Lactobacillus plantarum PC with functions of relaxing bowels and reducing blood sugar and application of lactobacillus plantarum PC

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum PC (Lactobacillus plantarum) and application of the lactobacillus plantarum PC. The lactobacillus plantarum PC strain provided by the invention has the characteristics of excellent acid resistance, bile salt resistance and the like, and is a multifunctional strain. The strain itself and metabolites thereof have multiple effects of relaxing bowel, reducing blood sugar, resisting oxidation and resisting bacteria, can shorten the defecation time of mice, improve the defecation number, promote the propulsion rate of small intestines, improve the water content of excrement, inhibit alpha-amylase and alpha-glucosidase, and can remove hydroxyl free radicals, DPPH free radicals and O2 <->. Multiple pathogenic bacteria and putrefying bacteria such as escherichia coli and staphylococcus aureus can be inhibited. The lactobacillus plantarum PC and the metabolite thereof provided by the invention can be applied to preparation of food, health care products, medicines and feeds with the characteristics of relaxing bowel, reducing blood sugar, resisting oxidation and resisting bacteria, and have wide application prospects and application values.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a plant lactobacillus ( Lactobacillus plantarum ) PC and its applications. Background Art

[0002] Constipation is a common digestive system symptom, which is mainly manifested by reduced bowel movement frequency, difficulty in defecation, dry and hard stool, small amount of stool, anal discomfort, heaviness and blood in stool during defecation, etc., accompanied by various discomfort symptoms, which seriously affect human health. If the human body is in a state of constipation for a long time, toxins in the body cannot be removed in time, and are repeatedly absorbed and utilized by the intestines and enter the blood circulation, thereby causing harm to the organs in the body, leading to neurasthenia, toxemia, metabolic diseases and other related diseases, and is a complication of many diseases. With the acceleration of the pace of life, factors such as eating habits, mental stress, and age can induce constipation.

[0003] At present, the commonly used clinical treatments for constipation are drugs and surgery. Drug treatment includes taking laxatives, lubricants, and gastrointestinal prokinetic drugs, such as lactose, polyethylene glycol, and lubiprostone. Although drug treatment has a certain effect, long-term medication has great side effects. The body may develop drug resistance, drug dependence, nausea, and severe diarrhea, causing intestinal microecological imbalance in patients. Once discontinued, constipation symptoms will be aggravated. Surgical treatment only treats the symptoms but not the root cause. It is easy to relapse if you don't pay attention to it. It can be seen that clinical treatment still has the characteristics of high difficulty and easy recurrence. Therefore, it is very necessary to find a safe and effective diet or health intervention measure to prevent and treat constipation. The common cause of constipation is intestinal flora imbalance. Lactobacillus, as a probiotic, performs well in regulating intestinal flora and improving intestinal environment, and has a significant effect on relieving constipation. After entering the human body, lactic acid bacteria can benefit constipation patients by regulating the balance of intestinal flora, promoting intestinal peristalsis, changing the intestinal environment, affecting intestinal movement and secretion of intestinal hormones, and improving immunity. Lactobacillus plantarum is a type of lactic acid bacteria. Its strain morphology is straight or curved rod-shaped, generally single, sometimes in pairs or chains, with an optimal growth temperature of 30-35°C. It is an anaerobic or facultative anaerobic bacterium. Lactobacillus plantarum has been reported to be effective in relieving and improving constipation. For example, a strain of Lactobacillus plantarum Lp18 with a deposit number of CCTCCNO: M20231524 disclosed in Chinese invention patent application 202311446476.3 improves constipation. However, the prior art has the disadvantages of a single function of Lactobacillus plantarum in relieving constipation and weak antibacterial ability. Therefore, the art still needs to develop new strains of Lactobacillus plantarum with rich constipation relief functions and strong antibacterial ability.

[0004] Diabetes is a disease characterized by high blood sugar. The main cause is the defect of insulin secretion, or the impaired biological action of insulin, or the combination of the two. There are many causes of diabetes. For example, congenital factors are genetic factors, which lead to impaired biological action of insulin and cause high blood sugar. There are also acquired factors, such as obesity. Obesity is caused by the lack of self-control in diet, which in turn causes high blood sugar. Bad living habits can also lead to high blood sugar in the body, such as high-fat diet, staying up late for a long time, lack of exercise, etc. In addition, the decline of insulin function with age can also cause high blood sugar in the body.

[0005] Probiotics can improve the ecological balance of host microorganisms, help the absorption of nutrients in the human body, and produce important nutrients. Probiotics help the human body lower blood sugar concentration, thereby inhibiting the symptoms of pre-diabetes. Lactic acid bacteria in probiotics can successfully arrive, adhere and successfully colonize in the ileum and colon with a large number of live bacteria. Lactic acid bacteria regulate the body's enzyme-linked immune system and improve the body's immunity. Lactic acid bacteria can also produce active substances to remove excess free radicals in the body, reduce oxidative stress, and improve antioxidant capacity, thereby improving the level of glycosylated hemoglobin in diabetic patients and achieving the function of lowering blood sugar. At the same time, probiotics can inhibit a series of enzymes in the human body that cause increased blood sugar content, such as α-glucosidase and α-amylase, thereby reducing the sugar content in the body and achieving the effect of preventing and solving the symptoms of hyperglycemia in the body. Probiotics inhibit the activity of α-glucosidase, the speed at which high-sugar compounds break down glucose will slow down, the absorption of glucose in the intestine will decrease, postprandial blood sugar will decrease, and the occurrence of complications will decrease. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Lactobacillus plantarum PC which has the functions of moistening the intestines and relieving constipation, regulating blood sugar, and simultaneously having antibacterial and antioxidant properties and application thereof.

