CN118516280B

CN118516280B - Lactobacillus johnsonii XY9 and application thereof in preparation of hypoglycemic and anti-aging medicines

Info

Publication number: CN118516280B
Application number: CN202410834405.9A
Authority: CN
Inventors: 许敏青; 陆勇军; 葛振煌
Original assignee: Guangdong Yuechuang Biotechnology Co ltd
Current assignee: Guangdong Yuechuang Biotechnology Co ltd
Priority date: 2024-06-26
Filing date: 2024-06-26
Publication date: 2024-12-17
Anticipated expiration: 2044-06-26
Also published as: CN118516280A

Abstract

The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus johnsonii XY9 and application thereof in preparation of hypoglycemic and anti-aging foods and medicines. The invention separates and purifies a strain of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 from a fecal sample of a healthy adult in Guangdong area of China, and the strain has excellent protease activity, can generate and secrete gamma-aminobutyric acid, can inhibit alpha-glucosidase activity, can remove DPPH free radical, and has excellent cellulase activity. Therefore, the strain has the functions of promoting digestion and absorption of protein food, improving protein allergy, resisting depression and sleep, reducing blood sugar and weight, resisting oxidation and whitening, delaying aging and inflammation, promoting absorption of cellulose, improving constipation and the like, can be used in the fields of reducing blood sugar and resisting aging and has important application value and economic value.

Description

Lactobacillus johnsonii XY9 and application thereof in preparation of hypoglycemic and anti-aging medicines

Technical Field

The invention belongs to the technical field of probiotics and application thereof, and particularly relates to lactobacillus johnsonii XY9 and application thereof in preparation of hypoglycemic and anti-aging medicines.

Background

Lactobacillus johnsonii (Lactobacillus johnsonii) is a gram positive bacillus belonging to the genus Lactobacillus of the family Lactobacillus, the size is generally 0.5-1.2 μm×0.5-1.5 μm, the thallus is in the shape of short rod, two ends are round, and usually appear singly, and few pairs are in short chain. No spore, no capsule and no movement. Lactobacillus johnsonii was first found in the human gland and is a primary species in the human digestive tract. The method has no special requirement on oxygen, is facultative anaerobic, can grow well when the oxygen pressure is reduced, and can grow at 10 ℃ at a temperature of 30-37 ℃ suitable for growth. The bacterium Lactobacillus johnsonii is a normal strain in the human body, and is widely found in the human body because it is detected in almost all the digestive tracts of the human body. In addition, lactobacillus johnsonii has good acid and bile salt resistance, can survive in intestines and stomach of human and animals, and widely exist in intestines and stomach of human and animals.

Lactobacillus johnsonii is one of the producible lactobacillus species that is widely present in the intestinal tract of humans and animals and excreted with faeces, and it is important to maintain the balance of intestinal micro-ecology as an important component of the normal microbial system of the intestinal tract, along with the host for life. Meanwhile, as probiotics in intestinal flora, the lactobacillus johnsonii is also edible lactobacillus, can also play a role in assisting intestinal peristalsis, and can also enhance the digestion capability of human bodies and improve immunity. Thus, lactobacillus johnsonii has become a hot spot in recent research as a probiotic lactobacillus with great potential, and in recent years, lactobacillus johnsonii has been widely used as a probiotic in the production of probiotic products and is being continuously used to make probiotic preparations suitable for human and animals.

