bioactive substances. Whole grains can improve constipation symptoms, help maintain a feeling of fullness for a longer period, regulate blood sugar and insulin levels, and help reduce risk factors such as high cholesterol and high blood pressure. However, because whole grains contain anti-nutritional factors and a large amount of dietary fiber, they can put some pressure on the gastrointestinal tract, and their palatability is poor, resulting in low market acceptance. How to address the issues associated with whole grains and improve their effectiveness in relieving constipation requires further research. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a strain of Lactobacillus plantarum ZENIYOUTH-01 and its applications.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum ZENIYOUTH-01, the Lactobacillus plantarum ZENIYOUTH-01, is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20251778.

[0006] This invention provides the application of the Lactobacillus plantarum ZENIYOUTH-01 in fermented whole grains.

[0007] Preferably, the *Lactobacillus plantarum* ZENIYOUTH-01 is applied in the form of a bacterial suspension, and the concentration of the *Lactobacillus plantarum* ZENIYOUTH-01 bacterial suspension is 1×10⁻⁶. 8 ~1×10 10 The CFU / ML of Lactobacillus plantarum ZENIYOUTH-01 bacterial culture is used at a concentration of 3% to 15% of the whole grain weight.

[0008] This invention provides a method for fermenting whole grains using *Lactobacillus plantarum* ZENIYOUTH-01, comprising the following steps: 1) Grind the whole grains into a pulp, mix with water, and sterilize to obtain whole grain slurry; 2) Amplify Lactobacillus plantarum ZENIYOUTH-01 to a bacterial concentration of 1×10⁻⁶. 8 ~1×10 10 After CFU / ML, the mixture is inoculated into whole grain slurry and fermented at 27-35℃ for 24-48 h to obtain fermentation broth; 3) Sterilize, homogenize, and separate the fermentation broth to obtain fermented grains; Step 2) The mass of the inoculated Lactobacillus plantarum ZENIYOUTH-01 bacterial solution is 3% to 15% of the mass of the whole grain.

[0009] Preferably, the mass ratio of whole grain to water in step 1) is 1:1 to 4.

[0010] Preferably, the sterilization temperature in step 3) is 110~135℃ and the sterilization time is 10~20 s.

[0011] Preferably, the homogenization pressure in step 3) is 500~1000 bar, and the number of refluxes is 1~7.

[0012] Preferably, the separation in step 3) includes centrifugation and filtration; The centrifugal force is 10000~15000 g to obtain the supernatant; The filtration process involves filtering the supernatant obtained from centrifugation using a 50-150 nm ceramic membrane to obtain a filtrate, i.e., fermented grain, at a pressure of 1-3 bar.

[0013] Preferably, the process of obtaining fermented grains also includes a drying process; The drying process includes spray drying, with an inlet air temperature of 160~180℃ and an outlet air temperature of 75~85℃.

[0014] This invention provides the application of the method in improving the quality of whole grains, the application including increasing the content of any one or more of the following in whole grains: bioactive peptides, short-chain fatty acids, extracellular polysaccharides, polyphenols, or flavonoids.

[0015] This invention provides the application of the aforementioned Lactobacillus plantarum ZENIYOUTH-01 in the preparation of food products that help promote bowel movements.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarumZENIYOUTH-01, with accession number CCTCC NO: M 20251778, is a fermented whole grain prepared using *Lactobacillus plantarum* according to this invention. It effectively retains nutrients such as B vitamins, vitamin E, niacin, and minerals in the whole grain. Utilizing the rich enzyme system secreted by *Lactobacillus plantarum*, it degrades macromolecular proteins and dietary fiber in the raw materials to produce bioactive peptides, short-chain fatty acids, extracellular polysaccharides, and flavor compounds, promoting the release of polyphenols and flavonoids. The contents of protein peptides, lactic acid, acetic acid, citric acid, and β-glucan are 14±0.8g / 100g, 3.5±0.26g / 100g, 0.08g / 100g, 0.11g / 100g, and 42±1.5g / 100g, respectively. It can effectively proliferate intestinal flora, promote intestinal peristalsis, and has a significant effect on improving constipation. Attached Figure Description

[0017] Figure 1 Photograph of Lactobacillus plantarum ZENIYOUTH-01 colonies; Figure 2 Photograph of Lactobacillus plantarum ZENIYOUTH-01 cells; Figure 3 The curve showing the change in TCA-SN content during the fermentation of Lactobacillus plantarum ZENIYOUTH-01; Figure 4 The curve showing the change in lactic acid content during the fermentation of Lactobacillus plantarum ZENIYOUTH-01.

