CN113462613A - Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof - Google Patents
Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof Download PDFInfo
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- CN113462613A CN113462613A CN202110888458.5A CN202110888458A CN113462613A CN 113462613 A CN113462613 A CN 113462613A CN 202110888458 A CN202110888458 A CN 202110888458A CN 113462613 A CN113462613 A CN 113462613A
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Abstract
The invention relates to the technical field of food microorganisms, and discloses lactobacillus plantarum ZJUIDS04 with the preservation number of CGMCC NO. 22609. The lactobacillus plantarum has the functions of strongly reducing blood sugar and strongly improving the activity of bile salt hydrolase; the strain has obvious advantages in acid resistance and bile salt resistance compared with other lactic acid bacteria, is suitable for gastrointestinal environment and has proliferation capacity; has no antibiotic resistance and has antibacterial activity; the antioxidant activity is strong and is obviously superior to that of the conventional lactobacillus plantarum, so that the lactobacillus plantarum can be widely used for developing products with blood sugar-reducing related probiotic functions.
Description
Technical Field
The invention relates to the technical field of food microorganisms, in particular to lactobacillus plantarum ZJUIDS04 with a blood sugar function and application thereof in the fields of food, feed, medicines and the like.
Background
Diabetes has now become the most abundant metabolic disease in the Chinese and world population. High serum blood glucose levels are considered to be a major factor in metabolic diseases such as diabetes. Therefore, lowering serum glucose levels is directly related to the health of the human body. So far, a great number of experiments prove that the partial taking of the lactic acid bacteria and the related products thereof has the effects of reducing the blood sugar content of serum of a human body and reducing complications caused by diabetes. The lactobacillus is an important probiotic in human intestinal tracts, the quantity and the composition of the lactobacillus play an important role in maintaining the micro-ecological balance of a host and improving the function of an immune system, and researches show that the lactobacillus has the effects of reducing the activity of alpha-glucosidase and the DPP-IV inhibition rate in vitro and the like, thereby achieving the effect of reducing blood sugar.
Lactobacillus plantarum is closely related to human life, and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Lactobacillus plantarum is a kind of lactobacillus, and it can be added into food to improve the taste, texture and flavor of the food. The lactobacillus plantarum can also be planted in a human body to play a probiotic role, such as regulating intestinal flora, inhibiting the growth of intestinal pathogenic bacteria, reducing the blood sugar content in the animal body, enhancing the immunity of the organism, improving lactose digestion, resisting tumors, resisting oxidation and the like.
CN 105053202A, a preparation method of high-quality fermented camel milk with auxiliary hypoglycemic function, teaches that: the fermented camel milk with the auxiliary blood sugar reducing function is prepared by using Lactobacillus plantarum Grx16(Lactobacillus plantarum Grx16) and combining other strains as a leavening agent.
CN111733111A entitled "Lactobacillus plantarum NX-1 and application thereof in preparation of hypoglycemic drugs" discloses Lactobacillus plantarum NX-1, wherein fermentation supernatant, bacterial suspension and cell disruption supernatant of the Lactobacillus plantarum NX 1 which is not inactivated and inactivated have strong inhibition effect on alpha glucosidase and alpha amylase; and the glucose content in the zebra fish hyperglycemia model can be obviously reduced.
CN105105145A A Lactobacillus plantarum strain and its application in preparing functional food for reducing blood sugar and blood fat discloses Lactobacillus plantarum Sc52, the main functions of Lactobacillus plantarum Sc52 are: reducing blood glucose and/or blood lipid; promote the growth of probiotics in the intestinal flora and/or inhibit the growth of pathogenic bacteria in the intestinal flora.
CN110964659A Lactobacillus plantarum for reducing blood sugar, a screening method thereof, an application medicament and food thereof provides Lactobacillus plantarum with a preservation number of CCTCC M2019591 and a Latin literature name of Lactobacillus plantarum CT 152; the lactobacillus plantarum is used for preparing a medicament with the function of reducing blood sugar.