[0007] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a plant lactobacillus ( Lactobacillus plantarum )PC, its deposit number is CGMCC No. 27298.

[0008] In a second aspect, the present invention provides a bacterial agent containing the Lactobacillus plantarum PC.

[0009] In a third aspect, the present invention provides a fermentation product obtained by fermenting the Lactobacillus plantarum PC or the bacterial agent.

[0010] In a fourth aspect, the present invention provides any of the following applications of the Lactobacillus plantarum PC, the bacterial agent or the fermentation product: 1) Used to prepare products for moistening the intestines and relieving constipation; 2) Used for preparing products for lowering blood sugar; 3) Used for preparing antioxidant products; 4) Used to prepare antibacterial products; 5) Used for laxative purposes other than disease treatment; 6) For non-disease treatment purposes, such as lowering blood sugar; 7) Antioxidant for non-disease treatment purposes; 8) Antibacterial for non-disease treatment purposes.

[0011] Preferably, the bowel-moistening and laxative effect includes at least one of the following functional performances: shortening defecation time, increasing the number of stool particles, promoting small intestinal propulsion rate, and increasing feces water content.

[0012] Preferably, the blood sugar lowering includes at least one of the following functional manifestations: inhibiting the enzymatic activity of α-amylase, inhibiting the enzymatic activity of α-glucosidase, improving blood sugar, improving glucose tolerance, and reducing the content of glycosylated hemoglobin.

[0013] Preferably, the anti-oxidation comprises at least one of the following functional performances: scavenging hydroxyl free radicals, scavenging superoxide anions, and scavenging DPPH free radicals.

[0014] Preferably, the antibacterial activity includes at least one of the following functional performances: inhibiting or killing Staphylococcus aureus, inhibiting or killing Listeria monocytogenes, inhibiting or killing Bacillus cereus, inhibiting or killing Escherichia coli, inhibiting or killing Salmonella, and inhibiting or killing Pseudomonas aeruginosa.

[0015] In a fifth aspect, the present invention provides a product having laxative, blood sugar lowering, anti-oxidation and antibacterial functions, wherein the effective active ingredients of the product include the plant lactobacillus PC, the bacterial agent or the fermentation product.

[0016] Preferably, the product is food, medicine, health product, feed, food additive or feed additive.

[0017] Beneficial effects: The present invention provides a plant lactobacillus ( Lactobacillus plantarum ) PC, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 9, 2023, with a deposit number of CGMCC No. 27298. The plant lactobacillus PC strain provided by the present invention exhibits excellent acid resistance, bile salt resistance and other characteristics, and is a multifunctional strain. The strain itself and its metabolites have multiple functions of laxative, hypoglycemic, antioxidant and antibacterial, which can promote the shortening of defecation time, increase the number of defecation particles, promote small intestinal propulsion rate, increase fecal water content, inhibit α-amylase and α-glucosidase, and can scavenge hydroxyl free radicals, DPPH free radicals and O2- ·, can inhibit multiple pathogenic bacteria and spoilage bacteria such as Escherichia coli and Staphylococcus aureus. The plant lactobacillus PC and its metabolites provided by the present invention can be used to prepare foods, health products, medicines and feeds with laxative, blood sugar lowering, antioxidant and antibacterial properties, and have broad application prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0019] Figure 1 This is the morphology of Lactobacillus plantarum PC.

[0020] Figure 2 This is the phylogenetic tree of Lactobacillus plantarum PC.

[0021] Figure 3 This is the hemolysis result of Lactobacillus plantarum PC.

[0022] Figure 4 This is the growth curve of Lactobacillus plantarum PC.

[0023] Figure 5 This is the effect of Lactobacillus plantarum PC on the first defecation time, the number of defecation particles in 5 hours, fecal water content, and small intestinal propulsion rate in constipated mice.

[0024] Figure 6 This is the effect of Lactobacillus plantarum PC on the body weight of diabetic mice.

[0025] Figure 7 The changes of blood glucose levels before and after meals in each group of mice.

[0026] Figure 8 The effect of Lactobacillus plantarum PC on oral glucose tolerance and AUC in mice.

[0027] Fig. 9 This is the effect of Lactobacillus plantarum PC on serum glycosylated hemoglobin in mice. DETAILED DESCRIPTION

[0028] The present invention provides a strain of Lactobacillus plantarum PC with laxative, blood sugar regulating, antioxidant and antibacterial effects. The strain was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Committee on May 9, 2023, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 27298.

