Plant lactobacillus MG360 capable of improving constipation, compound probiotic fermentation preparation and application of compound probiotic fermentation preparation

Abstract

The invention provides a lactobacillus plantarum MG360 with the efficacy of improving constipation, the preservation number of the lactobacillus plantarum MG360 is CGMCC NO 32953, and the lactobacillus plantarum MG360 is preserved in China General Microbiological Culture Collection Center on February 14, 2025. Meanwhile, the invention further provides a compound probiotic composition of the plant lactobacillus MG360 and the lactobacillus rhamnosus Eupro, the compound probiotic composition can be used for fermenting the coix seed enzymatic hydrolysate, and a fermentation product of the compound probiotic composition is rich in ferulic acid, vanillic acid, short-chain fatty acid, glutamic acid and the like. The fermentation product can obviously shorten the first black excrement discharge time of constipation mice, obviously improve the small intestine propulsion rate and the excrement water content, obviously improve the level of motility hormone (MTL, Gas, AchE, SP and 5-HT) in serum of the constipation mice and obviously reduce the level of inhibitory hormone (SS and VIP), and it is indicated that the fermentation product can relieve constipation by adjusting the gastrointestinal hormone peptide level. The preparation method of the probiotic preparation containing the plant lactobacillus MG360 and the compound probiotic fermentation preparation of the compound probiotic composition is simple, large-scale production can be achieved, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a composite probiotic fermentation preparation having the effect of improving constipation and an application thereof. Background Art

[0002] Constipation is a common gastrointestinal disorder characterized by infrequent and painful bowel movements, accompanied by abdominal pain and bloating. The prevalence of constipation increases with age, reaching 23.0% in people over 70 years old and 38.0% in those over 80 years old.

[0003] Constipation occurs due to slowed intestinal motility, so the goal of treating constipation is to restore the body's normal intestinal motility and normalize bowel movements. Currently, the treatment of constipation is mainly based on medication, which can be roughly divided into (1) DA receptor antagonists (domperidone, metoclopramide), (2) 5-hydroxytryptamine receptor agonists (mosapride), (3) motilin receptor agonists (erythromycin) and (4) laxatives. Laxatives can quickly relieve constipation symptoms and are therefore widely chosen by constipation patients. However, long-term use of medications can easily lead to drug dependence. In addition, medication has many disadvantages, such as long-term use of stimulant laxatives can lead to colon melanosis, lubricating laxatives are inconvenient to use, and bulk laxatives can easily cause flatulence. Probiotic treatment has the characteristics of low side effects, effectiveness and multiple benefits to the body, and has become a new treatment concept in constipation treatment. Clinical studies have shown that there is severe bacterial imbalance in the intestines of constipated patients. By regulating the local microecological environment through the administration of probiotics, the purpose of relieving constipation can be achieved. In addition to regulating intestinal flora, probiotics may also improve constipation by regulating certain metabolites, bile acids, and the immune system in the intestine, acting through multiple targets.

[0004] Coix seed is the dried, mature kernel of the grass plant Coix lachryma-jobi. In 2002, it was listed as a food and medicine. It is primarily distributed in most of China, including Liaoning, Hebei, Henan, and Shaanxi, and is a prime example of a food and medicine that combines both medicinal and medicinal properties. It is rich in dietary fiber, polysaccharides, protein, and various vitamins and minerals. Due to its unique benefits, coix seed is widely used to relieve constipation. Its dietary fiber absorbs water in the intestines, softening and increasing stool volume while stimulating intestinal peristalsis and accelerating stool excretion. Its rich polysaccharide content helps regulate intestinal flora, optimize the intestinal environment, and enhance intestinal function, thereby alleviating constipation symptoms. For example, patents CN119564800A and CN118787713A describe a traditional Chinese medicine composition for treating constipation, both of which include coix seed as the primary active ingredient.

[0005] As the research value of synergistic effects between edible and medicinal substances and probiotics becomes increasingly prominent, researchers are gradually shifting their focus to innovative combinations of the two. Using probiotic fermentation technology to biotransform edible and medicinal matrices not only significantly improves the bioavailability of their active ingredients but also promotes the breakdown of macromolecular nutrients, converting them into smaller molecules that are more easily absorbed by the body. This technological integration offers diverse application prospects in the food industry and can be widely applied to the development of innovative products such as functional beverages and fermented dairy products, imbuing traditional foods with novel health properties that enhance bodily functions. Currently, there are few reports on combining probiotics with edible and medicinal substances and other ingredients to alleviate constipation. Patent CN119522977A prepares a health-care yogurt that helps alleviate constipation. It uses edible and medicinal substances with constipation-relieving properties, such as hemp seed, and adds bifidobacteria for complex fermentation. However, the patent focuses solely on evaluating the product's sensory and experiential effects, ignoring its specific mechanism of action in alleviating constipation. Summary of the Invention

[0006] The first object of the present invention is to provide a Lactobacillus plantarum MG360 that effectively improves constipation.

