CN117384790B - Pediococcus pentosaceus KS5 and application thereof in preparation of sleep-aiding drugs - Google Patents
Pediococcus pentosaceus KS5 and application thereof in preparation of sleep-aiding drugs Download PDFInfo
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- CN117384790B CN117384790B CN202311322668.3A CN202311322668A CN117384790B CN 117384790 B CN117384790 B CN 117384790B CN 202311322668 A CN202311322668 A CN 202311322668A CN 117384790 B CN117384790 B CN 117384790B
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Abstract
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to pediococcus pentosaceus KS5 and application thereof in preparation of sleep-aiding medicines. In order to further develop and utilize the probiotic functions of Pediococcus pentosaceus, the invention separates a Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain from the feces of a healthy adult in Guangdong province of China, and the strain has various probiotic effects, including excellent protease activity, excellent cellulase activity, and can produce and secrete 3-hydroxybutyric acid and gamma-aminobutyric acid. Thus, the KS5 strain has the effects of promoting digestion and absorption of protein foods and improving protein allergy; promoting absorption of cellulose and improving constipation; improving intestinal flora; the multifunctional health food has the functions of resisting depression, dispelling the effects of alcohol, assisting sleep and the like, can be used in the fields of assisting sleep and the like, and has important application value and economic value.
Description
Technical Field
The invention belongs to the technical field of probiotics and application thereof, and particularly relates to pediococcus pentosaceus KS5 and application thereof in preparation of sleep-aiding medicines.
Background
Pediococcus pentosaceus (Pediococcus pentosaceus), which is round, milky white, smooth, convex, neat in edge, opaque, gram-positive, belonging to cocci. The bacterial cells are spherical and have a diameter of 0.8-1.0 μm. The optimal growth temperature is 35 ℃, and the maximum growth temperature is 42-45 ℃. Pediococcus pentosaceus is a widely existing lactobacillus in human body, and is a normal strain existing in human body. In addition, pediococcus pentosaceus has good acid and bile salt resistance, can survive in intestines and stomach of human and animals, and is widely present in intestines and stomach of human and animals.
Pediococcus pentosaceus is one of the strains of production and processing, has become a hot spot in recent years as a probiotic with great potential, and is being continuously used to make probiotic preparations suitable for humans and animals. Pediococcus pentosaceus is widely present in the intestinal tract of humans and animals and excreted with feces, and is an important component of the normal microbial system of the intestinal tract and accompanies the host for life, and is of great importance in maintaining the intestinal microecological balance. In recent years pediococcus pentosaceus has been widely used as a probiotic for the production of human and animal probiotic formulations. As probiotics in intestinal flora, the pediococcus pentosaceus is also an edible probiotic, can play a role in helping intestinal peristalsis, and can also enhance the digestion capacity of a human body, improve immunity and the like.
Studies have shown that different pediococcus pentosaceus strains have different probiotic functions: (1) Pediococcus pentosaceus SMM914 of the breast milk flora can activate antioxidant signaling pathways in the liver of piglets, reduce albumin ALT and total protein TP levels in plasma, and thereby reduce the potential liver damage of piglets during weaning. In addition, oral pediococcus pentosaceus SMM914 can also increase heart coefficients in piglets, activate liver Nrf2-Keap1 pathway, interfere with the level of amino acid metabolites in plasma, and increase beneficial microbiota in the colon. (2) The combined use of Pediococcus pentosaceus CECT8330 and Bifidobacterium longum CECT 7894 can effectively reduce the daily crying time and crying times of infants, has no adverse reaction, and can effectively relieve the intestinal colic symptoms of infants. (3) Pediococcus pentosaceus CECT8330 can promote intestinal flora to protect colitis induced by inflammatory drugs, and regulate the composition and function, immune morphology and intestinal barrier function of intestinal microorganisms. Thus, this study suggests that the pentose sheet strain may have the potential for IBD therapeutic defenses. (4) Pediococcus pentosaceus also has effects of promoting digestion, enhancing immunity, and inhibiting bacterial growth. Pediococcus pentosaceus is a probiotic capable of promoting digestion, can effectively promote gastrointestinal peristalsis, and helps food to be absorbed and digested as soon as possible, so that the effect of promoting digestion is achieved. The Pediococcus pentosaceus contains polysaccharide component, and can stimulate immune cells to produce antibodies, so as to improve immunity and prevent diseases. Pediococcus pentosaceus can also inhibit bacterial growth, can be used for treating gastrointestinal discomfort such as diarrhea clinically, and can also play a role in sterilization. The pediococcus pentosaceus has the effect of treating scalds.