CN108085285A entitled Lactobacillus plantarum DM-50 and application thereof provides a Lactobacillus plantarum DM 50, and the Lactobacillus plantarum DM 50 plays a role in reducing blood sugar by promoting secretion of glucagon-like peptide 1(GLP 1), repairing insulin secretion function, regulating blood lipid metabolism, reducing chronic low-grade inflammation and inhibiting activity of dipeptidyl peptidase 4.
CN106834196A Lactobacillus plantarum CH126 and application thereof in preparing functional food for reducing blood sugar discloses Lactobacillus plantarum CH126, wherein Lactobacillus plantarum CH126 has strong effects of reducing fasting glucose and blood fat, and can be widely applied to the fields of food, health food and medical health.
CN112852662A discloses Lactobacillus plantarum YZX21, which has excellent blood sugar regulation effect and wide application prospect and can be used for preparing yoghourt, namely, an efficacy and application of Lactobacillus plantarum YZX21 for reducing blood sugar.
CN106520609A, "Lactobacillus plantarum YYS 06 from marine brown algae and applications thereof" discloses Lactobacillus plantarum with the collection number of CCTCC NO. M2016512. The lactobacillus plantarum is derived from sea brown algae, namely kelp, and is extremely beneficial to human health as probiotics and has good flavor; the lactobacillus plantarum has the advantages of salt resistance, acid resistance and bile salt resistance, and the survival rate in vivo after being ingested is high; the lactobacillus plantarum used for fermenting fruits and vegetables has a probiotic effect, and the fermentation liquor has alpha glucosidase inhibiting activity and a blood sugar reducing effect.
CN109694834A, "Lactobacillus plantarum and its use for removing body fat, reducing hepatomegaly and resisting inflammation", discloses Lactobacillus plantarum (Lactobacillus plantarum) GKM3, which has acid-resistant, alkali-resistant and/or cell adsorption capacity, and can achieve the effects of removing body fat, reducing blood lipid, reducing hepatomegaly, reducing liver function index, resisting inflammation, reducing uric acid, improving allergy and/or reducing blood glucose.
CN105132318A Lactobacillus plantarum Grx16 and applications thereof discloses Lactobacillus plantarum Grx16(Lactobacillus plantarum Grx16) which has acid and bile salt resistance and in vitro antioxidant capacity and can be used for preparing fermented milk with alpha-glucosidase activity inhibition capability.
Disclosure of Invention
The invention aims to solve the technical problem of providing a hypoglycemic lactobacillus plantarum ZJUIDS04 derived from fermented food and application thereof.
In order to solve the above technical problems, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) zjuid 04: the preservation number is CGMCC NO. 22609.
As an improvement of Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 of the present invention: the 16S rDNA complete sequence of the lactobacillus plantarum ZJUIDS04 is SEQ ID No: 1 is shown.
The invention also provides the application of the Lactobacillus plantarum ZJUIDS04 in preparing products with the function of reducing blood sugar; the product with the function of reducing blood sugar is, for example, food, medicine, health care product, feed and pet food with the function of reducing blood sugar.
The food with the function of reducing blood sugar comprises fermented fruit and vegetable juice (such as fermented pumpkin and dragon fruit juice) with the function of reducing blood sugar; functional fermented sour meat with hypoglycemia; yogurt with hypoglycemic function;
the medicine with blood sugar lowering effect comprises viable bacteria preparation with blood sugar lowering effect, wherein the viable bacteria number of Lactobacillus plantarum ZJUIDS04 is 1.0 × 1011～1×1012CFU/g；
The lactobacillus plantarum ZJUIDS04 is used for preparing silage with the function of reducing blood sugar and a pet milk powder product by fermenting alone or in a way of mixing with other strains.
The lactobacillus plantarum ZJUIDS04 of the invention has the preservation name: lactobacillus plantarum; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22609, preservation time 2021 year 05 month 26 days.
The Lactobacillus plantarum ZJUIDS04(Lactobacillus plantarum ZJUIDS04) is screened from traditional fermented dairy products in inner Mongolia, and is identified by the morphological, physiological and culture characteristics of bacteria and the combination of 16S rDNA sequencing and the like.
