CN107502575B - Lactobacillus plantarum with high alpha-glucosidase inhibition activity - Google Patents
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Abstract
The invention discloses a lactobacillus plantarum with high alpha-glucosidase inhibitory activity, which is classified and named as: lactobacillus plantarum, which has been deposited in China general microbiological culture Collection center (CGMCC) in 2017 at 8 and 29 months, with the preservation number of CGMCC NO. 14573; according to the screening of the lactobacillus plantarum, the lactobacillus plantarum has alpha-glucosidase in-vitro inhibitory activity; the survival rate of the lactobacillus plantarum reaches 71.04% under the acidic condition of pH2.0, the survival rate of the lactobacillus plantarum in 2.0% cholate deionized water is 77.14%, the tolerance to artificial saliva and intestinal juice is higher than 90%, the tolerance to gastric juice also reaches 52.49%, and the survival rate of the lactobacillus plantarum after being sequentially digested by the artificial saliva is 88.27%; the lactobacillus plantarum has good prevention and treatment effects on type 2 diabetes mellitus; the lactobacillus plantarum and a protective agent are prepared into freeze-dried bacterial powder with high viable count, and the viable count is up to 87 percent before freeze-drying; in addition, the lactobacillus plantarum also has a good fermentation effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to lactobacillus plantarum with high alpha-glucosidase inhibition activity and screening and application thereof.
Background
Diabetes is a metabolic disorder characterized by chronic hyperglycemia due to either inadequate insulin secretion (type 1) or insulin resistance (type 2). Diabetes has become a major disease at present, causing great damage to human life and health. According to data of the world health organization, the prevalence rate of Chinese diabetes is about 1.1 hundred million, which accounts for about one tenth of the total number of Chinese adults, in 2017. According to the existing trend, by 2040 years, more than 1.5 hundred million patients in China have diabetes mortality rate which is greater than the sum of the mortality rates of AIDS, tuberculosis and malaria. Therefore, researches on drugs and functional foods for diabetes become a current focus.
Carbohydrates in food are absorbed in the intestinal tract mainly in the form of monosaccharides. After polysaccharide substances such as starch and the like ingested by a human body are degraded by saliva and pancreatic alpha-amylase to generate oligosaccharide and disaccharide, the polysaccharide substances can be absorbed only after being subjected to enzymolysis under the action of alpha-glucosidase to generate glucose. Therefore, alpha-glucosidase becomes an effective target for inhibiting carbohydrate absorption. At present, the clinically applied alpha-glucosidase inhibitor hypoglycemic drugs mainly comprise acarbose, voglibose and miglitol, but almost all drugs cause certain adverse reactions or side effects. A great deal of research at present shows that the blood sugar reducing and oxidation resisting capacities of some probiotics play a beneficial role in treating type 2 diabetes of human beings, such as lactobacillus plantarum, lactobacillus acidophilus, bifidobacterium, lactobacillus casei and the like. The probiotic is taken to improve the intestinal function, and simultaneously, the absorption of monosaccharide substances such as glucose and the like can be delayed, thereby being beneficial to the development of functional foods and medicines thereof for diabetes patients and obesity people.
Probiotics (probiotics) are a class of microorganisms that are beneficial to hosts such as humans and animals. After being eaten by human or animals, the probiotic preparation can only play a probiotic role on a host when a large number of live bacteria successfully reach, adhere and are successfully planted in ileum and colon parts. During this digestion process, the environment in the mouth, the low pH environment of the stomach and the antibacterial substances such as pepsin in the gastric juice act as the first natural barrier preventing the probiotic from entering the intestine. Therefore, the higher tolerance of the somatic cells to the gastrointestinal environment is important for exerting the probiotic effect.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
Another purpose of the invention is to provide a Lactobacillus plantarum strain with high alpha-glucosidase inhibition activity;
still another object of the present invention is to provide a method for screening lactobacillus plantarum having a high α -glucosidase inhibitory activity, which comprises primarily screening a laboratory deposited strain for the high α -glucosidase inhibitory activity of lactobacillus plantarum; 16S rDNA sequencing is used for taxonomic identification; and performing probiotic property determination on the lactobacillus plantarum for a plurality of probiotic indicators;
the invention also aims to provide an application method of lactobacillus plantarum with high alpha-glucosidase inhibition activity, namely lactobacillus plantarum and a protective agent are added to prepare active freeze-dried bacterial powder, and the active freeze-dried bacterial powder can be used for probiotic supplements and adjuvant therapy of diabetes.