[0018] Biological Preservation Instructions The present invention relates to a strain of Lactobacillus plantarum ( Lactobacillus plantarum ZENIYOUTH-01, the Lactobacillus plantarum ZENIYOUTH-01, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO: M 20251778. Detailed Implementation

[0019] This invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum ZENIYOUTH-01, the Lactobacillus plantarum ZENIYOUTH-01, is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20251778.

[0020] This invention provides the application of the Lactobacillus plantarum ZENIYOUTH-01 in fermented whole grains.

[0021] In this invention, *Lactobacillus plantarum* ZENIYOUTH-01 is preferably used in the form of a bacterial suspension, and the concentration of the *Lactobacillus plantarum* ZENIYOUTH-01 bacterial suspension is preferably 1×10⁻⁶. 8 ~1×10 10 CFU / ML, further preferably 1×10 9 ~3.5×10 9 CFU / ML, further preferably 2.4 × 10⁻⁶ 9 The preferred mass of the CFU / mL Lactobacillus plantarum ZENIYOUTH-01 bacterial suspension used is 3% to 15% of the weight of the whole grain, more preferably 4% to 10%, and even more preferably 7.5%.

[0022] This invention provides a method for fermenting whole grains using *Lactobacillus plantarum* ZENIYOUTH-01, comprising the following steps: 1) Grind the whole grains into a pulp, mix with water, and sterilize to obtain whole grain slurry; 2) Amplify Lactobacillus plantarum ZENIYOUTH-01 to a bacterial concentration of 1×10⁻⁶. 8 ~1×10 10 After CFU / ML, the mixture is inoculated into whole grain slurry and fermented at 27-35℃ for 24-48 h to obtain fermentation broth; 3) Sterilize, homogenize, and separate the fermentation broth to obtain fermented grains; Step 2) The mass of the inoculated Lactobacillus plantarum ZENIYOUTH-01 bacterial solution is 3% to 15% of the mass of the whole grain.

[0023] In this invention, whole grains are ground into a slurry, mixed with water, and sterilized to obtain a whole grain slurry. The mass ratio of whole grains to water is preferably 1:1 to 4, more preferably 1:2 to 3.5, and even more preferably 1:3. The mixing includes soaking and grinding. The soaking time is preferably 8 to 16 hours, the slurry particle size is 5 to 20 μm, the sterilization temperature is preferably 100 to 130°C, more preferably 110 to 120°C, and even more preferably 115°C, and the sterilization time is preferably 10 to 20 minutes, more preferably 12 to 18 minutes, and even more preferably 15 minutes.

[0024] In this invention, *Lactobacillus plantarum* ZENIYOUTH-01 was amplified to a bacterial concentration of 1 × 10⁻⁶. 8 ~1×10 10 After CFU / mL, the mixture was inoculated into whole grain slurry and fermented at 27-35℃ for 24-48 h to obtain the fermentation broth; the concentration of Lactobacillus plantarum ZENIYOUTH-01 bacterial culture was 1×10⁻⁶. 8 ~1×10 10CFU / ML, preferably 1×10 9 ~3.5×10 9 CFU / ML, further preferably 2.4 × 10⁻⁶ 9 The CFU / ML of Lactobacillus plantarum ZENIYOUTH-01 bacterial culture is used at a rate of 3% to 15% of the total grain weight, preferably 4% to 10%, and more preferably 7.5%; the fermentation temperature is 27 to 35°C, preferably 28 to 32°C, and more preferably 30°C; the fermentation time is 24 to 48 hours, preferably 30 to 42 hours, and even more preferably 36 hours.