Pediococcus pentosaceus has source diversity, so that it has gene diversity and functional diversity. However, the current studies on the separation and identification, the probiotic characteristics and the metabolic mechanism of Pediococcus pentosaceus are still relatively few, which also affects the development and utilization of Pediococcus pentosaceus to a certain extent. Therefore, it is necessary to develop different functions according to different sources of Pediococcus pentosaceus, so that the Pediococcus pentosaceus can better play roles, for example, the functions of the strain or the probiotics metabolic products can be determined, and the application prospect of the Pediococcus pentosaceus can be clarified. In conclusion, the research and application of the probiotic pediococcus pentosaceus have a very wide development space.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention separates a Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain from the feces of a healthy adult in Guangdong province in China, and the strain has excellent protease activity, excellent cellulase activity, capability of producing and secreting 3-hydroxybutyric acid, capability of producing and secreting gamma-aminobutyric acid, and important potential application value.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The first aspect of the invention provides a pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain, which is characterized in that the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain is preserved in China center for type culture collection (China, with the preservation number: CCTCC NO: M20231155; the 16S rDNA complete sequence of the pediococcus pentosaceus (Pediococcus pentosaceus) KS5 strain is shown in SEQ ID No: 1.
In a second aspect, the invention provides the use of a Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to the first aspect in the production of proteases.
Preferably, the protease comprises a protease that degrades milk proteins.
The research shows that the probiotic Pediococcus pentosaceus KS5 strain can produce protease, which suggests that the Pediococcus pentosaceus KS5 strain is expected to be used for producing the protease, and the property of the protease is applied to the fields of promoting the digestion and absorption of human bodies on proteins in food, improving the absorption of small peptides and amino acids, resisting allergy and the like.
In a third aspect, the invention provides the use of a pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to the first aspect for producing cellulases.
The research shows that the probiotic Pediococcus pentosaceus KS5 strain can produce cellulase, which suggests that the Pediococcus pentosaceus KS5 strain is expected to be used for producing the cellulase, and the characteristic of producing the cellulase is applied to the fields of promoting digestion and absorption of dietary components, improving constipation and reducing cholesterol.
In a fourth aspect, the invention provides the use of a Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to the first aspect for the production of 3-hydroxybutyric acid.
The research shows that the probiotic Pediococcus pentosaceus KS5 strain can produce 3-hydroxybutyric acid, which suggests that the Pediococcus pentosaceus KS5 strain is expected to be used for producing 3-hydroxybutyric acid, and can be applied to the fields of providing energy for various activities of the body, resisting osteoporosis, preventing and treating chronic syndromes (hypertension, alcoholic fatty liver, enteritis, intestinal cancer and the like), improving brain cognitive functions (improving learning and memory capacity, protecting glial cells, improving Alzheimer's disease and the like), improving lipid metabolism and the like through the characteristic of producing 3-hydroxybutyric acid.
In a fifth aspect, the invention provides the use of a pediococcus pentosaceus (Pediococcus pentosaceus) KS5 strain according to the first aspect for the production of gamma-aminobutyric acid.
The research shows that the probiotic Pediococcus pentosaceus KS5 strain can produce gamma-aminobutyric acid, which suggests that the Pediococcus pentosaceus KS5 strain is expected to be used for producing gamma-aminobutyric acid, and can be applied to the fields 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 effects of alcohol), improving climacteric syndrome and the like through the characteristic of producing gamma-aminobutyric acid.