Still another object of the present invention is to provide a method for fermenting lactobacillus plantarum having a high α -glucosidase inhibitory activity, which is to use the lactobacillus plantarum in the fermentation of animal milk, soybean milk, peanut milk, vegetables or fruit juice to obtain functional food suitable for diabetics.
To achieve these objects and other advantages in accordance with the present invention, there is provided a lactobacillus plantarum having a high α -glucosidase inhibitory activity, wherein the strain is classified under the names: lactobacillus plantarum, which has been deposited in the China general microbiological culture Collection center in 8-29 th 2017 at the deposition address of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC NO. 14573.
A screening method of lactobacillus plantarum with high alpha-glucosidase inhibition activity, wherein the lactobacillus plantarum screen is selected from a laboratory preserved strain of agricultural product processing research institute of Chinese academy of agricultural sciences, the strain is deposited in a laboratory with the deposit number of BLP12, and the screening steps are as follows:
step one, primarily screening a strain BLP12 with the highest alpha-glucosidase inhibition activity from lactic acid bacteria preserved in a glycerol tube in a laboratory, wherein the primary screening comprises sample preparation and alpha-glucosidase inhibition activity determination;
step two, determining the 16S rDNA gene sequence of the strain BLP12 obtained in the step one by preliminary screening, and performing taxonomic identification;
and step three, measuring the probiotic characteristics of the strain BLP12 aiming at a plurality of probiotic indexes.
Preferably, the sample preparation in the first step includes preparation of cell metabolites and cell contents, and the cell metabolites and the cell contents are filtered through a 0.22 μm water system filter membrane and stored at-80 ℃ for later use.
Preferably, the preparation process of the cell content comprises ultrasonication, wherein the ultrasonication is carried out under ice bath conditions, and the power is 200W, and the ultrasonication is carried out for 10-12min by a pulse of 3-5 s.
Preferably, the step one of measuring the α -glucosidase inhibitory activity includes measuring the inhibition rate of the strain BLP12 on the intestinal α -glucosidase activity of the mouse.
Preferably, step two further comprises performing PCR amplification with bacterial 16S rDNA amplification universal primers 27F and 1492R.
Preferably, the probiotic indicators in step three include:
(1) acid tolerance, higher survival rate under pH2.0;
(2) bile salt tolerance, good growth condition after being cultured in deionized water containing 2% bile salt for 24 h;
(3) can survive in artificial simulated saliva, gastric juice and intestinal juice;
(4) after a series of digestions of artificial simulated saliva, gastric juice and intestinal juice in sequence, the biological enzyme still can survive well;
(5) the growth period is short.
Use of lactobacillus plantarum having a high alpha-glucosidase inhibitory activity, wherein the lactobacillus plantarum is used for the prevention and treatment of type 2 diabetes.
An application method of lactobacillus plantarum with high alpha-glucosidase inhibition activity is characterized in that lactobacillus plantarum is added with a protective agent to prepare active freeze-dried fungus powder, wherein the protective agent comprises 5% -15% of skim milk and 0.5% -1.5% of L-glutamic acid, and the active freeze-dried fungus powder is prepared through the following steps:
step A, inoculating the strain into an MRS broth culture medium with an inoculum size of 4% for mass fermentation, culturing for 16-20h at 36-38 ℃, after microscopic examination confirms no pollution, centrifuging the cultured bacterial liquid at 6000r/min at 4 ℃ for 10min, and collecting thalli;
and step B, mixing the protective agent and the bacteria according to the volume ratio of 1:1-3, and freeze-drying to obtain the bacterial powder containing the lactobacillus plantarum.
A fermentation method of lactobacillus plantarum with high alpha-glucosidase inhibition activity is characterized in that lactobacillus plantarum is inoculated into animal milk, soybean milk, peanut milk, green vegetables or fruit juice for fermentation, and the fermentation steps of the lactobacillus plantarum are as follows: before inoculation, heating the fermentation substrate to 65 ℃ and keeping the temperature for 30min or 75 ℃ for 10s for disinfection treatment; and (3) inoculating the lactobacillus plantarum when the fermentation substrate is cooled to about 40 ℃, and uniformly stirring until the fermentation substrate foams. Sealing, maintaining the temperature at 40-41 deg.C, and fermenting for 8 hr to obtain functional food suitable for diabetic patients.