[0007] The second object of the present invention is to provide a probiotic freeze-dried powder.

[0008] The third object of the present invention is to provide a composite probiotic composition that effectively improves constipation.

[0009] The fourth object of the present invention is to provide a fermentation concentrate obtained by fermenting coix seed hydrolysate with a composite probiotic composition.

[0010] The fifth object of the present invention is to provide a method for preparing the composite probiotic fermentation concentrate.

[0011] The sixth object of the present invention is to provide an application of the composite probiotic fermentation concentrate.

[0012] The present invention is achieved through the following technical solutions:

[0013] A strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) MG360, with accession number CGMCCNO 33527. This strain was deposited with the General Microbiology Center of the China Culture Collection Administration on February 14, 2025. The depository code is CGMCC. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The depository number is CGMCCNO 33527. The strain was alive at the time of deposit.

[0014] A probiotic freeze-dried powder comprises Lactobacillus plantarum MG360, Lactobacillus rhamnosus Eupro and a freeze-drying protective agent.

[0015] Specifically, Lactobacillus rhamnosus Eupro was deposited in the General Microbiology Center of the China Culture Collection Administration on January 18, 2024, with the deposit number CGMCCNO.29667, see the Chinese patent publication number CN118516250A.

[0016] The freeze-drying protective agent comprises the following components: 8-10% skim milk powder, 10-12% trehalose, 1-2% sodium glutamate, 0.5-1.5% ascorbic acid, 0.5-1.5% glycerol, 0.1M potassium dihydrogen phosphate and the balance water.

[0017] A composite probiotic composition,

[0018] The powder comprises the bacterial powder of Lactobacillus plantarum MG360 and the bacterial powder of Lactobacillus rhamnosus Eupro;

[0019] The mass ratio of the bacterial powder of Lactobacillus plantarum MG360 to the bacterial powder of Lactobacillus rhamnosus Eupro is 1:1;

[0020] The content of bacteria in the powder of Lactobacillus plantarum MG360 is 1.5×10 12 CFU / g;

[0021] The content of the bacteria in the powder of Lactobacillus rhamnosus Eupro is 1.1×10 12 CFU / g.

[0022] A compound probiotic fermentation concentrate,

[0023] The product is obtained by fermenting coix seed enzymatic hydrolysate with the composite probiotic composition as claimed in claim 4.

[0024] The preparation method of the coix seed enzymatic hydrolyzate comprises the following steps:

[0025] The coix seeds are mixed with water, and then enzymatically hydrolyzed with α-amylase, cellulase and saccharifying enzyme, sterilized at high temperature, and then chitosan solution and clarifier are added, and filtered to obtain the product.

[0026] The material-liquid ratio of coix seed to water is 1g:10mL to 1g:15mL;

[0027] The added amount of the α-amylase is 0.5-0.9 wt%;

[0028] The amount of cellulase added is 1.0-1.2 wt%;

[0029] The added amount of the saccharifying enzyme is 1.0-1.5 wt%;

[0030] The temperature of the enzymatic hydrolysis is 45-60°C;

[0031] The temperature of the high temperature sterilization is 85°C;

[0032] The clarifier includes ZTC natural clarifier;

[0033] The amount of the clarifier added is 0.1%;

[0034] The added amount of the chitosan is 0.05%.

[0035] The preparation method of the composite probiotic fermentation concentrate,

[0036] The steps include:

[0037] S1 adaptively evolving the probiotics in the composite probiotic composition according to claim 5;

[0038] S2 inoculates the adaptively evolved composite probiotics into a nutrient medium including coix seed enzymatic hydrolysate for fermentation to obtain a composite probiotic fermentation liquid.

[0039] 9. The method for preparing the composite probiotic fermentation concentrate according to claim 8, wherein:

[0040] The adaptive evolution in S1 includes the steps of sequentially inoculating the composite probiotics into nutrient media containing coix seed hydrolysate at concentrations of 1%, 2%, 5%, 10%, 20%, and 50% for fermentation;

[0041] In S1, during the adaptive evolution, the total viable count of probiotics was 1.3×10 12 CFU / g;

[0042] In S1, during the adaptive evolution, the concentration of the bacteria inoculated into the coix seed enzymatic hydrolyzate nutrient medium is 1 to 8×10 11 CFU / mL;

[0043] In S1, during the adaptive evolution, the inoculation ratio of probiotics is 2% to 5%;

[0044] In S2, the ratio of coix seed enzymatic hydrolysate to water in the nutrient medium of coix seed enzymatic hydrolysate is 1:50

[0045] In S2, the bacterial strain concentration in the nutrient medium of coix seed hydrolysate was 2×10 11 CFU / mL;

[0046] In S2, the inoculation ratio of the bacterial strain in the nutrient medium of coix seed enzymatic hydrolysate was 3%;

[0047] The nutrient medium comprises, by weight percentage, 1% sucrose, 2% soy peptone, 1% yeast extract, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.02% manganese sulfate, 0.2% Tween 80, 0.5% sodium acetate, and the balance water.