The colony morphology characteristics of the Lactobacillus plantarum ZJUIDS04(Lactobacillus plantarum ZJUIDS04) provided by the invention are as follows: obvious colonies are formed on an MRS agar culture medium, and the size of the colonies is 0.3-1.5 mm. The colony is round, the edge is neat, white, and the surface is moist and smooth. The morphological characteristics of the thallus are as follows: gram staining is positive, no spore is produced, and the bacillus circulans is single, paired or short chain.
The Lactobacillus plantarum ZJUIDS04(Lactobacillus plantarum ZJUIDS04) provided by the invention has the blood sugar reducing capacity of 35.1-48.6%. Besides the function of reducing blood sugar of the strain according to the activity of alpha-glucosidase, the invention also proves that the strain has the function of reducing blood sugar from the perspective of another mechanism (DPP-IV inhibition rate).
The Lactobacillus plantarum ZJUIDS04(Lactobacillus plantarum ZJUIDS04) of the invention also has the following capabilities:
1. has stronger antioxidant activity and is obviously higher than the prior similar strains.
2. Can resist acid and bile salt, and has certain proliferation ability; sensitivity to common antibiotics; has antibacterial activity; namely, it can resist the gastrointestinal environment, has no antibiotic resistance, and can inhibit pathogenic bacteria harmful in the intestine.
In conclusion, the lactobacillus plantarum with the function of strongly reducing blood sugar is screened from probiotics separated from traditional inner Mongolia fermented dairy products. The strain has strong bile salt hydrolase activity. The strain has obvious advantages in acid resistance and bile salt resistance compared with other lactic acid bacteria, is suitable for gastrointestinal environment and has proliferation capacity. Has no antibiotic resistance and has antibacterial activity. The antioxidant activity is strong and is obviously superior to that of the conventional lactobacillus plantarum, so that the lactobacillus plantarum can be widely used for developing products with blood sugar-reducing related probiotic functions.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum ZJUIDS 04.
FIG. 2 is a gram-stained bacterial morphology of Lactobacillus plantarum ZJUIDS 04.
FIG. 3 is an electrophoretic identification chart of 16S rDNA of Lactobacillus plantarum ZJUIDS 04;
the first band on the right is marker and the second band is the electrophoretic band of ZJUIDS 04.
FIG. 4 is an example of a plate of Lactobacillus plantarum ZJUIDS04 sensitive to antibiotics.
From left to right, compound sulfamethoxazole and gentamicin, cefazolin and amikacin, ampicillin and ciprofloxacin, penicillin G and erythromycin, chloramphenicol and norfloxacin are sequentially arranged;
FIG. 5 is an example of a plate of the antibacterial activity of Lactobacillus plantarum ZJUIDS 04;
in FIG. 5, S represents the supernatant of the bacterium; p represents a blank; j represents a bacterial liquid;
the inhibition zones of the lactobacillus plantarum ZJUIDS04 on staphylococcus aureus, Listeria monocytogenes, escherichia coli and salmonella enteritidis are represented from left to right in sequence.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 screening and characterization of lactobacillus plantarum ZJUIDS 04:
1. screening of lactic acid bacteria
1.1 sample sources
The strains used in the following cases of the invention are all separated from fermented dairy products (as samples) such as fermented mare milk, cheese, milk skin, milk lumps and the like which are self-made by inner Mongolian herdsmen. More than 30 samples are collected for subsequent separation, purification and identification.