The invention at least comprises the following beneficial effects:
the lactobacillus plantarum has high alpha-glucosaccharase in-vitro inhibitory activity, and when the concentration of CFS of the lactobacillus plantarum is 1 multiplied by 1010The inhibition rate of the activity of alpha-glucosidase in intestinal tracts of mice is 71.22% when cfu/mL is adopted, and the CFS concentration is 1 multiplied by 109The inhibition rate of cfu/mL on the activity of alpha-glucosidase in intestinal tracts of mice is 63.64 percent, and the fasting blood glucose value and the glucose of type 2 diabetic mice are simultaneously controlledThe glycosylated hemoglobin has good intervention effect; the lactobacillus plantarum has good ability of living in a human body digestion environment; the lactobacillus plantarum-containing bacterial powder disclosed by the invention has high activity, can be directly used for supplementing probiotics to human bodies, is simple in preparation method, and is convenient to store and transport; the lactobacillus plantarum has a short growth cycle and relatively high activity; the lactobacillus plantarum is applied to the production of dairy products or is added into functional food as a food ingredient, for example, the lactobacillus plantarum is inoculated into animal milk, soybean milk, peanut milk, green vegetables or fruit juice to ferment to obtain a fermentation product, so that the lactobacillus plantarum is suitable for being eaten by diabetics and can achieve good prevention and intervention effects on diabetes and obesity.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the inhibitory activity of Lactobacillus plantarum BLP12 of the present invention on mouse intestinal α -glucosidase;
FIG. 2 is a schematic diagram of the ultraviolet gel electrophoresis imaging of the 16S rDNA of Lactobacillus plantarum BLP12 according to the present invention;
FIG. 3 is a schematic diagram of the growth curve of Lactobacillus plantarum BLP12 according to the invention;
FIG. 4 is a schematic diagram showing the measurement of fasting blood glucose value of each group after feeding lactobacillus plantarum BLP12 of the present invention for 13 weeks in the experiment of prevention and treatment of type 2 diabetes in mice;
FIG. 5 is a schematic diagram showing the measurement of glycosylated hemoglobin value of each group after 13 weeks feeding in the experiment of prevention and treatment of type 2 diabetes in mice by Lactobacillus plantarum BLP12 according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
As shown in figures 1-5, the invention provides a lactobacillus plantarum with high alpha-glucosidase inhibitory activity, and the classification and designation of the strain are as follows: lactobacillus plantarum, which is preserved in the China academy of sciences microbial strain collection center in 2017 in 8-29 th day, with the preservation numbers as follows: CGMCC NO. 14573.
A screening method of lactobacillus plantarum with high alpha-glucosidase inhibition activity is provided, wherein the lactobacillus plantarum screen is selected from laboratory preservation strains of agricultural product processing research institute of Chinese academy of agricultural sciences.
The screening steps according to the lactobacillus plantarum having higher alpha-glucosidase inhibitory activity are as follows:
first, activation of bacterial species and determination of total number of bacterial colonies
Inoculating 77 strains of lactobacillus in glycerol tubes frozen at-80 ℃ into MRS broth, culturing at 37 ℃ for 18h, and activating for three generations for subsequent tests. Inoculating the activated strain into MRS broth culture medium at an inoculum size of 4% (v/v), standing at 37 deg.C for 18h, collecting bacterial liquid, measuring bacterial colony total number according to GB 4789.2-2010, and regulating bacterial liquid concentration to 1 × 109cfu/mL。
Second, sample preparation
(1) Preparation of cell-free metabolites (cell-free experimental superoxide CFS), standing and culturing the activated strain in MRS medium at 37 deg.C for 18h, centrifuging at 4 deg.C for 12000r/min, and collecting thallus. The collected cells were washed 3 times with sterile 0.1M PBS (pH6.8), resuspended in PBS, and the concentration of the cells was adjusted to 1X 109cfu/mL, mix well. Incubating at 37 deg.C for 12h, centrifuging at 4 deg.C for 15min at 12000r/min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain CFS, and storing at-80 deg.C;
(2) preparation of cell content (cell-free intracellular extract CFE), standing and culturing the activated strain in MRS culture medium at 37 deg.C for 18h, centrifuging at 4 deg.C for 12000r/min, and collecting thallus. Will receiveThe collected cells were washed 3 times with sterile 0.1M PBS (pH6.8), resuspended in PBS, and the concentration of the cells was adjusted to 1X 109cfu/mL, mixed well and sonicated. The conditions of ultrasonication were: the disruption was carried out with a pulse of 3-5s (3 s at work, 5s at 200W) for 12min under ice bath conditions. Centrifuging the liquid obtained after ultrasonic treatment at 4 deg.C and 12000r/min for 15min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain CFE, and storing at-80 deg.C.