[0048] The application of the composite probiotic composition,

[0049] The invention can be used to prepare food or medicine for increasing the levels of MTL, AchE, SP, Gas and 5-HT hormones and decreasing the levels of SS and VIP hormones.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The Lactobacillus plantarum MG360 provided by the present invention has strong prebiotic functions, including gastric juice and intestinal juice tolerance, adhesion and antibacterial ability, and also has strong BglA gene and Pal gene expression capabilities.

[0052] The Lactobacillus plantarum MG360, Lactobacillus rhamnosus Eupro and their composite probiotic powder provided by the present invention are all helpful in improving constipation, and the composite probiotic powder is more effective in improving constipation than single bacterial powder.

[0053] The composite probiotic composition provided by the present invention can effectively ferment coix seed, and more polyphenols such as vanillic acid are produced during the fermentation process. Vanillic acid can activate intestinal chromaffin cells to secrete 5-HT, accelerate colon propulsion, and is closely related to improving constipation; a certain amount of short-chain fatty acids are also produced, among which butyric acid can promote mucus secretion and intestinal epithelial cell proliferation, thereby relieving constipation and strengthening the intestinal barrier; and the content of glutamate is increased by 15.38%. Glutamate, as a neurotransmitter precursor, can directly stimulate intestinal ganglion cells, increase peristalsis frequency, and thus relieve constipation. After verification by a mouse model, it was found that the composite probiotic fermentation concentrate can significantly increase the water content of feces in constipated mice, significantly shorten the time to discharge the first black stool, and significantly increase the small intestinal propulsion rate, thereby helping to relieve constipation.

[0054] The preparation method of the composite probiotic fermentation concentrated liquid provided by the invention is simple and can be produced on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The relative expression levels of the BglA gene and the Pal gene before and after fermentation of Lactobacillus plantarum MG360 are shown.

[0056] Figure 2 The colonies and microscopic examination images of Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro are shown.

[0057] Figure 3Shown are the growth curves of Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro. DETAILED DESCRIPTION

[0058] The present invention will be described in more detail below with reference to Examples. However, these Examples are only for illustrative purposes only and the scope of the present invention is not limited to these Examples.

[0059] The present invention first selects to screen probiotics from traditional food fermentation broth. The specific steps are as follows:

[0060] (1) Strain isolation

[0061] Take 10 mL of traditional food fermentation broth, perform a gradient dilution, spread it onto MRS medium, and place it upside down in an anaerobic incubator at 37°C for 48 hours. Once colonies grow on the plate, select individual colonies of varying color, size, and shape and purify them until a single strain is obtained. Perform Gram staining, retaining Gram-positive bacteria for strain screening.

[0062] (2) Strain screening

[0063] The retained Gram-positive bacteria were streaked on an MRS plate containing 0.8% calcium carbonate for activation, and slightly larger bacteria with a calcium-dissolving zone were picked out.

[0064] (3) Nine strains of bacteria with different morphologies were selected and named MG360, MG362, MG368, PC01, PC05, BC03, BC05, BC19, and BC21 for the next step of artificial gastric juice and intestinal juice tolerance experiments.

[0065] (4) Artificial gastric juice tolerance test

[0066] The selected bacteria were activated. The treated MG360, MG362, MG368, PC01, PC05, BC03, BC05, BC19, and BC21 bacterial cultures were inoculated at a ratio of 1:10.0 into artificial gastric fluid (pH 3.0). The cells were incubated at 37°C for 3 hours. The viable bacterial count and survival rate were then determined. The results are shown in Table 1.

[0067] (5) Artificial intestinal fluid tolerance test

[0068] After 3 hours of treatment in artificial gastric fluid, the bacterial suspension was inoculated into artificial intestinal fluid (pH 6.8, containing 0.2% bile salts) at a ratio of 1:10.0 and placed in a 37°C incubator for 3 hours. The viable bacterial count and survival rate were determined. The results are shown in Table 1.

[0069] Table 1:

[0070]

[0071] As shown in Table 1, strains MG360, PC05, BC019, BC03, MG368, and BC21 have strong tolerance to artificial gastric juice and artificial intestinal juice, indicating that these strains have strong survival ability in the digestive system and have certain potential to reach the intestine and act, and can be used for the next screening experiment.

[0072] (6) Self-aggregation and hydrophobicity experiments

[0073] After the highly tolerant strains MG360, PC05, BC019, BC03, MG368, and BC21 were activated, their self-aggregation and hydrophobicity were measured. The specific results are shown in Table 2.