1.2 isolation and purification of the Strain
Approximately 5g of the sample was collected in a sterile tube and immediately sent to the laboratory for strain isolation. Putting 1g of sample into 9mL of MRS liquid culture medium, carrying out vortex mixing, and then carrying out enrichment culture at 37 ℃ for 48 h; then sucking 1mL of enrichment liquid in a super clean bench, performing tenfold gradient dilution by using sterile physiological saline, and selecting 10-6、10-7、10-8And (3) three dilution gradients, wherein 100 mu L of bacterial liquid of each gradient is taken and smeared on an MRS agar culture medium, and the bacterial liquid is cultured for 48h at 37 ℃. After the culture is finished, selecting a plate with 50-150 single colonies growing in an agar culture medium, picking typical colonies, carrying out streaking purification on an MRS agar plate for many times until the colony forms on the whole plate are consistent, and picking single colonies to an MRS liquid culture medium for enrichment culture. The obtained strains were all stored in MRS liquid medium containing 40% glycerol at-80 deg.C by freezing.
Selecting a strain which has good growth and good genetic stability, and naming the strain as ZJUIDS 04.
2. Identification of Lactobacillus plantarum ZJUIDS04
2.1 characteristics of the colonies
After lactobacillus plantarum ZJUIDS04 is cultured in an MRS agar culture medium for 48 hours, the diameter of the lactobacillus plantarum ZJUIDS04 is 0.3-1.5 mm, colonies are round, the edges of the lactobacillus plantarum ZJUIDS are neat, the lactobacillus plantarum ZJUIDS is white, and the surface of the lactobacillus plantarum ZJUIDS is moist and smooth, and is shown in figure 1.
2.2 microscopic morphology:
lactobacillus plantarum ZJUIDS04 colony smear: gram-positive, non-sporulating, rectus rotundus, single, paired, or short chain, see fig. 2.
2.316S rDNA identification
Extracting the target strain genome DNA by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out 16S rDNA PCR experiment by using bacterial universal primers 27F and 1492R, and after the PCR amplification reaction is finished, taking a PCR product to carry out agarose gel detection and photographing, wherein the length of an amplified fragment is about 1.5kb, and the figure is 3. The PCR product was sent to Beijing Liuhua Dageney (Wuhan) Co., Ltd for sequencing, and the results were shown in SEQ ID NO: 1, BLAST sequence alignment on NCBI website, showing that the sequence has over 99% homology with the 16SrDNA sequence of Lactobacillus plantarum.
Combining the sequence alignment result of the strain ZJUIDS04 with the physiological and biochemical results to determine that the screened Lactobacillus ZJUIDS04 is Lactobacillus plantarum (Lactobacillus plantarum).
The deposited information of the strain ZJUIDS04 is as follows: the preservation name is: lactobacillus plantarum; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22609, preservation time 2021 year 05 month 26 days.
Example 2 confirmation of the hypoglycemic Activity of Lactobacillus plantarum ZJuuds 04
1. Alpha-glucosidase inhibition rate
Preparation of Strain fermentation broth
Firstly, streaking and activating strains stored in a glycerol tube on an MRS agar plate for 2-3 times, then selecting a single strain, and carrying out amplification culture in an MRS liquid culture medium at 37 ℃ for 18h until the concentration of the strain reaches 107CFU/mL or so as to be used as bacterial suspension. Inoculating the bacterial suspension into MRS liquid culture medium with glucose (water-soluble) concentration of 100 mu g/mL in an inoculation amount of 2 percent (volume percent), and culturing at 37 ℃ for 48 hours to obtain strain fermentation liquor. The above cultivation is carried out at a rotation speed of 4000 r/min.
The strain fermentation liquor is centrifuged for 20min at 12000rpm to obtain supernatant and thalli, after a part of thalli are crushed by a cell crusher (the power of the crusher is 40 percent, the crushing time is 8 min), the supernatant obtained after centrifugation for 10min at 12000r/min is taken as the content.
A total volume of 205 μ L of a system was set, consisting of: to 50. mu.L of PBS buffer solution (concentration 0.1mol/L, pH 6.8), 50. mu.L of a 20mmol/L p-nitrophenol-alpha-D-glucopyranoside (PNPG) solution and 25. mu.L of a sample to be tested (supernatant/cells/contents) were added, the mixture was incubated at 37 ℃ for 10min, 30. mu.L of an alpha-glucosidase solution (20U/mL) was added, the reaction was continued for 20min, and 50. mu.L of Na having a concentration 1mol/L was added2CO3As a reaction termination solution.