According to research reports, different strains of lactobacillus johnsonii have different probiotic functions, namely (1) lactobacillus johnsonii BS15 can be planted in intestinal tracts of human beings, rats, mice, fishes, pigs and chickens, has better effect of improving the muscle quality of broilers, can reduce abdominal fat rate, avoids energy consumption and other (Qing X,Zeng D,Wang H,et al.Preventing subclinical necrotic enteritis through Lactobacillus johnsonii BS15by ameliorating lipid metabolism and intestinal microflorain broiler chickens.AMB Express.2017Dec;7(1):139.doi:10.1186/s13568-017-0439-5.Epub2017Jun26.PMID:28655217;PMCID:PMC5484656.). viable bacteria in production and is a necessary condition for the probiotics to act, and the lactobacillus johnsonii BS15 can simultaneously promote the development and digestion capacity of small intestines, and the probiotics are probably due to the fact that the BS15 strain has an effect of regulating the intestinal microecological structure. (2) Lactobacillus johnsonii GLJO02 is effective in alleviating apparent symptoms in a colitis mouse, increasing cecal SCFAs levels, reducing intestinal barrier permeability, and thereby inhibiting the passage of LPS into the blood, and reducing inflammatory conditions in the body (Wang Yu, huang Yina, caijie, etc. lactobacillus johnsonii GLJO02 relieves DSS-induced colitis in mice [ J ]. Journal of chinese microecology, 2023,35 (03): 284-290.Doi:10.13381/j.cnki.cjm.202303006. (3) Lactobacillus johnsonii can improve intestinal flora, increase the number of intestinal digestive cocci and clostridium, and can also remarkably improve daily gain of piglets and reduce diarrhea (Xin J,Zeng D,Wang H,et al.Probiotic Lactobacillus johnsonii BS15 Promotes Growth Performance,Intestinal Immunity,and Gut Microbiota in Piglets.Probiotics Antimicrob Proteins.2020Mar;12(1):184-193.doi:10.1007/s12602-018-9511-y.PMID:30617949.).(4) of piglets, lactobacillus johnsonii jjjb 3 shows good safety in vivo and in vitro tests, improves the oxidative stress of IPEC-J2 cells induced by H ₂O₂ by promoting down regulation of gene transcription levels of two oxidative stress related metabolic pathways, and has reliable effect on reducing the oxidative stress of intestinal epithelial cells of pigs (Wang Yanyan safety assessment of lactobacillus johnsonii jjjb 3 and antioxidant effect study on IPEC-J2 cells [ D ] university of Sichuan agriculture, 2023.doi: 10.27345/d.611 cnki.gsnyu.2023.000).

Lactobacillus johnsonii has genetic and functional diversity due to its source diversity. However, there are still few studies on the isolation and identification, probiotic properties and metabolic mechanism of lactobacillus johnsonii at present, which affects the development and utilization of lactobacillus johnsonii to some extent. Therefore, it is necessary to excavate more novel strains according to different sources of lactobacillus johnsonii, determine the efficacy of the strains according to the functions or the probiotics metabolites of the strains, and clear the application prospect of the strains so that the strains can better exert the probiotics efficacy. In conclusion, the research and application of the probiotic lactobacillus johnsonii have a very wide development space.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention separates and purifies a lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain from a fecal sample of a healthy adult in Guangdong area of China, and the strain has excellent protease activity and cellulase activity, can generate and secrete gamma-aminobutyric acid, can inhibit alpha-glucosidase activity, can remove DPPH free radicals, and has important potential application value in the fields of reducing blood sugar, resisting aging and the like.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

The invention provides a Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain, wherein the Lactobacillus johnsonii XY9 strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20232013 in the 10 th month of 2023, and the 16S rDNA sequence of the Lactobacillus johnsonii XY9 strain is shown as SEQ ID No. 1.

In a second aspect, the invention provides the use of the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of the first aspect in the production of a protease.

The research shows that the lactobacillus johnsonii XY9 strain of the probiotics can produce protease, which suggests that the lactobacillus johnsonii XY9 strain is hopeful to be used for producing the protease, and the characteristics of the protease are applied to the fields of promoting the digestion and absorption of human bodies to proteins in foods, resisting allergy, helping animals digest and absorb nutrition and the like.

In a third aspect, the invention provides the use of the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of the first aspect for producing cellulase.

The research shows that the lactobacillus johnsonii XY9 strain of the probiotics can produce cellulase, which suggests that the lactobacillus johnsonii XY9 strain can be used for producing the cellulase, and the characteristics of the cellulase can be used for promoting the digestion and absorption of protein in food by human body, improving the absorption of small peptide and amino acid, resisting allergy and other fields.