[0029] The accession number of the 16S rDNA sequence of the Lactobacillus plantarum PC of the present invention in NCBI is MW795696.

[0030] The plant lactobacillus PC of the present invention is separated from fermented sauce in Hebei Province, which is a traditional fermented food in China and has high safety.

[0031] In the hemolytic study, the Lactobacillus plantarum PC of the present invention did not show a hemolytic ring, which further proves that it has high safety and can be applied to the fields of food, medicine, health care products, feed, etc.

[0032] The biological characteristics of Lactobacillus plantarum PC of the present invention are as follows: (1) The colonies are round, smooth, with neat edges and white color. They are rod-shaped Gram-positive bacteria.

[0033] (2) It has excellent acid resistance, bile salt resistance, hydrophobicity and heat resistance.

[0034] (3) The plant grows fast, entering the rapid growth phase within 2 hours and rapidly growing to the stable phase within 2-10 hours.

[0035] The present invention also provides the use of plant lactobacillus PC in the preparation of food, medicine, health care product, feed, food additive or feed additive with laxative, blood sugar lowering, anti-oxidation or antibacterial functions.

[0036] The invention uses Lactobacillus plantarum PC or Lactobacillus plantarum PC containing its fermentation metabolites as food, medicine, health care product, feed, food additive or feed additive or their components. Among them, Lactobacillus plantarum PC or its fermentation liquid containing bacteria can be used as the only active ingredient or can be used as an active ingredient together with other active ingredients.

[0037] The present invention further provides a food, medicine, health product, feed, food additive or feed additive or a combination thereof, which contains the plant lactobacillus PC and can be used for moistening the intestines and facilitating bowel movements, preventing type II diabetes, and resisting oxidation or inhibiting bacteria.

[0038] Preferably, the intestinal moisturizing and laxative effect includes shortening the defecation time, increasing the number of stool particles, promoting the propulsion rate of the small intestine, and increasing the water content of feces.

[0039] Preferably, the prevention of diabetes includes the inhibition of α-amylase and α-glucosidase or the improvement and alleviation of blood sugar, glucose tolerance and glycosylated hemoglobin.

[0040] Preferably, the antioxidant activity includes scavenging one or more of hydroxyl radicals, superoxide anions or DPPH radicals.

[0041] Preferably, the antibacterial property includes inhibiting or killing one or more of Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Salmonella or Pseudomonas aeruginosa.

[0042] The plant lactobacillus PC of the present invention has good acid resistance, bile salt resistance, hydrophobicity, heat resistance and high safety. Through the study of intestinal lubrication and laxative in mice, plant lactobacillus PC can improve constipation, which is manifested in shortening the first defecation time, increasing the number of defecation grains in mice, promoting the small intestinal propulsion rate of mice, and increasing the feces water content of mice, effectively improving and alleviating the defecation of constipated mice. Through the study of hypoglycemic in mice, plant lactobacillus PC can alleviate the trend of weight loss in diabetic mice; during oral glucose tolerance test, the bacteria has the ability to improve the glucose tolerance of mice, alleviate the trend of increased blood sugar levels in mice, and reduce the pre-meal and post-meal blood sugar of mice; the bacteria can control the increase of organ index of mice and protect the organs of hyperglycemic mice; the bacteria significantly reduces the glycosylated hemoglobin content of mice, effectively improves the blood lipid level in the body, and has a certain effect on regulating the blood lipid metabolism of mice. At the same time, Lactobacillus plantarum PC showed inhibitory effects on several conditional pathogens; it had a scavenging effect on hydroxyl free radicals, superoxide anions or DPPH free radicals, with a scavenging rate of 10%-20%; the inhibition rate of α-glucosidase and α-amylase was 10%-12%.

[0043] On the other hand, Lactobacillus plantarum PC and its fermentation metabolites shortened fecal defecation time by 32%-49%, increased small intestinal propulsion rate by 35%-44%, increased fecal water content by 11%-19%, inhibited α-glucosidase and α-amylase by about 25%, and cleared hydroxyl radicals, superoxide anions or DPPH radicals by 20%-29%. The inhibition zone for several conditional pathogens was 10-20 mm.

[0044] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0047] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0048] Example 1 This example illustrates the screening and identification process of Lactobacillus plantarum PC.

[0049] 1. Sample separation and initial screening of lactic acid bacteria The traditional fermented sauce samples collected from Hebei Province were diluted and spread on MRS solid medium containing CaCO3 to separate the colonies that produced transparent circles. The colonies that produced transparent circles were further purified and a total of 6 lactic acid bacteria were screened out.

[0050] 2. Rescreening of probiotic strains The six lactic acid strains were screened for their probiotic properties, and the acid resistance, bile resistance and hydrophobicity of the strains were tested to ensure that the screened strains could adapt to the human environment and produce probiotic effects.