And measuring the light absorption value of the obtained reaction liquid at 405nm, wherein the light absorption value is in direct proportion to the free amount of the p-nitrophenol PNP, a PBS (phosphate buffer solution) with the pH value of 6.8 and the concentration of 0.1mol/L is adopted in the reaction system as a blank control of the alpha-glucosidase solution and a sample to be measured, and the inhibition of the lactic acid bacteria on the alpha-glucosidase is calculated after the reaction.
The inhibition rate calculation formula is as follows:
alpha-glucosidase inhibition rate ═ 100% (1- (a-B)/(C-D) ];
in the formula
A is a determination light absorption value of a solution containing alpha-glucosidase and a sample to be detected;
b is a determination light absorption value which does not contain an alpha-glucosidase solution but contains a sample to be detected;
c is the measured light absorption value of the solution containing the alpha-glucosidase and not containing the sample;
d is a determination light absorption value of the solution without the alpha-glucosidase and the sample to be detected;
that is to say that the first and second electrodes,
a corresponds to the above system;
changing 30 mu L of alpha-glucosidase solution into 30 mu L of LPBS buffer solution to obtain a system B;
changing 25 mul of sample to be detected into 25 mul of PBS buffer solution to obtain a system C;
changing 30 mu L of alpha-glucosidase solution into 30 mu L of PBS buffer solution, and changing 25 mu L of sample to be detected into 25 mu L of PBS buffer solution, wherein the obtained system is D.
Each strain is subjected to 3 parallels, each group of experiments is repeated for 3 times, meanwhile, MRS liquid culture medium is used as a negative control (the alpha-glucosidase inhibition rate is basically 0), and lactobacillus rhamnosus ATCC53103 is used as a positive control strain.
TABLE 1 alpha-glucosidase inhibition ratio (%), strain ZJUIDS04
Marked by significant difference, P <0.05
As shown in Table 1, the alpha-glucosidase inhibition rate of the supernate, thallus and broken contents of the lactobacillus plantarum ZJUIDS04 screened by the invention is obviously higher than that of the standard strain lactobacillus rhamnosus ATCC 53103. This indicates that the Lactobacillus plantarum ZJUIDS04 cells have excellent alpha-glucosidase inhibitory activity.
Inhibition ratio of DPP-IV
The fermentation supernatant, the cells and the cell contents were prepared as above.
The measurement method is as follows: in a 96-well microplate, 25. mu.l glycine-p-nitroaniline (0.2mM) and 25. mu.l of the bacterial sample (supernatant, bacterial suspension, contents, PBS as control) were added and preincubated at 37 ℃ for 10 minutes. Thereafter, 50. mu.l DPP-IV (0.01U/ml) was added and incubated at 37 ℃ for 60 minutes; the reaction was stopped by adding 100. mu.l of sodium acetate buffer (1M, pH 4.0), and the absorbance of the sample was measured at 405 nm.
In the reaction system, PBS solution with pH6.8 and 0.1mol/L is used as DPP-IV solution and blank control of a sample to be detected;
DPP-IV inhibition rate is [1- (A-B)/(C-D) ]. times.100%
Wherein:
a is a determination light absorption value containing DPP-IV solution and a sample to be detected;
b is a determination light absorption value which does not contain DPP-IV solution but contains a sample to be detected;
c is the measured absorbance value of the DPP-IV solution without the sample;
d is the measured light absorption value of the solution without DPP-IV and the sample to be measured.
Each strain was done in 3 replicates, each experiment was repeated 3 times, while MRS liquid medium was used as negative control and lactobacillus rhamnosus ATCC53103 was used as positive control strain.
TABLE 2 DPP-IV inhibition ratio (%)
Marked by significant difference, P <0.05
As shown in Table 2, the Lactobacillus plantarum ZJUIDS04 screened by the invention is significantly better than the standard strain Lactobacillus rhamnosus ATCC53103 in terms of thallus inhibition rate, supernatant inhibition rate, content inhibition rate and the like. The lactobacillus plantarum ZJUIDS04 is proved to have excellent DPP-IV inhibition effect and can be used for screening the hypoglycemic standard strain.