In a fourth aspect, the invention provides the use of a lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain according to the first aspect for the production of gamma-aminobutyric acid.

The research shows that the lactobacillus johnsonii XY9 strain can produce gamma-aminobutyric acid (GABA), which suggests that the lactobacillus johnsonii XY9 strain can be used for producing GABA and can be used for improving the sleeping quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism and other fields by producing GABA.

In a fifth aspect, the invention provides the use of the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of the first aspect for the preparation of an alpha-glucosidase inhibitor.

Through researches, the lactobacillus johnsonii XY9 strain of probiotics can effectively inhibit the activity of alpha-glucosidase, and the alpha-glucosidase is related to type 2 diabetes, and the inhibition of the alpha-glucosidase is one of methods for controlling postprandial hyperglycemia, which suggests that the lactobacillus johnsonii XY9 strain is expected to be applied to the fields of reducing blood sugar, inhibiting obesity and the like.

In a sixth aspect, the invention provides the use of a lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain according to the first aspect for the preparation of a DPPH radical scavenger.

The research shows that the probiotic Lactobacillus johnsonii XY9 strain can eliminate DPPH free radical to avoid harm to human body caused by the free radical, and can be applied in the fields of resisting aging, preventing cancer, protecting cardiac and cerebral vessels, etc.

In a seventh aspect, the invention provides a probiotic functional bacterial agent comprising the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of the first aspect.

Preferably, the microbial inoculum is a fermented product of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain.

Preferably, in the field of medical application, the microbial inoculum further comprises pharmaceutically acceptable auxiliary materials.

More preferably, the adjuvant comprises a carrier and an excipient. The excipient refers to diluents, binders, lubricants, disintegrants, cosolvents, stabilizers and the like which can be used in the pharmaceutical field and some medicinal matrixes. The carrier is a functional pharmaceutical adjuvant available in the pharmaceutical field and comprises a surfactant, a suspending agent, an emulsifying agent and a plurality of novel pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid and the like.

Preferably, in the field of medical application, the dosage forms of the microbial inoculum comprise tablets, granules, capsules, dripping pills, sustained release agents, oral liquid preparations and injections.

More preferably, the above-mentioned dosage forms refer to clinically usual dosage forms. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if some drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.

Compared with the prior art, the invention has the beneficial effects that:

The invention separates and purifies a strain of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 from a fecal sample of a healthy adult in Guangdong area of China, and the strain has a plurality of probiotics effects, including excellent protease activity, can generate and secrete gamma-aminobutyric acid, can inhibit alpha-glucosidase activity, can remove DPPH free radical, and has excellent cellulase activity. Therefore, the lactobacillus johnsonii XY9 strain has the functions of promoting digestion and absorption of protein food, improving protein allergy, resisting depression, improving sleep, reducing blood sugar and weight, resisting oxidation, whitening, delaying aging, resisting inflammation, promoting absorption of cellulose, improving constipation and the like. Therefore, the lactobacillus johnsonii XY9 strain newly separated by the invention has various probiotics effects, can be used in the fields of blood sugar reduction, aging resistance and the like, for example, can be prepared into medicines for reducing blood sugar and resisting aging, and has important application value and economic value.

Drawings

FIG. 1 is a phylogenetic tree of Lactobacillus johnsonii XY9 strain (the established tree strains are all derived from the Genome database of NCBI);

FIG. 2 shows the degradation of milk plates by Lactobacillus johnsonii XY9 strain (a is blank; b is experimental);

FIG. 3 shows that Lactobacillus johnsonii XY9 strain produces and secretes gamma-aminobutyric acid;

FIG. 4 is a graph showing that the secretion of substances by Lactobacillus johnsonii XY9 fermentation broth significantly inhibited the activity of alpha-glucosidase;

FIG. 5 shows that Lactobacillus johnsonii XY9 strain can scavenge DPPH free radicals;

FIG. 6 shows the degradation experiment (left, blank; right, experimental group) of Lactobacillus johnsonii XY9 strain on cellulose plates.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

The following examples relate to the following experimental materials:

(1) Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain was committed to the moisturizing laboratory of the intestinal microbiota from feces isolated from a healthy adult, guangdong, china and was cryopreserved in glycerol tubes at-80 ℃. Typically, colonies are obtained by inoculating them on the surface of a plate of MRS solid medium and culturing them upside down in a 37℃constant temperature anaerobic incubator for 24 hours, or fermentation broth is obtained by shaking culture in a 37℃constant temperature anaerobic incubator in MRS liquid medium for 24-48 hours.