[0051] (1) Acid resistance test Lactobacillus was added at 1% ( v / v ) were inoculated in MRS liquid medium with pH values ​​of 2.0, 3.0, and 4.0, respectively, and cultured at 37 °C. The bacterial suspension was taken at 0 h, 2 h, and 4 h for smearing and viable bacteria were counted. The survival rate of strain PC was the highest when incubated in a simulated environment of pH 2.0 for 4 h (Table 1).

[0052] (2) Bile salt tolerance test Lactobacillus was added at 1% ( v / v) were inoculated in MRS liquid culture medium containing bile salt concentrations of 0.1% and 0.3%, respectively, and cultured at 37 °C. The bacterial suspension was taken for smearing at 0 h, 2 h, and 4 h, and the survival rate was calculated. The survival rates of strains Z-1 and PC were the highest (Table 2).

[0053] (3) Hydrophobicity test Use PBS to adjust the absorbance of the bacterial suspension to OD 600nm = 0.25 ± 0.05. Take 3 mL of the bacterial suspension to be used, add an equal volume of xylene, vortex at high speed for 2 min, let stand for 20 min to separate the layers, take the lower aqueous phase and measure its absorbance at a wavelength of 600 nm.

[0054] .

[0055] Where: A0, A--absorbance of bacterial solution at 600nm before and after mixing with xylene.

[0056] Among the 6 strains of lactic acid bacteria, strain PC has the highest hydrophobicity, which can reach 62.35%. Good hydrophobicity means that it has a strong adhesion ability.

[0057] 3. Identification of strain PC (1) Morphological identification The strain PC was streaked onto MRS solid medium and cultured at 37 °C for 48 h. A single colony was picked for Gram staining to observe the strain and colony morphology. Figure 1 The colonies of strain PC were round, smooth, and white in color. Gram staining and microscopic observation showed that they were rod-shaped Gram-positive bacteria, which were consistent with the characteristics of lactic acid bacteria.

[0058] (2) Physiological and biochemical identification API identification reagent strips (bioMérieux) were used for physiological and biochemical identification of strain PC, and the results are shown in Table 3.

[0059] (3) 16S rDNA sequence identification The purified strain was sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the 16S rDNA of strain PC was uploaded to NCBI's GeneBank, obtaining the accession number MW795696. The sequencing results were subjected to Blast analysis in the GeneBank database, and a phylogenetic tree was constructed using MEGA7 software ( Figure 2 ), strain PC and Lactobacillus plantarum Lactobacillus plantarumThe homology was the highest, at 99.93%. Therefore, strain PC was identified as Lactobacillus plantarum and named Lactobacillus plantarum PC.

[0060] Example 2 In this example, the hemolytic activity of Lactobacillus plantarum PC strain was analyzed.

[0061] Lactobacillus plantarum PC was cultured to the second generation, diluted and spread on blood agar plates under a sterile environment, and cultured at 37 ℃ for 48 h. Staphylococcus aureus was used as a control, and the hemolysis of Lactobacillus plantarum PC and the control bacteria was observed.

[0062] Figure 3 The results showed that Lactobacillus plantarum PC did not show a hemolytic zone on the blood agar plate, while the control Staphylococcus aureus showed a hemolytic zone, further indicating that Lactobacillus plantarum PC has a higher safety.

[0063] Example 3 This example provides a growth curve of the Lactobacillus plantarum PC strain.

[0064] The growth curve of Lactobacillus plantarum PC was determined by turbidimetry. Figure 4 Lactobacillus plantarum PC grows fast, entering the logarithmic phase after 2 h of culture, growing rapidly during the 2-12 h period, and entering the stable phase after 14 h.

[0065] Example 4 This example analyzes the bowel-moistening and laxative effect of Lactobacillus plantarum PC strain in mice.

[0066] Cultivate Lactobacillus plantarum PC, collect the cells by centrifugation, and adjust the cell concentration to 1×10 9 CFU / mL.

[0067] 1. Mouse grouping and model establishment Thirty-six healthy 6-week-old male Kunming (KM) mice were fed with a basic diet and kept in stainless steel cages at room temperature of 24±2℃, relative humidity of 50±10%, and a 12-h light-dark cycle. After one week of adaptation, the 36 male KM mice were randomly divided into three groups, each with 12 mice, namely the normal group (N), the constipation control group (M), and the Lactobacillus plantarum PC group (P).

[0068] In the first week of the experiment, the mice were fed with basic feed. Six mice were randomly selected from each group for defecation experiment, and the remaining six mice were used for small intestinal motility experiment. The N group was gavaged with distilled water, and the M and P groups were gavaged with loperamide hydrochloride solution. The gavage dose for the defecation experiment mice was 7 mg / kg, and the gavage dose for the small intestinal motility experiment mice was 4 mg / kg. This was continued for 14 days to establish a mouse constipation model.

[0069] Starting from the third week, the rats were fed with a basic diet, and the N and M groups were gavaged with distilled water, and the P group was gavaged with a suspension of Lactobacillus plantarum PC for a total of 14 days. During the entire experimental period, the rats were supplied with normal diet and drinking water.