The two tests show that the lactobacillus plantarum ZJUIDS04 has good effects on DPP-IV and alpha-glucosidase inhibition, has obvious advantages compared with the standard strain lactobacillus rhamnosus ATCC53103, and can be used for later-stage research and product development.
Example 3 confirmation of antioxidant Activity of Lactobacillus plantarum ZJuuds 04
1. Total antioxidant capacity (FRAP method)
The method for measuring the total antioxidant capacity was slightly modified according to the method of Giuberti et al. To the microplate, 150. mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, mixed at V: V: V: 10:1:1, ready-to-use) and 20. mu.L of the sample were added, mixed by shaking, reacted at 37 ℃ for 10min, and the absorbance of the solution at 593nm was measured. The absorbance measured by the sample is substituted into a ferrous sulfate standard curve, and the antioxidant capacity of the sample is expressed by ferrous sulfate equivalent (mu mol FeSO4/mL sample). Each sample was repeated 3 times and averaged.
Ferrous sulfate standard curve: ferrous sulfate solutions with different mass concentrations (0. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 400. mu.M, 600. mu.M, 800. mu.M) were prepared, ferrous sulfate solutions with different molar concentrations, 10mM TPTZ buffer solution, and 0.3M acetate buffer solution were mixed at V: V: V ═ 1:1:10, 170. mu.L of the mixed solution was added to an ELISA plate, the reaction was carried out at 37 ℃ for 10min, and the absorbance of the solution at 593nm was measured. And drawing a standard curve by taking the absorbance as an ordinate and the ferrous sulfate mass concentration as an abscissa, and measuring.
2. Reducing power
The reduction ability was measured by the method of Lin et al with some modifications. 1mL of sample is taken out of a centrifuge tube, and 1mL of each of 0.2M PBS buffer solution with pH6.6 and 1% (w/v) potassium ferricyanide solution is added and mixed evenly. Water bath at 50 deg.c for 20min and ice bath cooling. Then adding 1mL of 10% trichloroacetic acid, centrifuging at 6000r/min for 5min, taking 1mL of supernatant, adding 1mL of 0.1% (w/v) ferric trichloride and 1mL of distilled water, mixing uniformly, standing for reaction for 10min, and measuring the absorbance at 700 nm. The samples were replaced with PBS buffer or MRS broth medium as a blank. Each sample was repeated 3 times and averaged.
Reducing power (%) [ (As-Ab)/Ab ]. multidot.100
In the formula: as-absorbance of the sample set;
ab-blank absorbance;
3. DPPH radical scavenging ability
The DPPH radical scavenging ability was determined by reference to Shimada et al with some modifications. Preparing 1000mg/ml VC standard solution, and diluting to different concentration gradients (0-30 mug/ml). Adding 100 μ L of sample to be tested (or VC standard solution) and 100 μ L of 0.2mM DPPH ethanol solution (prepared with absolute ethanol, stored at 4 deg.C in dark place, and used as prepared) into an ELISA plate, shaking, keeping dark for 30min at room temperature, and measuring the absorbance of the solution at 517 nm; replacing 100 mu L of ethanol solution of LDPPH with 100 mu L of absolute ethanol to obtain a blank group; a control group was prepared by replacing 100. mu.L of the test sample with 100. mu.L of PBS buffer (0.2M, PBS or MRS broth, pH 6.6) and blank-zeroing with a mixture of 100. mu.L of PBS buffer (or MRS broth) and absolute ethanol. Each sample was repeated 3 times and averaged.
DPPH radical scavenging capacity (%) [1- (As-Ab)/Ac ]. 100
In the formula: as-absorbance of the sample set;
TABLE 3 antioxidant Activity of Lactobacillus plantarum ZJUIDS04
Ab-blank absorbance;
ac-control absorbance;
indicates significant difference, P < 0.05; marked differences, P < 0.01.