(2) The kit is gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge), alpha-glucosidase inhibitor screening kit (abcam, cat: ab 284520), DPPH free radical scavenging ability kit (Nanjing built, cat: A153-1-1).

(3) The MRS flat plate is prepared by adding 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar, 1L of ddH ₂ O, and sterilizing at pH of 6.2-6.6,121 ℃ for 20min under high pressure.

(4) The MRS liquid culture medium is prepared by supplementing 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, adding 1L of ddH ₂ O, adjusting the pH to 6.2-6.6,121 ℃ and sterilizing for 20min under high pressure.

(5) MP plate, 10g of skimmed milk powder, 1g of sodium chloride, 10g of beef extract, 10g of peptone, 5g of yeast extract, 20g of glucose, 2g of triammonium citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar, and 1L of ddH ₂ O, adjusting the pH to 6.2-6.6,121 ℃ and sterilizing under high pressure for 20 min.

(6) CMC plate, sodium carboxymethylcellulose 10g, ammonium sulfate 1.5g, manganese sulfate 0.3g, calcium chloride 0.2g, sodium chloride 5g, urea 0.3g, beef extract 10g, peptone 10g, yeast extract 5g, glucose 20g, tri-ammonium citrate 2g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, tween 80.5 mL, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar 15g, ddH ₂ O are added to 1L, pH is adjusted to 6.2-6.6,121 ℃ and autoclaved for 20min, thus preparing CMC plate.

Example 1 isolation and identification of Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 Strain

Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain was isolated from faeces of a healthy adult (bmi=20.6) from guangdong province, china, and is specifically as follows:

The fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, 500uL of sterile water was added per 100mg of fecal sample, thoroughly ground to homogenate, and an appropriate amount of the grinding fluid was pipetted, spread on an MRS plate, and incubated at room temperature for 3 days. Colonies to be streaked and purified in the separation assay plates were then numbered with a marker and strain numbers were marked on the plates accordingly. After labelling, colonies were picked and inoculated onto MRS plates and the strains were purified by plate streaking. If the strain cannot be separated by the method, colonies need to be picked from the enrichment plate, and the colonies are coated on a culture medium after being subjected to gradient dilution by MRS liquid culture medium. Finally, reference is made to the "Berger's Manual of bacteria identification" (eighth edition) and the "manual of fungus classification identification", which identify strains belonging to bacteria first. A purified strain is obtained by preliminary separation, the strain number is XY9, and after 48 hours of culture, the colony of the strain is observed to be round, white and semitransparent.

Next, the isolated XY9 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACTT), and then the XY9 strain was subjected to whole genome sequencing by Beijing Baimeike Biotechnology Co. The resulting 16S rDNA sequence (SEQ ID No: 1) was subjected to BLAST alignment at NCBI' S Genome database. The results showed that the XY9 strain had >99% homology with the known Lactobacillus johnsonii (Lactobacillus johnsonii) 16S rDNA sequence and was analyzed by evolution with the homologous strain (FIG. 1) to confirm that the XY9 strain was a homologous, different strain of Lactobacillus johnsonii.

And finally, the strain XY9 is preserved, wherein the preservation information is as follows, the preservation time is 2023, 10 and 25 days, the preservation unit name is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M20232013, the preservation unit address is China university of Wuhan, and the classification naming is Lactobacillus johnsonii.

Lactobacillus johnsonii is a probiotic strain with wide probiotic efficacy, but strains of different sources have different efficacy, which means that the novel lactobacillus johnsonii XY9 strain separated from human feces according to the invention can be used as a probiotic and may have novel efficacy and function.