[0070] 2. Determination of the first defecation time, number of defecation pellets in 5 hours and feces water content in mice On the 29th day, mice in each group were fasted for 16 hours but not water. Mice in group N were gavaged with distilled water, and mice in groups M and P were gavaged with loperamide hydrochloride solution. 30 minutes after administration, groups N and M were gavaged with ink, and group P was gavaged with ink containing PC suspension of Lactobacillus plantarum, and placed in a clean cage with absorbent paper. All mice were housed in a single cage and fed with normal water. Starting from gavage with ink, the time of the first black stool of each mouse was recorded, and then feces were collected every 30 minutes for a total of 5 hours. The number of feces of each mouse within 5 hours was recorded, and the collected 5-hour feces were weighed on a balance, and the wet weight of the feces was recorded. After the collected feces were dried, the dry weight of the feces was recorded. The count was terminated 5 hours after the last mouse in group M collected feces.

[0071] .

[0072] 3. Determination of small intestinal propulsion rate in mice On the 29th day, mice in each group were fasted but not watered for 16 hours. Mice in group N were gavaged with distilled water, and mice in groups M and P were gavaged with loperamide hydrochloride solution. 30 minutes after administration, mice in groups N and M were gavaged with ink, and mice in group P were gavaged with ink containing PC suspension of Lactobacillus plantarum. Starting from gavage with ink, mice were killed by dislocation 25 minutes later. The abdominal cavity was opened to separate the mesentery, and the intestinal tube from the pylorus at the upper end to the ileocecal part at the lower end was cut and placed on a tray. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink propulsion length". The small intestine propulsion rate was calculated: .

[0073] The results showed that the number of stool particles, fecal water content and small intestinal propulsion rate in group M were significantly lower than those in group N, and the first defecation time was significantly higher than that in group N, indicating that the loperamide hydrochloride induced constipation model in mice successfully. The number of stool particles in group Lactobacillus plantarum PC was significantly higher than that in group M (P<0.05), the fecal water content and small intestinal propulsion rate were significantly higher than those in group M (P<0.001), and the first defecation time was significantly lower than that in group M (P<0.05), indicating that Lactobacillus plantarum PC effectively improved the constipation of mice and had a significant effect on restoring intestinal function ( Figure 5 ).

[0074] Example 5 This example analyzes the hypoglycemic effect of Lactobacillus plantarum PC strain.

[0075] To investigate the in vitro hypoglycemic properties of Lactobacillus plantarum PC and its metabolites, including their effects on α-glucosidase and α-amylase.

[0076] 1. Inhibition rate of α-glucosidase The determination method is as follows: take 50 μL PBS solution and mix it with an equal volume of PNPG, add 20 μL sample, incubate at 37 ℃ for 10 minutes, then add 30 μL α-glucosidase solution, continue incubation for 20 minutes, and finally add 50 μL NaCO3 to terminate the reaction, measure the absorbance at 405 nm, and use PBS solution instead of blank control to calculate the inhibition rate of Lactobacillus plantarum PC on α-glucosidase.

[0077] .

[0078] Where: A1 and A2 are the absorbance of the sample group and blank group at 405nm respectively.

[0079] 2. Inhibition rate of α-amylase The determination method is as follows: take an equal volume of the sample and mix it with α-amylase solution, incubate at 37 ℃ for 5 min, then add 0.5 mL of soluble starch solution, continue incubation for 5 min, and finally add 1 mL of DNS solution to terminate the reaction, water bath for 5 min, cool to room temperature, add distilled water to make up the system to 10 mL, and then measure the absorbance at 540 nm. At the same time, make a sample control to calculate the inhibition rate of Lactobacillus plantarum PC on α-amylase.

[0080] .

[0081] Where: A and B are the absorbance of the sample group and the blank group at 540 nm, respectively.

[0082] The inhibition of α-glucosidase and α-amylase by the bacterial suspension and fermentation metabolite supernatant of Lactobacillus plantarum PC was studied. The results in Table 4 show that Lactobacillus plantarum PC and its fermentation metabolites have inhibitory effects on α-glucosidase and α-amylase, and the fermentation metabolites have a good in vitro hypoglycemic effect.

[0083] Example 6 This example analyzes the hypoglycemic effect of Lactobacillus plantarum PC strain in mice.

[0084] Cultivate Lactobacillus plantarum PC, collect the cells by centrifugation, and adjust the cell concentration to 1×10 9 CFU / mL.

[0085] 1. Mouse grouping and model establishment The purchased 30 male KM mice were randomly divided into 5 groups, with 6 mice in each group, namely the normal group (N), the hyperglycemia group (M), the treatment group (P), the Lactobacillus plantarum PC high-dose group (H), and the Lactobacillus plantarum PC low-dose group (L).

[0086] In the first week of the experiment, each group of mice was fed a high-sugar and high-fat diet (except group N) to model the disease. From the second week, mice were gavaged with different doses of samples every day until the end of the experiment. The gavage conditions are shown in Table 5. During the entire experiment, diet and drinking water were supplied normally.