[0074] Table 2:

[0075]

[0076]

[0077] As shown in Table 2, strains MG360, PC05, and MG368 have strong self-aggregation and hydrophobicity, which means that these strains have strong colonization ability in the intestine and can better exert their probiotic effects.

[0078] Antibacterial experiment

[0079] After activation, the highly self-aggregating and hydrophobic strains MG360, PC05, and MG368 were tested for their antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella, and Shigella flexneri. The results are shown in Table 3.

[0080] Table 3:

[0081]

[0082] As shown in Table 3, strain MG360 has the best antibacterial ability and has strong antibacterial ability against common pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella and Shigella flexneri. This shows that MG360 can regulate the balance of intestinal flora and inhibit pathogen infection, making it the preferred strain.

[0083] (8) Molecular biological identification of strain MG360

[0084] A single colony of strain MG360 was placed in Lysis Buffer for Microorganism to Direct PCR and lysed in an 80°C water bath for 30 minutes to obtain a bacterial DNA template. The universal primers for bacterial 16S rRNA gene amplification, 27F: 5'-CAGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3', were used. The reaction system (25 μL) consisted of 1 μL each of the universal primers 27F and 1492R, 0.5 μL of bacterial DNA template, and 12.5 μL of 2× RapidTaq Master Mix. The total volume was made up to 25 μL with ddH2O.

[0085] The PCR reaction procedure was as follows: initial denaturation at 95°C for 3 min; 30 cycles (denaturation at 94°C for 25 s, annealing at 55°C for 25 s, and extension at 72°C for 25 s), followed by a 5-min hold at 72°C for complete extension; and storage at 4°C. After completion of PCR, the reaction mixture was electrophoresed on a 1.5% agarose gel in 1× TAE (50 mmol / L Tris-acetic acid, 1 mmol / L EDTA, pH 8.0) buffer.

[0086] (9) MG360 was sequenced and the obtained sequencing results were compared and identified as belonging to Lactobacillus plantarum.

[0087] The gene sequence of the bacterium is shown below:

[0088] GGGGCTGGCGGACATCTAGATTGATAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTG

[0089] AGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCG

[0090] CATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGC

[0091] TAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGA

[0092] CACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGT

[0093] GAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACG

[0094] GTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTA

[0095] TTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAA

[0096] ACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACAC

[0097] CAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCT

[0098] GGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCAT

[0099] TCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG

[0100] TTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGG

[0101] GACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC

[0102] CTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0103] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAG

[0104] TAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATC

[0105] GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCC

[0106] AAAGTCGGTGGGGTAACCTAATAGGAACCAGCCTACCTCATAGAGATCCGCCC

[0107] (10) In order to further explore the potential biological functions of Lactobacillus plantarum MG360, the present invention enriched and extracted the mRNA of Lactobacillus plantarum MG360 and combined it with transcriptome sequencing, and found that the β-glucosidase gene (BglA) and the phenylalanine ammonia lyase gene (Pal) in Lactobacillus plantarum MG360 were actively expressed. Among them, the β-glucosidase encoded by the BglA gene can specifically recognize and cut the β-glycosidic bonds in polysaccharide molecules, decomposing complex polysaccharides into monosaccharides or oligosaccharides; and the phenylalanine ammonia lyase expressed by the Pal gene is a key rate-limiting enzyme in the secondary metabolic pathway of plants. It can catalyze the deamination of phenylalanine to produce cinnamic acid, and then participate in the synthesis of polyphenols, giving the fermentation products richer biological activity. To verify the results of transcriptome sequencing, this study further used real-time fluorescence quantitative PCR (qPCR) technology to quantitatively analyze the expression dynamics of BglA and Pal genes during fermentation. The results are as follows Figure 1As shown in the data, the relative expression levels of BglA and Pal genes were significantly upregulated, indicating that Lactobacillus plantarum MG360 can activate the expression of related genes during the fermentation process, achieve effective degradation of polysaccharides and synthesis of polyphenols, thereby fully releasing the functional components in the raw materials. Therefore, it can be used in the field of functional fermented foods.

[0108] Table 4 qPCR analysis primers

[0109] Gene Upstream primer (5'-3') Downstream primer (5'-3') BglA CACTGCCAACTTTCCCTA TTCATTGCACAATAAGGAA Pal AATGGCCAACGGAGAGAGAG TGGGGACTTTACTTTCTCACCA

[0110] Since the Lactobacillus rhamnosus Eupro provided in CN118516250A has the function of promoting the synthesis of 5-hydroxytryptamine (5-HT), the combination of Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro may have a better effect in alleviating constipation.

[0111] The preferred Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro were subjected to a high-density fermentation experiment to obtain probiotic powder. The specific steps are as follows:

[0112] (1) Strain activation

[0113] Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro were streaked and cultured for 48 h, and single colonies on the plates were picked for microscopic examination to observe the morphology of the strains, such as Figure 2 Then, single colonies were picked and inoculated into MRS liquid culture medium, cultured at 37℃ for 24h, and set aside.