In a sixth aspect, the invention provides a probiotic functional bacterial agent comprising the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain of the first aspect.
Preferably, the microbial inoculum is a product of fermentation of the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain of the first aspect.
Preferably, in the field of medical application, the microbial inoculum further comprises pharmaceutically acceptable auxiliary materials. The auxiliary materials comprise 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 injection, tablet, granule, capsule, dripping pill, sustained release agent and oral liquid preparation.
Compared with the prior art, the invention has the beneficial effects that:
The invention separates a Pediococcus pentosaceus (Pediococcus pentosaceus) KS5 strain from the feces of a healthy adult in Guangdong province in China, and the strain KS5 has various probiotics effects including excellent protease activity, excellent cellulase activity, capability of producing and secreting 3-hydroxybutyric acid and capability of producing and secreting gamma-aminobutyric acid. Thus, pediococcus pentosaceus strain KS5 has the effects of promoting digestion and absorption of protein food and improving protein allergy; promoting absorption of cellulose and improving constipation; improving intestinal flora; anti-depression, anti-alcohol and sleep-aiding functions. Therefore, the pediococcus pentosaceus strain KS5 newly separated by the invention has various probiotics effects, can be used in the fields of sleep assistance and the like, for example, can be prepared into sleep-assisting medicines, and has important application value and economic value.
Drawings
FIG. 1 is a phylogenetic tree of Pediococcus pentosaceus KS5 strain;
FIG. 2 shows the degradation of milk plates by Pediococcus pentosaceus KS5 strain (left, blank; right, experimental group);
FIG. 3 shows the degradation experiments of Pediococcus pentosaceus KS5 strain on cellulose plates (left, blank; right, experimental group);
FIG. 4 shows that Pediococcus pentosaceus KS5 strain can produce and secrete 3-hydroxybutyric acid;
FIG. 5 shows that Pediococcus pentosaceus KS5 strain can produce and secrete gamma-aminobutyric acid.
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) Strains: pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain was isolated from a sample of intestinal faeces of a healthy adult (BMI=18.8) in Guangzhou, guangdong, china, and placed in a glycerol tube for cryogenic storage at-80 ℃. In general, the strain is inoculated on the surface of a MRS solid culture medium flat plate, and is inversely cultured in a constant temperature anaerobic incubator at 37 ℃ for 24 hours to obtain a bacterial colony, and simultaneously is shake-cultured in a MRS liquid culture medium for 24-48 hours in the constant temperature anaerobic incubator at 37 ℃ to obtain a fermentation liquor.
(2) The kit comprises: 3-hydroxybutyric acid (3-HB) detection kit (Cloud-Clone Corp., cat: CEB022 Ge), gamma-aminobutyric acid (GABA) detection kit (Cloud-Clone Corp., cat: CEA900 Ge).
(3) MRS plate: 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, 6.2-6.6 of pH value, and autoclaving at 121 ℃ for 20min to prepare an MRS plate.
(4) MRS liquid medium: 10g of beef extract, 10g of peptone, 5g of yeast extract, 2g of citric acid, 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, supplementing ddH 2 O to 1L, adjusting the pH to 6.2-6.6, and carrying out high-pressure sterilization at 121 ℃ for 20min.
(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 tri-ammonium 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, 1L of ddH 2 O, pH adjustment to 6.2-6.6, and high-pressure sterilization at 121 ℃ for 20min, thus preparing an MP plate.
(6) CMC plate: 10g of sodium carboxymethyl cellulose, 1.5g of ammonium sulfate, 0.3g of manganese sulfate, 0.2g of calcium chloride, 5g of sodium chloride, 0.3g of urea, 10g of beef extract, 10g of peptone, 5g of yeast extract, 20g of glucose, 2g of tri-ammonium 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, 1L of ddH 2 O, pH adjustment, 6.2-6.6, and autoclaving at 121 ℃ for 20min, and preparing into CMC plates.