Lactobacillus johnsonii XY916S rDNA sequence(1438bp):

GCAAGTCGAGCGAGCTTGCCTAGATGATTTTAGTGCTTGCACTAAATGAAACTAGATACAAGCGAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAAGAGACTGGGATAACACCTGGAAACAGATGCTAATACCGGATAACAACACTAGACGCATGTCTAGAGTTTGAAAGATGGTTCTGCTATCACTCTTGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATTACTTAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAAGTCTGATGTGAAAGCCTTCGGCTCAACCGGAGAATTGCATCAGAAACTGTTGAACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCAGTGCAAACCTAAGAGATTAGGTGTTCCCTTCGGGGACGCTGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGAAGCGAACCTGCGAAGGCAAGCGGATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTCTGTAACACCCAAAGCCGGTGGGATAACCTTTATAGGAGTCAGCC.

Example 2 function and use of Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 Strain

(1) Lactobacillus johnsonii XY9 strain capable of producing protease

The ability of lactobacillus johnsonii XY9 to secrete protease hydrolyzed protein was identified and measured according to the agar well diffusion assay using skim milk plate medium (MP plate). In the test, 3uL of Lactobacillus johnsonii XY9 bacteria liquid with the concentration of 10Abs is dripped into an MP plate of an experimental group, and 3uL of blank MRS culture medium is dripped into a control group, and then the culture is performed for 3 days in an anaerobic incubator with constant temperature at 37 ℃. The results show that strain XY9 can significantly degrade protein and form a distinct degradation circle compared to the control with the addition of the blank medium (fig. 2). Illustrating that Lactobacillus johnsonii XY9 strain produces proteases that degrade milk proteins.

The lactobacillus johnsonii XY9 strain can produce protease, and the protease can promote the digestion and absorption of protein in food by human body and improve the absorption of small peptide and amino acid when used as a probiotic strain. And can be used for resisting allergy (improving food allergy caused by protein dyspepsia or non-absorption). In addition, the method can also be used for extracting protease, applied to the production of protease in the washing industry and the like, and also can be used in microbial feed to help animals digest and absorb nutrition and improve the utilization rate of the feed.

(2) Lactobacillus johnsonii XY9 strain produces and secretes gamma-aminobutyric acid (GABA)

Lactobacillus johnsonii XY9 cultured with MRS liquid medium to stationary phase was expanded into new MRS liquid medium at a dilution factor of 1:30, bacterial suspension was harvested at 24h of culture to stationary phase, fermentation broth supernatant was harvested after centrifugation at 10,000×g at 4 ℃ for 10min, and GABA concentration of fermentation broth supernatant was then determined by GABA specific ELISA kit (CEA 900 Ge). The results showed that the concentration of GABA in the fermentation supernatant of strain XY9 was significantly increased compared to the low concentration of GABA in the blank medium MRS, and the accumulated amount was 13.38pg/mL, indicating that lactobacillus johnsonii XY9 can produce and secrete gamma-aminobutyric acid in the stationary phase (fig. 3).

Gamma-aminobutyric acid is an important central nervous system inhibitory neurotransmitter, and is widely present in animals, plants and microorganisms. It has been demonstrated that GABA, a small molecular weight non-protein amino acid, is food safe and can be used as a food additive. Research shows that intake of a certain amount of GABA has the physiological effects of improving sleeping quality of organisms, resisting depression, resisting anxiety, reducing blood pressure, improving lipid metabolism, enhancing memory and brain activity, accelerating brain metabolism, strengthening liver and kidney, promoting ethanol metabolism (dispelling alcohol effect), improving climacteric syndrome and the like.

Thus, the probiotic Lactobacillus johnsonii XY9 strain may serve several of the above purposes by producing gamma-aminobutyric acid.