[0114] (2) Probiotic growth curve determination

[0115] Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro were inoculated into MRS liquid medium at a 2% inoculum volume, cultured for 24 h, and the OD value of the bacterial solution was measured every 2 h to draw a growth curve. Figure 3 shown.

[0116] (3) Probiotic fermentation culture

[0117] Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro were inoculated into 5LMRS liquid culture medium at a 2% inoculum volume, cultured at 37° C., and centrifuged and freeze-dried after fermentation to the stable phase according to the growth curve of the strains.

[0118] (4) Probiotics centrifugal freeze-drying

[0119] The fermented broth was centrifuged at 6000 rpm and 4 ° C for 10 minutes to obtain the probiotic slurry. A freeze-drying protectant was added at a ratio of 1:1, mixed thoroughly, transferred to -80 ° C for pre-freezing for 2 hours, and then vacuum-freezed and freeze-dried to calculate the freeze-drying survival rate. The freeze-drying protectant composition is: skim milk powder 8-10%, trehalose 10-12%, sodium glutamate 1-2%, ascorbic acid 0.5-1.5%, glycerol 0.5-1.5%, potassium dihydrogen phosphate 0.1M (pH 6.8), and distilled water as the balance. The specific results are shown in Table 5.

[0120] Table 5:

[0121]

[0122] Experiment on the combination of Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro

[0123] (1) Antagonism experiment

[0124] The bacterial suspensions of Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro were adjusted to a concentration of 1.0 × 10 8 CFU / mL, and then spread on MRS plates at a concentration of 1.0×10 8 CFU / mL of Lactobacillus plantarum MG360 solution was added, and after standing for 30 min, the sterile Oxford cup was gently placed on the plate with sterile tweezers, and an equal amount of 1.0×10 8 CFU / mL of Lactobacillus rhamnosus Eupro culture was cultured for 48 hours and the presence of inhibition zone was observed.

[0125] The results showed that no inhibition zone appeared on the plate, indicating that there was no antagonism between Lactobacillus plantarum MG360 and Lactobacillus rhamnosus Eupro, and they could be combined.

[0126] (2) Preparation of composite probiotic composition

[0127] Lactobacillus plantarum MG360 powder (1.5×10 12 CFU / g and Lactobacillus rhamnosus Eupro powder (1.1×10 12 CFU / g) were mixed evenly at a mass ratio of 1:1 to prepare composite probiotic powder (1.3×10 12 CFU / g).

[0128] This study used a zebrafish model to further validate the constipation-relieving effects of the probiotic powder. Zebrafish share 87% genetic similarity with humans, and their intestines are also very similar to humans. The specific steps are as follows:

[0129] (1) Experimental design

[0130] The zebrafish were divided into six groups: blank control group, model control group, positive control group, Lactobacillus plantarum MG360 treatment group (1.5×10 8 CFU / mL), Lactobacillus rhamnosus Eupro-treated group (1.1×10 8 CFU / mL and the compound probiotic powder treatment group (1.3×10 8 CFU / mL). The blank control group received no treatment. The other five groups were treated with aluminum sulfate as a model. Domperidone served as a positive control. The sample-treated group was immersed in the sample solution for a period of time. The intestines of the zebrafish in different groups were fluorescently stained and observed and photographed using a fluorescence microscope. The images were analyzed with software, and the data analysis results of the six groups were compared.

[0131] (2) Data Analysis

[0132] The results of intestinal fluorescence intensity data analysis are shown in Table 6.

[0133] Table 6:

[0134]

[0135] As shown in Table 6, compared with the model control group, the intestinal fluorescence intensity of the Lactobacillus rhamnosus Eupro treatment group, the Lactobacillus plantarum MG360 treatment group, and the composite probiotic powder treatment group was significantly reduced, indicating that Lactobacillus rhamnosus Eupro, Lactobacillus plantarum MG360, and their composite probiotic powder have a certain effect on promoting intestinal motility and improving constipation in constipated mice, among which the composite probiotic powder treatment has the best effect. Compared with the positive control group, the fluorescence intensity of the Lactobacillus rhamnosus Eupro treatment group and the composite probiotic powder treatment group was significantly reduced, indicating that Lactobacillus rhamnosus Eupro and the composite probiotic powder are better than the positive control (domperidone) in promoting intestinal motility and improving constipation; while the intestinal fluorescence intensity of the Lactobacillus plantarum MG360 treatment group was not significantly different from that of the positive control group, indicating that the effect of Lactobacillus plantarum MG360 in promoting intestinal motility and improving constipation is similar to that of the positive control (domperidone). This shows that Lactobacillus plantarum MG360, Lactobacillus rhamnosus Eupro, and the composite probiotic powder can improve constipation with low toxic and side effects.