Example 1 isolation and identification of Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 Strain
Pediococcus pentosaceus Pediococcuspentosaceus KS was isolated from a sample of intestinal faeces from a healthy adult (BMI=18.8) in Guangzhou province, guangdong, china, as follows:
the fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, thoroughly homogenized by adding 500uL of sterile water per 100mg, and applied to MRS plates by pipetting with appropriate amounts of the slurry, 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 "Burjie's Manual of identification of bacteria" (eighth edition) and the "manual of identification of classification of fungi", which identify the strains belonging to bacteria first, and then observe the growth of the colonies: growth morphology, presence or absence of hyphae, uniformity or absence of color, colony individual surface and edge. Finally, a purified strain is obtained through preliminary separation, the strain number is KS5, and after 24 hours of culture, the bacterial colony morphology of the strain is observed to be circular bacterial colony, milky white, smooth convex, and clean-edged and opaque coccus.
Next, the isolated KS5 strain was subjected to molecular characterization by a 16S rDNA universal primer (27F: AGAGTTTGATCCTGGCTCAG,1492R: TACGGCTACCTTGTTACGACCTT), and then subjected to whole genome sequencing by Beijing Baimaike Biotechnology Co. BLAST alignment of the resulting 16S rDNA sequence (SEQ ID No: 1) at NCBI' S Genome database revealed that KS5 strain had >99% homology with the known 16S rDNA sequence of Pediococcus pentosaceus (Pediococcuspentosaceus), and that strain KS5 was a different strain from the same species by evolutionary analysis of the homologous strain (FIG. 1).
Finally, strain KS5 was deposited with the following information: preservation time: 2023, 7, 3; preservation unit name: china Center for Type Culture Collection (CCTCC); deposit number: CCTCC NO: M20231155; deposit unit address: chinese university of Wuhan; classification naming: pediococcuspentosaceus.
Pediococcus pentosaceus is a probiotic strain with wide probiotic effects such as anti-aging and cholesterol reduction, but different strains from different sources have different effects, which indicates that the Pediococcus pentosaceus KS5 strain separated from human excrement can be used as a probiotic and possibly has new effects and functions.
Pediococcuspentosaceus KS516S rDNAsequence(1446bp，SEQ ID No：1)：
AGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTAGGAGCTAGCCGTCTAAGT.
Example 2 Functions of Pediococcus pentosaceus (Pediococcuspentosaceus) KS5 Strain and uses thereof
(1) Pediococcus pentosaceus KS5 strain can produce protease capable of degrading milk protein
Pediococcus pentosaceus KS5 was assayed for its ability to secrete proteolytic proteins by the agar well diffusion assay using skim milk plate medium (MP plate). In the experiment, 3uL of Pediococcus pentosaceus KS5 bacterial liquid with the concentration of 10Abs is dripped into an MP plate of an experimental group, 3uL of blank MRS culture medium is dripped into a control group, and the culture is inverted and carried out for 3 days in a constant temperature anaerobic incubator at 37 ℃. The results showed that strain KS5 significantly degraded protein and formed a distinct degradation circle (FIG. 2) compared to the control with the blank medium, indicating that Pediococcus pentosaceus KS5 strain produced protease.
Therefore, the Pediococcus pentosaceus KS5 can produce protease as a probiotic bacterial strain to promote the digestion and absorption of protein in food by human body and improve the absorption of small peptide and amino acid. 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 and applied to the protease fields such as washing industry and the like; can also be used in microbial feed to help animals digest and absorb nutrition, and improve the utilization rate of the feed.
(2) Pediococcus pentosaceus KS5 strain can produce cellulase
The ability of Pediococcus pentosaceus KS5 to degrade cellulose was determined by agar well diffusion using a cellulose plate medium (MP plate). In the experiment, 3uL of Pediococcus pentosaceus KS5 bacterial solution with the concentration of 10Abs was added dropwise to MP plates of the experimental group, and 3uL of blank MRS medium was added dropwise to the control group. Congo red staining was performed after 3 days of inversion culture in an anaerobic incubator at 37 ℃. The results showed that strain KS5 significantly degraded cellulose compared to the control with the blank medium drop-wise, forming a distinct degradation circle (FIG. 3), indicating that Pediococcus pentosaceus KS5 strain produced cellulase.