(3) Lactobacillus johnsonii XY9 strain fermentation broth can effectively inhibit alpha-glucosidase activity

Lactobacillus johnsonii XY9 cultured with MRS liquid medium to stationary phase was expanded into new MRS liquid medium at a dilution factor of 1:30, bacterial suspension was collected at 24h of culture to stationary phase, fermentation broth supernatant was collected after centrifugation at 10,000×g at 4 ℃ for 10min, and then the effect of fermentation broth supernatant on the enzyme activity ability of α -glucosidase to hydrolyze glucose was determined by α -glucosidase inhibitor screening kit (ab 284520). The results showed that the fermentation supernatant of strain XY9 significantly inhibited the ability of alpha-glucosidase to hydrolyze glucose compared to the non-inhibitory effect of the blank medium MRS, with an inhibition rate of about 30.18%, indicating that the fermentation broth of Lactobacillus johnsonii XY9 can effectively inhibit the activity of alpha-glucosidase (FIG. 4).

Alpha-glucosidase, an enzyme that plays a role in carbohydrate breakdown, is associated with type 2 diabetes, and inhibition of alpha-glucosidase is one of the methods of controlling postprandial hyperglycemia, thereby contributing to the treatment of diabetes. Thus, α -glucosidase inhibitors help maintain blood glucose balance, and can improve diabetic complications. Furthermore, inhibition of α -glucosidase activity may control obesity.

Thus, the probiotic lactobacillus johnsonii XY9 strain has an alpha-glucosidase inhibitory activity, which makes it a potential hypoglycemic and obesity inhibiting probiotic.

(4) Lactobacillus johnsonii XY9 strain capable of scavenging DPPH free radical

Lactobacillus johnsonii XY9 strain cultured with MRS liquid medium to stationary phase was expanded into new MRS liquid medium at dilution ratio of 1:30, bacterial suspension was collected at 24h of stationary phase, and fermentation broth supernatant was collected after centrifugation at 10,000xg at 4 ℃ for 10min, and then the DPPH scavenging ability of fermentation broth supernatant was measured by DPPH radical scavenging ability kit (nanking, cat: a 153-1-1). The results show that compared with the blank control MRS, the fermentation supernatant of the strain XY9 has 317.0Trolox/mL of DPPH free radical scavenging capacity, is obviously improved (P is less than 0.01), which shows that the lactobacillus johnsonii XY9 has better DPPH free radical scavenging capacity and shows good antioxidant capacity (figure 5).

The term "radical" is also referred to as "radical" in chemistry, and refers to an atom or group having unpaired electrons formed by homolytic cleavage of a molecule of a compound under external conditions such as photothermal. Since free radicals contain unpaired electrons, they are extremely unstable (particularly hydroxyl radicals) and therefore abstract electrons from neighboring molecules (including fat, protein and DNA) and leave themselves in a stable state. In this way, the adjacent molecules become a new radical and then abstract electrons. Such a linkage reaction may damage the structure of the cell, resulting in loss of cell function, gene mutation, and even death.

Free radicals have a number of disadvantages. Such as (1) weakening the resistance of cells to make the body vulnerable to bacterial and bacterial infection, (2) generating chemical substances which destroy the cells to form cancerogenic substances, (3) hindering the normal development of the cells, interfering with the recovery function of the cells to make the cell turnover rate lower than the withering rate, (4) destroying genetic (DNA) tissues in the body to disturb the operation and regeneration functions of the cells to cause gene mutation and evolve into cancers, (5) destroying mitochondria (energy storage body) in the cells to cause oxidative fatigue, (6) destroying proteins to destroy enzymes in the body to cause inflammation and aging, (7) destroying fat to cause lipid peroxidation to cause atherosclerosis, cardiovascular and cerebrovascular diseases and the like.

In addition, DPPH free radical scavenging has various effects, such as ① anti-aging, namely, the free radical has obvious effect on human body, and free radical scavenging can prevent the free radical from damaging cells and avoid the aging of the body caused by aging and decay, so the free radical scavenging has anti-aging effect. ② Preventing cancer, that is, when free radicals damage cell membrane proteins, the function and shape of cells can be promoted, and other cells around the cells are damaged, when free radicals damage DNA, the mutation of protooncogene and cancer suppressor gene can be caused, and the generation of cancer cells can be caused, so that the free radicals are eliminated, and the effect of preventing cancer is achieved. ③ Protecting cardiac and cerebral vascular diseases, which is to deposit free radical on vascular wall to destroy vascular elastic fiber, harden vascular cell and form atherosclerosis, and to oxidize lipid, so as to cause thrombus and other cardiac and cerebral vascular diseases easily. The free radicals can be removed to prevent the free radicals from depositing on the vessel wall, so that the composition has the function of protecting cardiovascular and cerebrovascular vessels.