Determination of tri, alpha-glucosidase inhibitory Activity
Adding 25 μ L20 mM PNPG and 25 μ L CFS or CFE into reaction well with 96-well enzyme-labeled plate by micropipette, culturing at 37 deg.C for 10min, adding 50 μ L0.01U/mL alpha-glucosidase, reacting at 37 deg.C for 15min, and adding 100 μ L0.1M Na2CO3The reaction was terminated and the absorbance of the reaction solution was measured at 405nm using a microplate reader. Acarbose was used as a positive control, and the calculation formula (1) was as follows: alpha-glucosidase inhibition rate is abbreviated as alpha-GIR
A: samples containing no enzyme were replaced with 0.1M PBS (pH6.8)
B: samples containing no enzyme were replaced by 0.1M PBS (pH6.8) for both samples
C: enzyme-containing sample
D: samples containing no enzyme were replaced with 0.1M PBS (pH6.8)
TABLE 1 screening results for alpha-glucosidase inhibitory Activity
(Note: PNPG is hydrolyzed by alpha-glucosidase to produce glucose and PNP, and the absorbance of PNP is measured at 405 nm; ND (not detected) in the table indicates that no inhibition is detected.)
The screening results are shown in table 1, wherein the CFS of 12 lactic acid bacteria has an inhibiting effect on the activity of alpha-glucosidase, and the inhibiting rate can reach 2.530-15.101%. Wherein, the inhibition rate of the acarbose of the positive control group is the highest and is 26.75 percent. And the CFE of all lactic acid bacteria had no inhibitory effect on α -glucosidase. According to the results of the inhibition rates in the table, the strain BLP12 showed the highest inhibitory activity against alpha-glucosidase.
In a preferred embodiment, the α -glucosidase inhibitory activity is determined by determining the inhibition rate of strain BLP12 on the intestinal α -glucosidase activity of mouse, as follows:
(1) extraction of alpha-glucosidase from intestinal tract of mouse
A clean-grade male Balb/c mouse is killed by decapitation after fasting for 12h, the upper segment of the small intestine is taken for 30cm, the small intestine is washed by sterile PBS at 4 ℃, cut into pieces and placed in a glass homogenizer. Adding PBS with about 4 times of volume, grinding and homogenizing under ice bath, and pouring into a centrifuge tube. Centrifuging at 4 deg.C and 4000r/min for 20min, and collecting supernatant and packaging. Storage at-20 ℃.
(2) Determination of intestinal alpha-glucosidase activity of mouse
According to the specification of the BCA kit, the protein content of the diluted 10-fold alpha-glucosidase in the intestinal tract of the mouse is 1.290mg/mL, and the enzyme activity of the diluted 10-fold alpha-glucosidase in the intestinal tract of the mouse is 0.057U/mL through experiment.
(3) The inhibitory activity of the strain BLP12 on mouse intestinal alpha-glucosidase
Adding 25 μ L of 20mM PNPG and 25 μ L of CFS of strain BLP12 with different concentrations into reaction well with 96-well enzyme-labeled plate by micropipette, culturing at 37 deg.C for 10min, adding 50 μ L of mouse intestinal alpha-glucosidase (0.057U/mL), reacting at 37 deg.C for 15min, and adding 100 μ L of 0.1M Na2CO3The reaction was terminated and the absorbance of the reaction solution was measured at 405nm using a microplate reader. Acarbose is used as positive control, and the calculation formula adopts the formula (1)
A: samples containing no enzyme were replaced with 0.1M PBS (pH6.8)
B: samples containing no enzyme were replaced by 0.1M PBS (pH6.8) for both samples
C: enzyme-containing sample
D: samples containing no enzyme were replaced with 0.1M PBS (pH6.8)
TABLE 2 Effect of the concentration of CFS strain on the inhibition of alpha-glucosidase in mouse intestinal tract
As shown in FIG. 1 and Table 2, the concentration of the bacterial suspension has a certain influence on the activity of alpha-glucosidase in mouse intestinal tract, and the higher the concentration of the bacterial suspension, the higher the CFS inhibitory activity against alpha-glucosidase in mouse intestinal tract, and the lower the CFS concentration is, the higher the CFS inhibitory activity is, the more the CFS inhibitory10The inhibition rate of the activity of alpha-glucosidase in intestinal tracts of mice is 71.22% when cfu/mL is adopted, and the CFS concentration is 1 multiplied by 109The inhibition rate of cfu/mL on the activity of alpha-glucosidase in intestinal tracts of mice is 63.64 percent, and the concentration is 1 multiplied by 106The inhibition rate of the alpha-glucosidase activity of the intestinal tract of the mouse can still reach 41.26% at cfu/mL.