[0136] Furthermore, in order to obtain more effective ingredients, the present invention prepares a composite probiotic fermentation concentrate. The specific steps are as follows:

[0137] (1) Preparation of coix seed enzymatic hydrolysate

[0138] Select mold-free, plump coix seeds, remove impurities, and clean thoroughly. Soak in clean water for 4-6 hours, drain, and dry at 60-70°C until the moisture content is ≤8%. Grind through a 60-80 mesh sieve. Add deionized water at a material-to-liquid ratio of 1:10-1:15 (g / mL). Gelatinize at 95°C for 1.5 hours. Cool and adjust the pH to 6.5. Add 0.5-0.9% α-amylase. Hydrolyze at 50-60°C for 45 minutes, then adjust the pH to 5.0. Add 1.0-1.2% cellulase, hydrolyze at 45-50°C for 1.5 hours, adjust the pH to 4.5, and finally add 1.0-1.5% saccharifying enzyme. Hydrolyze at 60°C for 2 hours. Autoclave at 85°C for 10 minutes to inactivate the enzymes, then rapidly cool. Chitosan solution (final concentration 0.05%) was then added. Chitosan can remove some impurities in the hydrolyzate through adsorption and complexation, acting as a pre-clarifier. The mixture was then stirred for 30 minutes, allowed to stand for 1 hour, and finally, 0.1% ZTC natural clarifier (added stepwise at an A:B ratio of 1:2) was added. After standing for 2 hours, the mixture was centrifuged (4000 rpm for 20 minutes), filtered, and then filtered through a 0.45 μm microporous membrane to obtain a clarified hydrolyzate.

[0139] (2) Adaptive evolution of compound probiotics

[0140] First, the composite probiotics (Lactobacillus plantarum MG360: Lactobacillus rhamnosus Eupro mass ratio of 1:1, total viable bacteria count of 1.3×10 12 CFU / g) were inoculated into MSR culture medium for activation, and then the composite probiotic solution (total viable bacteria count of 1-8×10 11 CFU / g) is inoculated in the nutrient base that contains coix seed enzymolyte concentration and is 1% and ferments, inoculation ratio is 2%~5%, and regulating fermentation pH is about 6.0, and temperature is 36.9 ℃, and normal pressure fermentation is 24h.After fermentation finishes, by fermentation liquid enrichment in MRS culture medium, enrichment is inoculated in the nutrient base that contains coix seed enzymolyte concentration and is 2% and ferments again after 24h, according to the above method, progressively improving coix seed enzymolyte concentration is 5%, 10%, 20%, and finally inoculated in the nutrient base that contains coix seed enzymolyte concentration and is 50%, strengthens the adaptability of bacterial strain.Nutrient base is 1% sucrose, 2% soy peptone, 1% yeast extract, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.02% manganese sulfate, 0.2% Tween 80, 0.5% sodium acetate.

[0141] (3) Preparation of compound probiotic fermentation concentrate

[0142] The adaptively evolved composite probiotics were inoculated into a nutrient medium containing 2% coix seed hydrolysate for fermentation. The strain concentration was 2×10 11CFU / mL, with an inoculation ratio of 3%, the fermentation pH was adjusted to approximately 6.0, the temperature was 36.9°C, and fermentation was carried out at normal pressure for 24 hours. After the fermentation, rotary evaporation and concentration were performed to obtain a composite probiotic fermentation broth. The nutrient medium consisted of 1% sucrose, 2% soy peptone, 1% yeast extract, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.02% manganese sulfate, 0.2% Tween 80, and 0.5% sodium acetate.

[0143] The active ingredients of the raw materials before and after fermentation were analyzed, and the specific results are shown in Table 7.

[0144] Raw materials before fermentation: coix seed enzymatic hydrolysate;

[0145] Fermented raw materials: composite probiotic fermentation liquid obtained by fermenting coix seed hydrolysate with composite probiotics.

[0146] Table 7:

[0147] project Before fermentation After fermentation Range of change Total polysaccharides 1.67 mg / mL 1.36 mg / mL ↓18.56％ Total polyphenols 0.09mgGAE / mL 0.16mgGAE / mL ↑77.78％ Ferulic acid 0.02mg / mL 0.03mg / mL ↑50.00％ Vanillic acid Not detected 0.03mg / mL ↑100.00％ Total short-chain fatty acids Not detected 7.00mmoL / L ↑100.00％ Butyric acid Not detected 2.70mmoL / L ↑100.00％ Acetic acid Not detected 2.50mmoL / L ↑100.00％ Propionic acid Not detected 1.60mmoL / L ↑100.00％ Total free amino acids 0.53 mg / mL 0.65 mg / mL ↑22.64％ glutamate 0.13 mg / mL 0.15mg / mL ↑15.38％ Alanine 0.08 mg / mL 0.11 mg / mL ↑37.50％