[0087] Weigh the mice in each group every week. Measure and record the blood glucose (before and after meals) of the mice on the last day of each week. Test the oral glucose tolerance of the mice one week before the end of the experiment. Anesthetize the mice and draw blood from the eyeballs. Kill the mice by dislocating the neck, dissect the mice, take out the liver, kidney and spleen of the mice, dry them with absorbent paper and weigh them on a scale. Detect the HbA1c content of the mice. For relevant experimental methods, refer to the instructions in the kit. Detect the contents of T-CHO, HDL-C, LDL-C and TG in the serum of the mice. For experimental methods, refer to the instructions of the relevant ELISA kits.

[0088] 2. Changes in mouse body weight Figure 6 It was shown that at the beginning, mice were evenly distributed to different groups according to their weight, so there was no significant difference in the weight of mice in each group at the beginning (p>0.05). After adapting to the living environment for a week, the food intake of mice in each group increased. After 6 weeks, the weight of mice in group N was the largest, and the weight of group M was significantly lower than that of group N (p<0.05). Studies have shown that the main characteristics of type 2 diabetic mice are polydipsia, polyuria, polyphagia and weight loss, the so-called "three more and one less" phenomenon. At the end of the experiment, the weight of mice H and L gavaged with Lactobacillus plantarum PC was not significantly different from that of group P (p>0.05), among which the weight growth trend of each group gavaged with high-dose strains was significantly higher than that of group M (p<0.05). From the above, it can be seen that gavage with Lactobacillus plantarum PC can effectively improve the trend of weight loss in diabetic mice.

[0089] 3. Changes in blood glucose in mice The fasting blood glucose and postprandial blood glucose of mice were measured during the entire experimental period. Except for group N, the blood glucose levels of other groups injected with STZ rose rapidly. This was because after the injection of STZ, STZ acted on the pancreatic islet cells of mice, causing the fasting blood glucose of each group of mice to rise rapidly. The fasting blood glucose of each modeling group was above 14.7 mmol / L, and the blood glucose value two hours after the meal reached above 21.2 mmol / L.

[0090] After two weeks of continuous gavage, the pre-meal blood glucose of mice in group M continued to rise, while the pre-meal blood glucose of group P decreased significantly. At the end of the experiment, the fasting blood glucose value of group P was 8.0 mmol / L. It may be due to the shorter feeding time that the pre-meal blood glucose value of group P was significantly different from that of group N (p>0.05), but from the trend of blood glucose decrease, the blood glucose of group P quickly approached the normal value. From the third week of the experiment to the end of the experiment, the blood glucose values ​​of mice in each group gavaged with lactic acid bacteria decreased compared with group M, and maintained a slowly decreasing trend. At the end of the experiment, there were significant differences between groups H, L and M (p<0.05), indicating that lactic acid bacteria inhibited the increase in pre-meal blood glucose values ​​of mice. Based on this speculation, as the gavage time increases, the fasting blood glucose of mice will be greatly improved.

[0091] 4. Detection of oral glucose tolerance in mice The blood glucose level in group N dropped to 6.8 mmol / L after 120 min, which was close to the normal value. This also indicated that the glucose tolerance of mice in group N was normal ( Figure 7 ). For mice in group M, the blood glucose level reached a maximum of 28.1 mmol / L at 30 min, and the blood glucose level was still 20.4 mmol / L after 120 min, which was always at a high blood glucose level, indicating that blood glucose regulation in the mice was out of control at this time. There was no significant difference in the AUC value of group P with that of group N (p>0.05), indicating that metformin had a significant effect in lowering blood glucose. There was a significant difference between the high-dose group H and group M (p<0.05). Although the AUC value of group L and Lactobacillus plantarum PC failed to significantly reduce the glucose tolerance of diabetic mice, it can be seen that the two strains of lactic acid bacteria have the ability to improve the glucose tolerance of mice and alleviate the trend of increased blood glucose levels in mice, and this trend is related to the dose of the bacteria ( Figure 8 ).

[0092] 5. Effects on mouse organ coefficients The liver index of group M was 6.60, which was significantly greater than 5.69 of group N (p<0.05); there was no significant difference in the liver index between the normal group N and the oral lactic acid bacteria groups (p>0.05), indicating that oral administration of plant lactobacillus PC played a certain protective role on the liver of mice. There was no significant difference in the kidney index between the positive control group P and oral administration groups H and L and group N (p>0.05), indicating that plant lactobacillus PC can also reduce the impact of hyperglycemia on the kidneys of the body (Table 6).

[0093] 6. Effects on Glycosylated Hemoglobin in Mice At the end of the experiment, there was no significant difference in glycosylated hemoglobin between the N group and the P group (p>0.05), which once again proved that the metformin treatment group had a better effect in lowering blood sugar ( Fig. 9 ). The glycosylated hemoglobin content in the H and L groups decreased significantly and was significantly different from that in the M group (p<0.05). After a certain period of intragastric administration, the ability of H and L to reduce the glycosylated hemoglobin content was not as great as that of the P group, but from the results of the intragastric administration of each strain group, it can be seen that the high dose of Lactobacillus plantarum PC reduced the glycosylated hemoglobin content more significantly.