(4) The inhibition rate of the strain having α -glucosidase activity of mammalian origin was searched, compared with the inhibition rate of the lactobacillus plantarum BLP12 for α -glucosidase activity derived from intestinal tract of mouse.
TABLE 3 summary of strains with inhibitory activity on mammalian alpha-glucosidase
As a result, as shown in Table 3, the concentration of Lactobacillus plantarum BLP12 in the bacterial liquid was 1X 1010cfu/mL and bacterial liquid concentration of 1X 109The inhibition rate of CFS on the activity of alpha-glucosidase is higher than that of other strains at cfu/mL, so that the lactobacillus plantarum has high alpha-glucosidase inhibition activity.
Sequencing of four, 16S-rDNA genes
The selected strain was inoculated into MRS broth at an inoculum size of 4% (v/v), and cultured at 37 ℃ for 18 h. And 2mL of bacterial liquid is taken to extract a genome according to the bacterial genome DNA extraction kit. The purity and concentration of the analyzed DNA were determined by agarose gel electrophoresis and UV spectrophotometry, and PCR was performed using bacterial 16S rDNA amplification universal primers 27F and 1492R, the base sequences of which are shown in Table 4. And detecting the amplification product by an agarose gel electrophoresis method. Finally, the amplified product is exchanged with Beijing Hua large gene research center to complete sequencing.
TABLE 4 bacterial 16S rDNA amplification Universal primers 27F and 1492R
(1) Amplification reaction system
(2) Reaction conditions
35 cycles, final extension at 72 ℃ for 10min, and agarose gel electrophoresis of the PCR amplification product are shown in FIG. 2. (Marker band compositions from bottom to top are respectively 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, electrophoresis direction from top to bottom.)
Blast results showed that the strain BLP12 was most closely related to Lactobacillus plantarum and belongs to Lactobacillus plantarum.
In a preferred embodiment, the probiotic indicator comprises:
(1) acid tolerance, higher survival rate under pH2.0;
(2) bile salt tolerance, good growth condition after being cultured in deionized water containing 2% bile salt for 24 h;
(3) can survive in artificial simulated saliva, gastric juice and intestinal juice;
(4) can still survive well after a series of digestions which artificially simulate saliva, gastric juice and intestinal juice in sequence.
(5) The growth period is short.
The following experiment was carried out on the probiotic properties of strain BLP12 for the above probiotic index.
(1) Acid resistance test of Lactobacillus plantarum BLP12
Inoculating the strain into MRS broth with an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. Resuspending the collected thallus in MRS broth with pH of 2.0, 3.0, 7.0, and adjusting the concentration of the bacteria solution to 1 × 109cfu/mL. Incubate at 37 ℃ for 3h, collect the bacterial solution, count viable bacteria according to GB 4789.2-2010. The tolerance is calculated as follows: survival rate (Survival rate)
Survival rate (%) (log N1/log N0) × 100 formula (2)
N1: viable count under the condition of enduring treatment
N0: pH 6.4 (Normal) MRS Broth Medium
The result shows that the survival rate of the strain BLP12 reaches 96.93 percent after being cultured for 3 hours under the acidic condition of pH 3.0; the survival rate of BLP12 was 71.04% at pH2.0, which also showed high survival, indicating that the strain was well resistant to acidic conditions.
(2) Tolerance of Lactobacillus plantarum BLP12 to bile salts
Inoculating the strain into MRS broth with an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. Resuspending the collected thallus in sterile deionized water with pH of 8.0 and 2% of bile salt, and adjusting the concentration of the bacteria solution to 1 × 109cfu/mL. Incubate at 37 ℃ for 24h, collect the bacterial solution, count the viable bacteria according to GB 4789.2-2010. The calculation formula is the same as the formula (2).
The result shows that the bacterial strain BLP12 has good growth condition and high survival rate of 77.14 percent after being cultured in deionized water containing 2 percent of bile salt for 24 hours, and has good tolerance to the bile salt.
(3) Growth assay of Lactobacillus plantarum BLP12 in simulated saliva, gastric juice and intestinal juice
The simulated digestive juice is prepared by referring to the method of Versantvorort and the like, and the digestive environment in the oral cavity and the gastrointestinal tract of a human body is fully simulated.