[0025] In this invention, the fermentation broth is sterilized, homogenized, and separated to obtain fermented grains; the sterilization temperature is preferably 110~135℃, more preferably 121~131℃, even more preferably 128℃, and the sterilization time is preferably 10~20 s, more preferably 12~18 s, even more preferably 15 s. The homogenization pressure is preferably 500-1000 bar, more preferably 700-950 bar, and even more preferably 850 bar; the reflux number is preferably 1-7 times, more preferably 2-6 times, and even more preferably 5 times; the separation preferably includes centrifugation and filtration; the centrifugation force is preferably 10000-15000 g, more preferably 11500-13500 g, and even more preferably 12800 g; the centrifuge is a horizontal screw centrifuge or a disc centrifuge, and a supernatant is obtained; the filtration preferably includes filtering the supernatant obtained by centrifugation with a ceramic membrane to obtain a filtrate, i.e., fermented grain; the pore size of the ceramic membrane is preferably 50-150 nm, more preferably 80-120 nm, and even more preferably 100 nm; the filtration pressure is preferably 1-3 bar, more preferably 1.5-2.5 bar, and even more preferably 2 bar.

[0026] In this invention, after obtaining fermented grains, a drying process is also included; the drying preferably includes spray drying, the inlet air temperature is preferably 160~180℃, more preferably 165~175℃, even more preferably 170℃, and the outlet air temperature is preferably 75~85℃, more preferably 78~82℃, even more preferably 80℃.

[0027] This invention provides the application of the method in improving the quality of whole grains, wherein the application preferably includes increasing the content of any one or more of the following in whole grains: bioactive peptides, short-chain fatty acids, extracellular polysaccharides, polyphenols, or flavonoids.

[0028] This invention provides the application of the aforementioned Lactobacillus plantarum ZENIYOUTH-01 in the preparation of food products that help promote bowel movements.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Feces were collected from healthy young adults with no history of constipation or other intestinal-related diseases, and lactic acid bacteria in the feces were screened using calcium carbonate plates. Calcium carbonate plate culture medium: 10g peptone, 5g beef powder, 4g yeast powder, 100g tomato juice, 20g calcium carbonate, 10g glucose, 1mL Tween 80, pH 6.5, distilled water to a final volume of 1000ml; Lactic acid bacteria with strong acid-producing ability were isolated based on the size of the calcium-soluble zone and microscopic examination. Furthermore, lactic acid bacteria with strong protein-degrading ability were screened by detecting the trichloroacetic acid soluble nitrogen content (TCA-SN) in the fermentation products. Figure 3 ; Fermentation medium (MRS): 10g peptone, 5g yeast extract, 10g beef extract, 10g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g anhydrous sodium acetate, 1ml Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, dissolved in distilled water and brought to a final volume of 1000ml. A fast-producing and adaptable lactic acid bacteria strain was screened and named zeniyouth-01. Morphological, physiological, biochemical, and 16S rDNA identification confirmed it to be *Lactobacillus plantarum*. The lactic acid content after 24 hours of fermentation was 0.78g / L (see...). Figure 4 The acid production rate and quantity were higher than those of *Lactobacillus plantarum* LP15 CGMCC 28253 (from *Lactobacillus plantarum* LP15 fermented bovine bone paste, *Acta Microbiologica Sinica* 2024, 64(10): 3749–3761). http: / / journals.im.ac.cn / actamicrocn DOI: 10.13343 / j.cnki.wsxb.20240191), colony diagram see [link to colony diagram]. Figure 1 See cell image Figure 2 .