[0148] As shown in Table 7, after fermentation, only vanillic acid was detected in the fermentation broth. Related studies have shown that vanillic acid can activate intestinal chromaffin cells to secrete 5-HT, accelerating colonic propulsion and being closely related to improving constipation. Total polysaccharide content decreased by 18.56% after fermentation, indicating that some polysaccharides were degraded by probiotics, generating low-molecular-weight fragments that are more easily digested and absorbed by the intestine. After fermentation, the fermentation broth also contained a certain amount of short-chain fatty acids, among which butyric acid can promote mucus secretion and intestinal epithelial cell proliferation, thereby alleviating constipation and strengthening the intestinal barrier. Glutamate content increased by 15.38% after fermentation. As a neurotransmitter precursor, glutamate can directly stimulate intestinal ganglion cells, increase peristalsis frequency, and thus alleviate constipation. This indicates that fermentation of coix seed hydrolysate with compound probiotics produces more active ingredients, thereby helping to alleviate constipation.

[0149] Finally, the present invention evaluated the effects of compound probiotics, coix seed hydrolysate and compound probiotic fermentation liquid on improving constipation using a constipation mouse model.

[0150] (1) Experimental design

[0151] Fifty 6-week-old SPF-grade C57BL / 6J male mice were selected and fed for one week at room temperature (25±2℃) with 12h light / 12h dark environment. They were then randomly divided into normal control group, model group, compound probiotic powder treatment group, coix seed enzymatic hydrolysate treatment group, and compound probiotic fermentation liquid treatment group. The compound probiotic powder treatment group was gavaged with 200μL compound probiotic aqueous solution (1×10 9CFU), the coix seed enzymatic hydrolysate treatment group was gavaged with 200 μL coix seed enzymatic hydrolysate every day, the compound probiotic fermentation liquid treatment group was gavaged with 200 μL compound probiotic fermentation liquid every day, and the other treatment groups were replaced with 200 μL sterile pure water every day for 6 days. Starting on day 7, mice were induced to constipate. Except for the normal control group, which was gavaged with sterile water, all groups received 0.25 mL of loperamide hydrochloride suspension (1 mg / mL) orally once daily for three consecutive days. After the induction period ended on day 9, the mice were fasted for 18 hours. All mice were then gavaged with 0.1 mL / 10 g (b / w) of ice water containing 10% activated carbon. Each treatment group was then divided equally into two groups. The time of first black stool excretion was recorded, and mice were sacrificed 25 minutes after gavage. Serum was collected and assayed for changes in constipation-related gastrointestinal hormone peptides. The mice were then dissected, and the small intestine from the pylorus to the cecum was removed. The total intestinal length and the distance the activated carbon advanced in the small intestine were measured, and the intestinal propulsion rate was calculated. Mice had free access to water and food throughout the experiment. Feces were collected daily, and the fecal moisture content was calculated.

[0152] (2) Result analysis

[0153] The time to the first black stool, small intestinal propulsion rate, and fecal water content are the most intuitive indicators for evaluating constipation in mice. The results are shown in Table 8.

[0154] Table 8:

[0155]

[0156] As shown in Table 8, after treatment with loperamide hydrochloride, the fecal water content of the model group mice was significantly reduced, the time to first black stool was significantly prolonged, and the small intestinal propulsion rate was significantly slowed compared to the control group, indicating that loperamide hydrochloride has successfully established a constipation mouse model. Compared with the model group, treatment with compound probiotic powder, coix seed enzymatic hydrolyzate, and compound probiotic fermentation liquid increased fecal water content, significantly shortened the time to first black stool, and increased small intestinal propulsion rate. This indicates that all three treatment groups can improve constipation in mice, with the compound probiotic fermentation liquid treatment group showing the greatest effect.

[0157] Compared with the control group, the levels of prokinetic hormones (MTL, Gas, AchE, SP, 5-HT) in constipated mice were reduced, while the levels of inhibitory hormones (SS, VIP) were increased. The constipation group had the lowest levels of prokinetic hormones and the highest levels of inhibitory hormones. After treatment with compound probiotic powder, coix seed hydrolysate, and compound probiotic fermentation concentrate, the gastrointestinal hormone levels of mice in each treatment group tended to return to normal levels. Among them, treatment with compound probiotic powder significantly increased the levels of MTL, Gas, AchE, and 5-HT hormones, and significantly decreased the levels of SS and VIP hormones; treatment with coix seed hydrolysate significantly increased the levels of MTL and SP hormones in constipated mice, and significantly decreased the level of VIP hormones, thereby improving constipation; and treatment with compound probiotic fermentation concentrate had the best effect, showing a certain improvement effect on the levels of all hormones.