Inoculating the strain into MRS broth with an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. The collected bacteria were resuspended in artificial simulated saliva (pH 7.0), gastric juice (pH 2.0), and intestinal fluid (pH 8.0) respectively, and the concentration of the bacteria solution was adjusted to 1 × 109cfu/mL. Incubating for 5min, 3h and 24h at 37 ℃ respectively, collecting bacterial liquid, and counting viable bacteria according to GB 4789.2-2010. The calculation formula is the same as the formula (2).
The results show that the strain BLP12 grew well in simulated saliva. When the number of viable bacteria in the MRS broth culture medium with the pH value of 7.0 is taken as a control, the survival rate is 94.32 percent; when the number of live bacteria in the MRS broth with pH 6.4 (normal) is taken as a control, the survival rate reaches 97.22%.
The survival rate of strain BLP12 in simulated gastric fluid was 52.49%, which was 50%.
In simulated intestinal juice, the growth condition is good, and the survival rate reaches 94.83 percent.
(4) In vitro simulated digestion test
Inoculating the strain into MRS broth with an inoculum size of 4% (v/v), culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. The collected bacterial cells were resuspended in simulated saliva at pH 7.0, and the bacterial liquid concentration was adjusted to 1X 109cfu/mL. Incubating at 37 deg.C for 5min, and at 4 deg.C for 6000r/min, centrifuging for 10min, and collecting thallus; suspending the collected thallus in simulated gastric fluid with pH of 2.0, incubating at 37 deg.C for 3h, centrifuging at 4 deg.C for 10min, and collecting thallus; finally, the collected thalli are resuspended in simulated intestinal fluid with the pH value of 8.0, incubated for 24 hours at 37 ℃, and bacterial fluid is collected and viable bacteria count is carried out according to GB 4789.2-2010. The calculation formula is the same as the formula (2).
The result shows that the strain BLP12 has good growth condition after being digested by simulated saliva, gastric juice and intestinal juice, the survival rate is 88.27 percent and basically reaches 90 percent, and the strain can well survive in intestinal tracts after being digested by a series of human bodies.
(5) Determination of the growth Curve of the Strain BLP12
Lactobacillus plantarum BLP12 in logarithmic growth phase was inoculated into MRS broth at an inoculum size of 4% (v/v), cultured at 37 ℃ for 18h, sampled every 1h, and the OD600nm value of the resulting culture was determined.
As shown in FIG. 3, the growth cycle of the strain was short, and the strain entered the logarithmic growth phase after 2 hours of culture and entered the stationary phase after 14 hours.
In a preferred embodiment, the lactobacillus plantarum is used for the prevention and treatment of type 2 diabetes.
The experiment for preventing the diabetes of the mice in vivo by the lactobacillus plantarum BLP12 specifically illustrates that the lactobacillus plantarum has good prevention and treatment effects on the type 2 diabetes, and the experiment adopts a type 2 diabetes model caused by high-fat feed feeding and injection of Streptozotocin (STZ).
The experimental mice were Balb/c mice, male, 4-5 weeks old, purchased from Beijing Wittingle Ltd, and certified card number: SCXK (Jing) 2013-. The animal breeding stock is bred in SPF animal house of pharmaceutical plant institute of Chinese medical science institute, and the license number is as follows: SYXK (Kyoto) 2013-0023 at 22. + -. 2 ℃ and 60% RH relative humidity. The mouse cage is regularly cleaned by lighting for 12 hours and darkness for 12 hours, drinking water is freely taken for eating, padding is regularly replaced, and the mouse cage is regularly cleaned.
40 were randomly divided into 4 groups by weight: normal group, model group, positive drug group and BLP12 group (all fed high fat diet except normal group, 10 per group). The BLP12 group was administered by prophylactic gavage (the lactobacillus plantarum-like) two weeks later, and was modeled after fasting one day without water deprivation for 12 hours, and was intraperitoneally injected with 100mg/kg streptozotocin (STZ, SIGMA), and repeatedly injected every 3 days. The normal group was fed with basal diet (crude protein content 19.2%, crude fat content 4.6%, crude fiber 4.0%, crude ash content 6.3%, moisture 8.8%, nitrogen-free extract 55.9%); the model group, positive drug group (acarbose (bayer)) and BLP12 group were fed with high-fat diet (crude protein content 17.5%, crude fat content 17.9%, crude fiber 3.1%, crude ash content 4.5%, moisture 8.5%, no nitrogen content)Extract 48.5%); BLP12 group lactic acid bacteria for intragastric administration 1 × 10 per day10CFU/mouse.