[0031] Example 2 Whole grains quinoa, oats, buckwheat, and black rice were mixed in a mass ratio of 2:4:3:1. After soaking, the mixture was ground into a homogenate using a colloid mill. Water was added until the solid content reached 25%. The mixture was then flash-sterilized (115℃, 15 min) and cooled to 30℃. Lactobacillus plantarum ZENIYOUTH-01 was pre-cultured to a bacterial concentration of 2.4 × 10⁻⁶. 9The culture medium for expansion was composed of: glucose 40 g / L, soybean peptone 15 g / L, yeast powder 5 g / L, sodium acetate 7 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, Tween 80 1 ml / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.25 g / L, pH 6.8, seed culture inoculation amount of 2%, culture temperature of 30℃, culture time of 6 h. The expanded bacterial culture was inoculated into whole grain slurry for anaerobic fermentation, the inoculation amount of which was 7.5% of the mass of whole grain, the fermentation temperature of 30℃, and the fermentation time of 36 h. After fermentation, the fermentation broth was subjected to ultra-high temperature instantaneous sterilization at 128℃ for 15 seconds. During the sterilization process, the thermal lysis of Lactobacillus plantarum cells was completed. The thermally pyrolyzed fermentation broth was homogenized using a high-pressure homogenizer at a pressure of 850 bar and refluxed 5 times. Microscopic examination revealed no intact cell structure. The homogenized fermentation broth was centrifuged using a horizontal screw centrifuge with a separation factor of 11600×G to obtain a supernatant. This supernatant was then filtered through a 100 nm ceramic membrane to obtain a clear filtrate. The ceramic membrane filtration pressure was 2 bar. After the filtration cycle was reduced to the minimum volume, pure water was added to wash the filter until the total volume was consistent with that before filtration. The ceramic membrane filtrate was spray-dried to obtain a powdered product. The inlet air temperature of the spray dryer was 165℃ and the outlet air temperature was 75℃. The finished product had good flowability and water solubility. The protein peptide content was 13.5 g / 100 g, the lactic acid content was 3.3 g / 100 g, and the β-glucan content was 42 g / 100 g.

[0032] Example 3 Whole grains corn, sorghum and millet were mixed in a mass ratio of 2:3:5. After soaking, the mixture was ground into a homogenate by a colloid mill. Water was added until the solid content was 40%. The mixture was then flash-sterilized (115℃, 15min) and cooled to 28℃. Lactobacillus plantarum ZENIYOUTH-01 was pre-cultured to a bacterial concentration of 3.5 × 10⁻⁶. 9 The culture medium for expansion was composed of: glucose 40 g / L, soybean peptone 15 g / L, yeast powder 5 g / L, sodium acetate 7 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, Tween 80 1 ml / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.25 g / L, pH 6.8. The seed culture inoculum was 2%, the culture temperature was 30℃, and the culture time was 8 h. The expanded culture was then inoculated into whole grain slurry for anaerobic fermentation at an inoculum size of 3% of the whole grain slurry mass, the fermentation temperature was 28℃, and the fermentation time was 30 h. After fermentation, the fermentation broth was subjected to ultra-high temperature instantaneous sterilization at a temperature of 130℃ for 11 seconds. During the sterilization process, the thermal lysis of Lactobacillus plantarum cells was completed. The thermally pyrolyzed fermentation broth was homogenized using a high-pressure homogenizer at a pressure of 950 bar and with two reflux cycles. Microscopic examination revealed no intact cell structures. The homogenized fermentation broth was centrifuged using a disc centrifuge with a separation factor of 12800×G to obtain a supernatant. The supernatant was then filtered through a plate and frame filter press pre-coated with diatomaceous earth. Initially, the filtration pressure was maintained at 0.5 bar. After the filtrate was refluxed until it was completely clear, the pressure was gradually increased to 6 bar. After filtration, pure water was added to wash the filter cake so that the total volume of the filtrate remained the same as before filtration. The clarified filtrate was spray-dried to obtain a powdered product. The inlet air temperature of the spray dryer was 170℃ and the outlet air temperature was 80℃. The finished product had good flowability and water solubility. The protein peptide content was 14.5 g / 100 g, the lactic acid content was 3.6 g / 100 g, and the β-glucan content was 39 g / 100 g.