[0094] 7. Effects on blood lipids in mouse serum There were significant differences in blood lipid levels (p<0.05). Table 7 shows that except for the significant difference in the level of C and N group, the other three blood lipid levels were not significantly different from those of N group (p>0.05). The TC, TG and LDL-C levels of group H were 3.02, 1.31 and 1.40 mmol / L, respectively, which were significantly reduced by 25.4%, 38.2% and 25.1% compared with group M (p<0.05); the HDL-C content of group H was 2.62 mmol / L, which was significantly increased by 29.4% compared with group M. There were significant differences in TC, TG and HDL-C between group L and group M (p<0.05), while there was no significant difference in LDL-C between group L and group M (p>0.05). Comprehensive analysis shows that although the effect of lowering blood lipids is still somewhat different from that of metformin, it can also be explained that oral administration of high-dose plant lactobacillus PC can effectively improve the blood lipid level in the body and has a certain effect on regulating blood lipid metabolism in mice.

[0095] Example 7 In this example, the antioxidant activity of Lactobacillus plantarum PC strain was analyzed.

[0096] 1. Ability to scavenge hydroxyl free radicals Take a sterilized 10 mL centrifuge tube, add 2 mL of 0.05 mmol / L PBS buffer (pH 7.4), 1 mL of 0.75 mmol / L FeSO4, 1 mL of 2.5 nmol / L o-phenanthroline and 1 mL of 20 mmol / L hydrogen peroxide, mix well, then add 1 mL of Lactobacillus plantarum PC sample (bacterial suspension, fermentation metabolite), mix well again, place the centrifuge tube in a 37 °C water bath for 1 h, measure the absorbance at 536 nm for the sample group (denoted as A a ), the sample in the above mixed solution was replaced with 1 mL of H2O2 as the blank group (denoted as A c ), the control group did not contain H2O2 (denoted as A b). The calculation formula for scavenging ability of hydroxyl free radicals is as follows: .

[0097] 2. Ability to scavenge DPPH free radicals Add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution and 2 mL of the sample to be tested (Lactobacillus plantarum PC bacterial suspension, fermentation metabolite) to a sterilized centrifuge tube, mix the two evenly and react at room temperature in the dark for 30 minutes, then centrifuge the mixture at 6000 r / min for 10 minutes, take the supernatant after centrifugation and measure its absorbance at 517 nm, and record the absorbance of the sample group (denoted as A a ), 2 mL of DPPH in the mixture was replaced with an equal volume of anhydrous ethanol as the control group (denoted as A b ), replace 2 mL of the sample to be tested with an equal volume of anhydrous ethanol as the blank group (denoted as A c ), the calculation formula for DPPH free radical scavenging ability is as follows: .

[0098] 3. Ability to remove superoxide anions The determination was carried out using the pyrogallol method. First, 0.1 mL of the sample to be tested (bacterial suspension of Lactobacillus plantarum PC, fermentation metabolite) was pipetted with a pipette, 4.5 mL of 150 mmol / L Tris-HCl buffer and 0.4 mL of 25 mmol / L pyrogallol solution were mixed evenly, and the mixture was placed in a water bath at 25 °C for 10 min. After the reaction was completed, 0.1 mL of hydrochloric acid was pipetted into the mixture, and finally the absorbance of the mixture was measured at 323 nm. The absorbance value of the sample group (denoted as A a ), replace 0.1 mL of the sample to be tested with an equal volume of distilled water as the blank group (denoted as A b ), the calculation formula for the scavenging ability of superoxide anions is as follows: .

[0099] The results are shown in Table 8. Lactobacillus plantarum PC and its metabolites have scavenging effects on hydroxyl radicals, superoxide anions or DPPH radicals, and the antioxidant capacity of fermentation metabolites is better than that of bacterial suspension. The free radical scavenging rate of Lactobacillus plantarum PC is 10%-20%, and the free radical scavenging rate of fermentation metabolites is 20%-29%.

[0100] Example 8 In this example, the antibacterial activity of Lactobacillus plantarum PC strain was analyzed.

[0101] The indicator bacteria selected in the antibacterial analysis were Staphylococcus aureus ATCC 25923, Listeria monocytogenes 10403s, Bacillus cereus ATCC 11778, Escherichia coli ATCC 44752, Pseudomonas aeruginosa ATCC 9027 and Salmonella typhimurium ATCC 14028.

[0102] 1. Inhibitory effect of bacteria on indicator bacteria The antibacterial activity of Lactobacillus plantarum PC was determined by the double-layer plate method. The bottom layer was MRS solid medium. Lactobacillus plantarum PC was inoculated on the medium and cultured at 37°C for 4 h. 6 The nutrient agar medium of CFU / mL indicator bacteria was poured onto the MRS medium and cultured for 12-24 h. The results in Table 9 showed that Lactobacillus plantarum PC had a certain antagonistic effect on the selected indicator bacteria.