Since Pediococcus pentosaceus KS5 can produce cellulase, it is seen that the probiotic Pediococcus pentosaceus KS5 strain can exert the following multiple purposes by producing the efficacy of cellulase: (1) for promoting the digestive absorption of dietary components; (2) The constipation is improved, and probiotics decompose cellulose in intestinal tracts to generate certain moisture, so that the constipation relieving tea has the effect of softening stool; (3) Cholesterol reduction is the most important effect of cellulose, because the soluble fiber can inhibit the absorption of cholesterol by human body after being absorbed by human body, can be combined with cholesterol in intestinal tract, quickens the metabolism of cholesterol in human body, prevents human body from inducing hyperlipidemia due to hypercholesteremia, and can reduce the incidence rate of arteriosclerosis and coronary heart disease; (4) The non-sweet 'sugar' of cellulose can slow down the absorption of glucose by blood, balance the concentration of blood sugar and promote the sensitivity of muscle and fat cells to insulin, thereby preventing and assisting in treating diabetes.
In addition, pediococcus pentosaceus KS5 can also be applied to the fermentation extraction of cellulase; applied to degrading cell walls of pathogenic fungi and controlling diseases; the method is applied to the decomposition of cellulose in compost; the method is applied to preparing livestock and poultry raising feeds, such as monogastric animal feeds for pigs, chickens and the like, so as to overcome the defect that cellulose cannot be utilized.
(3) Pediococcus pentosaceus KS5 strain can produce 3-hydroxybutyric acid (3-HB)
Pediococcus pentosaceus KS5 cultured to a stationary phase by using an MRS liquid culture medium is expanded and cultured into a new MRS liquid culture medium at a dilution ratio of 1:30, bacterial suspension is collected when the culture is carried out for 24 hours in the stationary phase, fermentation thalli are collected after centrifugation for 10 minutes at 10,000 Xg and 4 ℃, the obtained thalli are dissolved in 800mL of distilled water by using buffer PBS (8 g NaCl, 0.2g KCl and 1.44g Na 2HPO4、0.24g KH2PO4, the solution is regulated to 7.2 by using HCl, finally distilled water is added to a volume of 1L, so that buffer PBS is obtained, and after the thalli lysate is prepared, the concentration of 3-HB in the thalli lysate is measured by using a 3-HB specific ELISA kit (CEB 022 Ge). As a result, it was found that the concentration of 3-HB in the cell lysate of the strain KS5 was 243.10. Mu.g/mL, compared with the case where 3-HB was not present in the cell lysis buffer PBS, indicating that Pediococcus pentosaceus KS5 can produce 3-hydroxybutyric acid in the stationary phase (FIG. 4).
3-HB can provide energy for various physical activities and is a potential energy/functional food that has been added to athlete drinks, so the probiotic Pediococcus pentosaceus KS5 can be used as an additive for energy type products. Meanwhile, in view of the fact that 3-HB can effectively resist osteoporosis, prevent and treat chronic syndromes (hypertension, alcoholic fatty liver, enteritis, intestinal cancer and the like), improve brain cognitive functions (improving learning and memory capacity, protecting glial cells, improving Alzheimer's disease and the like), and improve lipid metabolism. Thus, the probiotic Pediococcus pentosaceus KS5 strain can exert the above multiple uses by the effect of producing 3-hydroxybutyric acid.
In addition, 3-hydroxybutyric acid is an endogenous small molecule substance naturally produced by the body, has an important role in maintaining the integrity of colorectal tissues, and has the functions of maintaining intestinal health, preventing colonic diseases and diminishing inflammation and productivity. The 3-HB treatment can promote the proliferation of beneficial intestinal bacteria, relieve the symptoms of multiple sclerosis, and has great potential in regulating flora and improving health. Thus the probiotic Pediococcus pentosaceus KS5 strain also helps to improve intestinal flora and alleviate intestinal inflammation.