[0033] Experimental Example 1 To study the effects of different implementation methods and comparative examples, 50 female mice were randomly divided into a blank control group, a model group, a sample group, and a positive control group, with 10 mice in each group. After one week of acclimatization, the blank control group was administered an equal volume of physiological saline by gavage, while the other groups were given a single gavage administration of 1 mL / 100 g loperamide hydrochloride suspension to induce constipation, and this administration continued for one week. From day 8... Initially, the sample group was administered 0.05 g / 100 g of the sample in the example via gavage, the blank group and the model group were administered an equal volume of physiological saline via gavage, and the positive control group was administered 0.05 g / 100 g of lactulose via gavage. The administration was continued for 14 days.

[0034] Fourteen days after drug administration, mice were housed separately, and fecal moisture content was collected and analyzed. The number of fecal pellets excreted by the mice over 6 hours was also counted. Fecal moisture content was determined using a rapid moisture analyzer at 105℃ using the drying method. Fecal moisture content = (wet weight - dry weight) / wet weight × 100% The patient was fasted for 24 hours but allowed free access to water. Then, 0.2 mL of 5% carbon black suspension was administered by gavage. Timing began immediately after gavage. The patient was sacrificed 30 minutes later, and the small intestine was collected. The length was measured: the distance from the pylorus to the ileocecal junction was recorded as the total intestinal length; the distance from the pylorus to the tip of the carbon black suspension was recorded as the carbon black propulsion length. The small intestinal propulsion rate was calculated. Intestinal propulsion rate = Carbon black propulsion length (cm) / Total intestinal length (cm) × 100% Table 1. Fecal water content, intestinal propulsion rate, and number of defecations within 6 hours in different groups of mice.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum ZENIYOUTH-01, characterized in that, The *Lactobacillus plantarum* ZENIYOUTH-01 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251778.

2. The application of the Lactobacillus plantarum ZENIYOUTH-01 as described in claim 1 in fermented whole grains.

3. The application according to claim 1, characterized in that, The *Lactobacillus plantarum* ZENIYOUTH-01 was applied in the form of a bacterial suspension, with a concentration of 1×10⁻⁶. 8 ~1×10 10 The CFU / ML of Lactobacillus plantarum ZENIYOUTH-01 bacterial culture is used at a concentration of 3% to 15% of the whole grain weight.

4. A method for fermenting whole grains using *Lactobacillus plantarum* ZENIYOUTH-01 as described in claim 1, characterized in that, Includes the following steps: 1) Grind the whole grains into a pulp, mix with water, and sterilize to obtain whole grain slurry; 2) Amplify Lactobacillus plantarum ZENIYOUTH-01 to a bacterial concentration of 1×10⁻⁶. 8 ~1×10 10 After CFU / ML, the mixture is inoculated into whole grain slurry and fermented at 27-35℃ for 24-48 h to obtain fermentation broth; 3) Sterilize, homogenize, and separate the fermentation broth to obtain fermented grains; Step 2) The mass of the inoculated Lactobacillus plantarum ZENIYOUTH-01 bacterial solution is 3% to 15% of the mass of the whole grain.

5. The method according to claim 4, characterized in that, Step 1) The mass ratio of whole grains to water is 1:1 to 4.

6. The method according to claim 4, characterized in that, Step 3) The sterilization temperature is 110~135℃ and the sterilization time is 10~20 s.

7. The method according to claim 4, characterized in that, Step 3) The homogenization pressure is 500~1000 bar, and the number of refluxes is 1~7. The separation includes centrifugation and filtration; The centrifugal force is 10000~15000 g, and a supernatant is obtained; The filtration process involves filtering the supernatant obtained from centrifugation using a 50-150 nm ceramic membrane to obtain a filtrate, i.e., fermented grain, at a pressure of 1-3 bar.

8. The method according to claim 4, characterized in that, After obtaining fermented grains, a drying process is also included; The drying process includes spray drying, with an inlet air temperature of 160~180℃ and an outlet air temperature of 75~85℃.

9. The application of the method according to any one of claims 4 to 8 in improving the quality of whole grains, characterized in that, The application includes increasing the content of any one or more of the following in whole grains: bioactive peptides, short-chain fatty acids, extracellular polysaccharides, polyphenols, or flavonoids.

10. The use of the Lactobacillus plantarum ZENIYOUTH-01 of claim 1 in the preparation of food products that help with bowel movements.

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