[0103] 2. Inhibitory effect of fermentation metabolites on indicator bacteria The antibacterial activity of the fermentation metabolite of Lactobacillus plantarum PC was measured by the Oxford cup agar diffusion method. The concentration of indicator bacteria was adjusted to 10 6 CFU / mL, draw the indicator bacteria into the melted solid nutrient agar medium, mix well, pour 20 mL of solid nutrient agar medium with indicator bacteria into the plate to prepare the antibacterial assay plate. Pipette 100 μL of the fermentation supernatant of Lactobacillus plantarum PC, connect it to the Oxford cup on the plate, diffuse at 4ºC for 10-12 h, and culture at 37ºC for 6-12 h, and measure the size of the inhibition zone. The results in Table 9 show that the fermentation metabolites of Lactobacillus plantarum PC have a significant inhibitory effect on the selected indicator bacteria, and the diameter of the inhibition zone is 10-20 mm.

[0104] Example 9 In this example, the acid production ability of Lactobacillus plantarum PC strain was detected.

[0105] Plant Lactobacillus PC was cultured in MRS medium for 24 h, and the fermentation supernatant was obtained by centrifugation. After filtration through a 0.22 μm membrane, HPLC detection was performed (γ-aminobutyric acid determination required derivatization: 1 mL of NaHCO3 and 1 mL of 1% DNFB derivatization agent were added to the supernatant, and the supernatant was shaken evenly at 60 °C. o (C constant temperature water bath for 1 h in the dark, dilute to 10 mL, and filter through a 0.22 μm filter membrane).

[0106] Lactic acid, acetic acid, phenyllactic acid and γ-aminobutyric acid were prepared at different concentrations of 1-500 µg / mL, and their peak areas were detected by HPLC. The horizontal axis was the organic acid content, and the vertical axis was the peak area. The standard curve was drawn, and the content of each organic acid in the fermentation broth was calculated based on the standard curve. The chromatographic conditions of lactic acid, acetic acid and phenyllactic acid were: Waters e2695 high performance liquid chromatograph and AminexHPX-87H column (300mm × 7.8mm), the mobile phase was 5mmol / L dilute sulfuric acid solution, isocratic elution, flow rate 0.5mL / min, detection wavelength 210nm, column temperature 50ºC, injection volume 20µL. The chromatographic conditions of γ-aminobutyric acid were: mobile phase A was 1:1 acetonitrile and ultrapure water, mobile phase B was 0.02mol / L PB solution, column temperature 35℃, flow rate 1.0 mL / min, injection volume 20 μL, wavelength 360 nm.

[0107] The results showed that the γ-aminobutyric acid production of Lactobacillus plantarum PC was 291.6 mg / 100mL, the lactic acid production was 753.42 mg / 100mL, the acetic acid production was 230.67 mg / 100mL, and the phenyllactic acid production was 4.5 mg / 100mL (Table 10).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum )PC, its deposit number is CGMCC No. 27298.

2. A bacterial agent containing the plant lactobacillus PC according to claim 1.

3. A fermentation product, characterized in that The method is prepared by fermenting the plant lactobacillus PC described in claim 1 or the bacterial agent described in claim 2.

4. Any of the following uses of the Lactobacillus plantarum PC according to claim 1, the bacterial agent according to claim 2, or the fermentation product according to claim 3: 1) Used to prepare products for moistening the intestines and relieving constipation; 2) Used for preparing blood sugar lowering products; 3) Used for preparing antioxidant products; 4) Used to prepare antibacterial products; 5) Used for laxative purposes other than disease treatment; 6) For non-disease treatment purposes, such as lowering blood sugar; 7) Antioxidant for non-disease treatment purposes; 8) Antibacterial for non-disease treatment purposes.

5. The application according to claim 4, characterized in that: The intestinal moistening and laxative effect includes at least one of the following functional manifestations: shortening defecation time, increasing the number of stool particles, promoting small intestinal propulsion rate, and increasing feces water content.

6. The use according to claim 4, characterized in that: The hypoglycemic effect includes at least one of the following functional manifestations: inhibiting the enzymatic activity of α-amylase, inhibiting the enzymatic activity of α-glucosidase, improving blood sugar, improving glucose tolerance, and reducing the content of glycosylated hemoglobin.

7. The use according to claim 4, characterized in that: The anti-oxidation includes at least one of the following functional performances: scavenging hydroxyl free radicals, scavenging superoxide anions, and scavenging DPPH free radicals.

8. The use according to claim 4, characterized in that: The antibacterial activity includes at least one of the following functional performances: inhibiting or killing Staphylococcus aureus, inhibiting or killing Listeria monocytogenes, inhibiting or killing Bacillus cereus, inhibiting or killing Escherichia coli, inhibiting or killing Salmonella, and inhibiting or killing Pseudomonas aeruginosa.

9. A product with laxative, blood sugar lowering, antioxidant and antibacterial functions, characterized in that: The effective active ingredients of the product include the plant lactobacillus PC described in claim 1, the bacterial agent described in claim 2 or the fermentation product described in claim 3.

10. The product according to claim 9, characterized in that: The product is food, medicine, health product, feed, food additive or feed additive.

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