(4) Pediococcus pentosaceus KS5 strain can produce and secrete gamma-aminobutyric acid (GABA)
Pediococcus pentosaceus KS5 cultured in MRS liquid medium to stationary phase was expanded into new MRS liquid medium at a dilution ratio of 1:30, bacterial suspension was collected at 24h of stationary phase, and after centrifugation at 10,000Xg and 4℃for 10min, the supernatant of the fermentation broth was collected, and then GABA concentration of the supernatant of the fermentation broth was measured by GABA specific ELISA kit (CEA 900 Ge). The results showed that the concentration of GABA in the fermentation supernatant of strain KS5 was significantly increased compared to the MRS control, and the cumulative amount was 148.45pg/mL, indicating that pediococcus pentosaceus KS5 can produce and secrete γ -aminobutyric acid in the stationary phase (fig. 5).
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, which is a small molecular weight non-protein amino acid, is food safe. Research shows that the intake of a certain amount of GABA has the physiological effects of improving the sleep 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 the effects of alcohol), improving climacteric syndrome and the like.
Thus, the probiotic Pediococcus pentosaceus KS5 strain can exert the above multiple uses by the effect of producing gamma-aminobutyric acid.
Taken together, the newly isolated pediococcus pentosaceus strain KS5 strain of the present invention has various probiotic effects: (1) has excellent protease activity; (2) has excellent cellulase activity; (3) 3-hydroxybutyric acid may be produced and secreted; (4) gamma-aminobutyric acid can be produced and secreted. Therefore, the pediococcus pentosaceus strain KS5 obtained by the novel separation has important application value and economic value.
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 (8)
1. A pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain, wherein the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain is deposited with the chinese collection for typical cultures at month 7 and 3 of 2023 under the accession number: CCTCC NO: M20231155; the 16S rDNA complete sequence of the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain is shown in SEQ ID No: 1.
2. Use of a pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to claim 1 for the production of a protease, wherein the protease is a protease that degrades milk proteins.
3. Use of the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain of claim 1 for producing cellulases.
4. Use of a pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to claim 1 for the production of 3-hydroxybutyric acid.
5. Use of a pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to claim 1 for the production of gamma-aminobutyric acid.
6. A probiotic functional bacterial agent, characterized in that it comprises the pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain according to claim 1.
7. The probiotic functional bacterial agent according to claim 6, characterized in that it is the product of fermentation of pediococcus pentosaceus (Pediococcuspentosaceus) KS5 strain.
8. The probiotic functional bacterial agent according to claim 6, characterized in that in the field of medical applications, the bacterial agent further comprises pharmaceutically acceptable excipients.
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| JP2005218324A (en) * | 2004-02-04 | 2005-08-18 | Eromlife Co Ltd | Pediococcus pentosaceus erom101, its mutant or variant, immunopotentiating composition, anticancer composition, antimicrobial composition, composition for controlling intestinal function, composition for live bacterium agent, composition for feed, composition for food addition and fermented product containing them, method for culturing them and method for controlling microbial growth |
| CN111705028A (en) * | 2012-06-04 | 2020-09-25 | 基因组股份公司 | Microorganisms and methods for making 4-hydroxybutyrate, 1, 4-butanediol, and related compounds |
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| JP2005218324A (en) * | 2004-02-04 | 2005-08-18 | Eromlife Co Ltd | Pediococcus pentosaceus erom101, its mutant or variant, immunopotentiating composition, anticancer composition, antimicrobial composition, composition for controlling intestinal function, composition for live bacterium agent, composition for feed, composition for food addition and fermented product containing them, method for culturing them and method for controlling microbial growth |
| CN111705028A (en) * | 2012-06-04 | 2020-09-25 | 基因组股份公司 | Microorganisms and methods for making 4-hydroxybutyrate, 1, 4-butanediol, and related compounds |
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| Title |
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| Antimicrobial properties of Pediococcus acidilactici and Pediococcus pentosaceus isolated from silage;Joanna Ivy Irorita Fugaban;J Appl Microbiol;20210808;第132卷(第1期);全文 * |
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