One week after the STZ induction molding, the mice of each group are fasted for 12 hours without water prohibition, and the blood glucose value of the tail vein is measured by a glucometer to be the fasting blood glucose value. The fasting blood glucose value of the model group is 11.3mmoL/L, and the blood glucose value of the normal group is 3.7 mmoL/L. The blood sugar value of the blood sugar group of the model group exceeds that of the normal group by more than 50 percent, which is the success of model building of the type 2 diabetes.
After the molding is successful, the positive drug group is gavaged with 10mg/kg every day, and the BLP12 group is gavaged with 250 mu L of the lactobacillus plantarum strain sample every day until the end of the 13-week experiment. Blood is taken in fasting state, fasting blood glucose value is measured (as shown in figure 4), after 13 weeks, the fasting blood glucose value of the model group is 12.74mmoL/L, the fasting blood glucose value of the positive drug group is 8.885mmoL/L, and the fasting blood glucose value of the BLP12 group is 7.94mmoL/L, which is obviously reduced or even exceeds that of the positive drug group. Glycosylated hemoglobin is a gold index in the evaluation index of diabetes and is also used for the therapeutic effect of long-term blood sugar regulation. The glycosylated hemoglobin values were measured from fasting blood samples (as shown in fig. 5), and after 13 weeks, the glycosylated hemoglobin had a tendency similar to that of fasting blood glucose values, and the glycosylated hemoglobin values of the BLP12 group were significantly reduced compared to the model group. The lactobacillus plantarum provided by the invention has a certain hypoglycemic effect on type 2 diabetes.
An application method of lactobacillus plantarum with high alpha-glucosidase inhibition activity is characterized in that lactobacillus plantarum and a protective agent are added to prepare active freeze-dried fungus powder, the preparation method of the active freeze-dried fungus powder is simple, and the active freeze-dried fungus powder is convenient to store and transport.
The preparation method of the active lyophilized powder is further described by the following specific examples.
Example 1
The protective agent comprises 5% of skim milk and 0.5% of L-glutamic acid, and the preparation of the active freeze-dried fungus powder comprises the following steps:
step A, inoculating the strain into an MRS broth culture medium with an inoculum size of 4% for mass fermentation, culturing for 16h at 36 ℃, after microscopic examination confirms no pollution, centrifuging the cultured bacterial liquid at 6000r/min at 4 ℃ for 10min, and collecting thalli;
and step B, mixing the protective agent and the thalli according to the volume ratio of 1:1, and freeze-drying to obtain the lactobacillus plantarum-containing bacterial powder.
Example 2
The protective agent comprises 10% of skim milk and 1.0% of L-glutamic acid, and the preparation of the active freeze-dried fungus powder comprises the following steps:
step A, inoculating the strain into an MRS broth culture medium with an inoculum size of 4% for mass fermentation, culturing at 37 ℃ for 18h, performing microscopic examination to confirm no pollution, centrifuging the cultured bacterial liquid at 4 ℃ for 10min at 6000r/min, and collecting thalli;
and step B, mixing the protective agent and the thalli according to the volume ratio of 1:2, and freeze-drying to obtain the lactobacillus plantarum-containing bacterial powder.
Example 3
The protective agent comprises 15% of skim milk and 1.5% of L-glutamic acid, and the preparation of the active freeze-dried fungus powder comprises the following steps:
step A, inoculating the strain into an MRS broth culture medium with an inoculum size of 4% for mass fermentation, culturing at 38 ℃ for 20h, performing microscopic examination to confirm no pollution, centrifuging the cultured bacterial liquid at 4 ℃ for 10min at 6000r/min, and collecting thalli;
and step B, mixing the protective agent and the thalli according to the volume ratio of 1:3, and freeze-drying to obtain the lactobacillus plantarum-containing bacterial powder.
In the scheme, the lactobacillus plantarum in the active freeze-dried bacterial powder has high alpha-glucosidase inhibition activity, can prevent the blood sugar of a diabetic from being rapidly increased when taken within one hour after a meal, can maintain the blood sugar level within 11.1mmol/L after 2 hours of the meal, can reduce the possibility that the diabetic induces various complications due to high blood sugar level after a long time of the meal, and can be used for long-term adjuvant therapy of the diabetes; in addition, the lactobacillus plantarum in the freeze-dried bacterial powder can be planted and survived in the intestinal tract of a human body, metabolic active substances are generated through rapid propagation, the intestinal environment health of a diabetic patient can be well adjusted, the intestinal environment of the human body is acidic, digestion and absorption are facilitated, and intestinal peristalsis is enhanced.