[0158] In summary, compound probiotic powder, coix seed hydrolysate, and compound probiotic fermentation concentrate all significantly increased fecal moisture content and small intestinal propulsion rate in constipated mice, significantly shortened the time to first black stool excretion, and effectively improved constipation symptoms. However, their effects and mechanisms differed. Compound probiotic powder improved constipation by regulating levels of gastrointestinal hormones MTL, Gas, AchE, 5-HT, SS, and VIP; coix seed hydrolysate improved constipation by restoring levels of MTL, SP, and VIP. Overall, compound probiotic fermentation concentrate had the best effect on improving constipation.

Claims

1. A Lactobacillus plantarum MG360, characterized in that: Its accession number is CGMCC NO 32953.

2. A probiotic freeze-dried powder, characterized in that: The invention comprises the plant lactobacillus MG360, rhamnosus lactobacillus Eupro and a freeze-drying protective agent.

3. The probiotic freeze-dried powder according to claim 2, wherein: The freeze-drying protective agent comprises the following components: 8-10% skim milk powder, 10-12% trehalose, 1-2% sodium glutamate, 0.5-1.5% ascorbic acid, 0.5-1.5% glycerol, 0.1M potassium dihydrogen phosphate and the balance water.

4. A composite probiotic composition, characterized in that: The method comprises the bacterial powder of Lactobacillus plantarum MG360 and the bacterial powder of Lactobacillus rhamnosus Eupro as claimed in claim 1; The mass ratio of the bacterial powder of Lactobacillus plantarum MG360 to the bacterial powder of Lactobacillus rhamnosus Eupro is 1:1; The content of bacteria in the powder of Lactobacillus plantarum MG360 is 1.5×10 12 CFU / g; The content of the bacteria in the powder of Lactobacillus rhamnosus Eupro is 1.1×10 12 CFU / g.

5. A composite probiotic fermentation concentrate, characterized by: The product is obtained by fermenting coix seed enzymatic hydrolysate with the composite probiotic composition as claimed in claim 4.

6. The composite probiotic fermentation concentrate according to claim 5, wherein: The preparation method of the coix seed enzymatic hydrolyzate comprises the following steps: The coix seeds are mixed with water, and then enzymatically hydrolyzed with α-amylase, cellulase and saccharifying enzyme, sterilized at high temperature, and then chitosan solution and clarifier are added, and filtered to obtain the product.

7. The composite probiotic fermentation concentrate according to claim 6, wherein: The material-liquid ratio of coix seed to water is 1g:10mL to 1g:15mL; The added amount of the α-amylase is 0.5 to 0.9 wt%; The amount of cellulase added is 1.0-1.2 wt%; The added amount of the saccharifying enzyme is 1.0-1.5 wt%; The temperature of the enzymatic hydrolysis is 45-60°C; The temperature of the high temperature sterilization is 85°C; The clarifier includes ZTC natural clarifier; The amount of the clarifier added is 0.1%; The added amount of the chitosan is 0.05%.

8. The method for preparing the composite probiotic fermentation concentrate according to claim 7, wherein: The steps include: S1 adaptively evolving the probiotics in the composite probiotic composition according to claim 5; S2 inoculates the adaptively evolved composite probiotics into a nutrient medium including coix seed enzymatic hydrolysate for fermentation to obtain a composite probiotic fermentation liquid.

9. The method for preparing the composite probiotic fermentation concentrate according to claim 8, wherein: The adaptive evolution in S1 includes the steps of sequentially inoculating the composite probiotics into nutrient media containing coix seed hydrolysate at concentrations of 1%, 2%, 5%, 10%, 20%, and 50% for fermentation; In S1, during the adaptive evolution, the total viable count of probiotics was 1.3×10 12 CFU / g; In S1, during the adaptive evolution, the concentration of the bacteria inoculated into the coix seed enzymatic hydrolyzate nutrient medium is 1 to 8×10 11 CFU / mL; In S1, during the adaptive evolution, the inoculation ratio of probiotics is 2% to 5%; In S2, the ratio of coix seed enzymatic hydrolyzate to water in the nutrient medium of coix seed enzymatic hydrolyzate is 1:50; In S2, the bacterial strain concentration in the nutrient medium of coix seed hydrolysate was 2×10 11 CFU / mL; In S2, the inoculation ratio of the bacterial strain in the nutrient medium of coix seed enzymatic hydrolysate was 3%; The nutrient medium comprises, by weight percentage, 1% sucrose, 2% soy peptone, 1% yeast extract, 0.05% magnesium sulfate, 0.1% dipotassium hydrogen phosphate, 0.02% manganese sulfate, 0.2% Tween 80, 0.5% sodium acetate, and the balance water.

10. The use of the composite probiotic composition according to claim 4, characterized in that: The invention can be used to prepare food or medicine for increasing the levels of MTL, AchE, SP, Gas and 5-HT hormones and decreasing the levels of SS and VIP hormones.

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