Thus, the probiotic Lactobacillus johnsonii XY9 strain can avoid the harm of DPPH free radical to the organism by eliminating the function of the DPPH free radical.

(5) Lactobacillus johnsonii XY9 strain capable of producing cellulase

The ability of lactobacillus johnsonii XY9 to degrade cellulose was identified and determined according to the agar well diffusion method using cellulose plate medium (CMC plate). In the test, 3uL of Lactobacillus johnsonii XY9 strain solution with a concentration of 10Abs was added dropwise to CMC plates of the experimental group, while 3uL of blank MRS medium was added dropwise to the control group. Congo red staining was performed after 3 days of inverted culture in a 37℃constant temperature anaerobic incubator. The results show that strain XY9 can significantly degrade cellulose, forming a distinct degradation loop, compared to the control with the addition of the blank medium (fig. 6).

The lactobacillus johnsonii XY9 strain can produce cellulase, so that the lactobacillus johnsonii XY9 strain can play a plurality of roles in promoting digestion and absorption of dietary components, improving constipation, decomposing cellulose by probiotics in intestinal tracts to generate certain moisture and softening stool, reducing cholesterol is the most important effect of cellulose, because soluble fiber can inhibit the absorption of cholesterol by human bodies after being absorbed by human bodies, can be combined with cholesterol in intestinal tracts, quickening the metabolism of cholesterol in the bodies, preventing hyperlipidemia caused by excessive cholesterol of the human bodies, and reducing the incidence of arteriosclerosis and coronary heart disease, and (4) the unsweetened sugar of cellulose can slow down the absorption of glucose by blood, balance blood glucose concentration, promote the sensitivity of muscle and fat cells to insulin, thereby preventing and assisting in treating diabetes.

In addition, lactobacillus johnsonii XY9 strain can be applied to fermentation and extraction of cellulase, degradation of cell walls of pathogenic fungi to control diseases, decomposition of cellulose in compost, and preparation of livestock and poultry raising feeds, such as monogastric animal feeds of pigs, chickens and the like, so as to overcome the defect that cellulose cannot be utilized.

In summary, the novel isolated Lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of the invention has a plurality of probiotics effects, namely (1) has superior protease activity, (2) can generate and secrete gamma-aminobutyric acid, (3) can inhibit alpha-glucosidase activity, (4) can remove DPPH free radical, and (5) has superior cellulase activity. Therefore, the lactobacillus johnsonii XY9 strain obtained by the novel separation has important application value and economic value in the fields of blood sugar reduction, aging resistance and the like.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims

1. The lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain is characterized in that the lactobacillus johnsonii XY9 strain is preserved in China center for type culture collection (China center for type culture collection) 10 months 2023, the preservation number is CCTCC NO: M20232013, and the 16S rDNA sequence of the lactobacillus johnsonii XY9 strain is shown as SEQ ID No. 1.

2. Use of the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of claim 1 for the production of proteases for the degradation of milk proteins.

3. Use of the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of claim 1 for the production of cellulases.

4. Use of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain as claimed in claim 1 for the production of gamma-aminobutyric acid.

5. Use of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain as claimed in claim 1 in the preparation of an alpha-glucosidase inhibitor.

6. Use of lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain as claimed in claim 1 in the preparation of DPPH radical scavenger.

7. A probiotic functional bacterial agent, characterized in that it comprises the lactobacillus johnsonii (Lactobacillus johnsonii) XY9 strain of claim 1.

8. The probiotic functional microbial agent of claim 7, wherein in the field of pharmaceutical applications, the microbial agent further comprises pharmaceutically acceptable excipients.