A fermentation method of Lactobacillus plantarum with high alpha-glucosidase inhibition activity comprises inoculating Lactobacillus plantarum into animal milk, soybean milk, peanut milk, green vegetables or fruit juice, and fermenting to obtain functional food suitable for diabetic patients.
The fermentation step of Lactobacillus plantarum is further illustrated by the following specific examples.
Example 4
Inoculating the lactobacillus plantarum into cow milk, goat milk or camel milk for fermentation, wherein the fermentation steps are as follows: before inoculation, cow milk, goat milk or camel milk is heated to 75 ℃, kept for 10s and disinfected. After the cow milk, the goat milk or the camel milk is cooled to about 40 ℃, the lactobacillus plantarum is inoculated and stirred uniformly until the milk foams. And (3) under a sealed condition, keeping the temperature at 40-41 ℃, and fermenting for 8h to obtain the fermented cow milk, the fermented goat milk or the fermented camel milk suitable for the diabetics.
Example 5
The bacterial powder is put into soybean milk or peanut milk for fermentation, and the fermentation steps are as follows: before inoculation, the soybean milk or peanut milk is heated to 65 ℃, kept for 30min and disinfected. After the soybean milk or the peanut milk is cooled to about 40 ℃, the lactobacillus plantarum is inoculated and stirred uniformly until the milk foams. And (3) under a sealed condition, keeping the temperature at 40-41 ℃ for 8h, and fermenting to obtain the fermented soybean milk or the fermented peanut milk suitable for the diabetics.
Example 6
The bacterial powder is put into green vegetables or fruit juice for fermentation, and the fermentation steps are as follows: before inoculation, the green vegetable or fruit juice is heated to 65 deg.C, and maintained for 30min for sterilization. And (3) inoculating the lactobacillus plantarum when the green vegetables or fruit juice is cooled to about 40 ℃, and uniformly stirring until the green vegetables or fruit juice foams. Sealing, maintaining the temperature at 40-41 deg.C, and fermenting for 8 hr to obtain fermented vegetable or fruit juice suitable for diabetic patients.
In the scheme, the lactobacillus plantarum is applied to the production of dairy products (such as animal milk, soybean milk, peanut milk or other milk products), or is added into functional foods (such as green vegetables, fruit juice or other foods) as a food ingredient, so that the lactobacillus plantarum is suitable for being eaten by diabetics, and can achieve good prevention and intervention effects on diabetes and obesity.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is thus not limited to the details given herein and to the illustrations shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (2)
1. A lactobacillus plantarum with high alpha-glucosidase inhibition activity, wherein the classification of the strain is named as: lactobacillus plantarum, which has been deposited in the China general microbiological culture Collection center in 8-29 th 2017 by the culture Collection of microorganisms with the following deposition numbers: CGMCC NO. 14573.
2. The application method of lactobacillus plantarum having a high alpha-glucosidase inhibitory activity according to claim 1, wherein lactobacillus plantarum is added with a protectant to prepare active lyophilized powder, the protectant comprises 5% -15% skim milk and 0.5% -1.5% L-glutamic acid, and the preparation method of the active lyophilized powder comprises the following steps:
step A, inoculating the strain into an MRS broth culture medium with an inoculum size of 4% for mass fermentation, culturing for 16-20h at 36-38 ℃, after microscopic examination confirms no pollution, centrifuging the cultured bacterial liquid at 6000r/min at 4 ℃ for 10min, and collecting thalli;
and step B, mixing the protective agent and the bacteria according to the volume ratio of 1:1-3, and freeze-drying to obtain the bacterial powder containing the lactobacillus plantarum.
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| CN115125163B (en) * | 2022-05-20 | 2023-11-14 | 北京农学院 | Lactobacillus casei and application thereof |
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| CN115322931B (en) * | 2022-08-19 | 2023-04-25 | 华南农业大学 | Bacillus coagulans Wei Ciman capable of inhibiting activity of alpha-glucosidase and/or alpha-amylase and application thereof |
| CN116555075B (en) * | 2023-03-13 | 2023-12-01 | 广东悦创生物科技有限公司 | Lactobacillus plantarum JF1 and application thereof in preparation of anti-aging food and drug |
| CN116814464B (en) * | 2023-03-13 | 2024-02-02 | 广东悦创生物科技有限公司 | Fermented lactobacillus mucilaginosus JF5 and application thereof in preparation of lipid-reducing and digestion-aiding foods and medicines |
| CN116676239B (en) * | 2023-07-26 | 2023-10-27 | 杭州微致生物科技有限公司 | Lactobacillus plantarum VB165 and application thereof |
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