As shown in Table 3, the total antioxidant capacity, the reducing capacity and the DPPH free radical of the fermentation supernatant of the lactobacillus plantarum ZJUIDS04 screened by the invention are all obviously higher than those of the standard strain ATCC 53103. Thus, the lactobacillus plantarum ZJUIDS04 fermentation supernatant and bacterial suspension have high antioxidant capacity.
Example 4 confirmation of bile salt hydrolase Activity of Lactobacillus plantarum ZJuuds 04
1. Qualitative determination of bile salt hydrolase produced by lactobacillus plantarum ZJUIDS04
To a freshly prepared MRS agar medium were added 0.3% (m/v, i.e., 3g/1000ml) sodium deoxycholate, 0.2% (m/v) sodium thioglycolate, 0.37g/L CaCl2It was completely dissolved. Sterilizing at 121 deg.C for 15min, pouring into sterile plate, placing sterile filter paper sheet into the plate after solidification, and dripping 10 μ L Lactobacillus plantarum ZJuuds 04 bacterial suspension (about 10 μ L) onto the filter paper sheet8CFU/mL) with 10 μ L of sterile phosphate buffer added dropwise as a blank. The plates were incubated anaerobically in anaerobic jars (OXOID) at 37 ℃ for 72 h. Bile salt hydrolase activity is considered to be present if there is a white precipitate around the filter paper sheet.
Description of the drawings: the ZJuIDS04 bacterial cells obtained in example 2 were resuspended in PBS buffer (0.2M, pH 7.0) to obtain a ZJuIDS04 bacterial suspension (about 10)8CFU/mL)。
2. Quantitative determination of the bile salt hydrolase Activity of Lactobacillus plantarum ZJUIDS04
Preparation of cells and cell suspension etc. referring to example 2, 0.1mL of cell disruption supernatant solution of Lactobacillus plantarum ZJUIDS04 was taken, and 1.8mL of 0.1mol/L PBS buffer solution (0.2M, pH 7.0) and 0.1mL of 6mmol/L sodium taurocholate were added thereto. These mixtures were incubated at 37 ℃ for 30min, after which 0.5mL of 15% trichloroacetic acid was added to stop the enzymatic reaction. The mixture was centrifuged, and 0.5mL of the supernatant was added to 1mL of ninhydrin color-developing solution. Mixing with vortex, and boiling for 35 min. After cooling, the absorbance at 570nm was determined. One bile salt hydrolase activity unit is defined as the amount of enzyme required to release 1. mu. moL taurine from the substrate per minute.
3. The activity of the bile salt hydrolase was measured using Lactobacillus rhamnosus ATCC53103 as a positive control. Protein concentration was determined using bovine serum albumin as a standard and all experiments were repeated 3 times.
4. Table 4 shows the results of quantitative determination of the activities of bile salt hydrolase of the two strains. As can be seen from Table 4, the activities of bile salt hydrolase of both strains were 1.0U/mg or more. Experimental results show that the lactobacillus plantarum ZJUIDS04 has high bile salt hydrolase activity.
TABLE 4 bile salt hydrolase assay results of the strains
Note: + indicates the formation of a precipitation ring, -indicates the absence of a precipitation ring.
5. The bile salt hydrolase can hydrolyze in-vivo bound bile salt into free bile salt, and the free bile salt does not participate in liver and intestine circulation and is discharged out of a body along with excrement, so that the activity of the bile salt hydrolase is a key factor for reducing in-vivo blood sugar. The lactobacillus plantarum ZJUIDS04 provided by the invention has stronger bile salt hydrolase activity.
Example 5 confirmation of acid and bile salt resistance of Lactobacillus plantarum ZJuuds 04
1. Acid resistance test
Selecting a single strain of lactobacillus plantarum ZJUIDS04, carrying out amplification culture at 37 ℃ for 18h in an MRS liquid culture medium, inoculating the expanded bacterial suspension into the MRS liquid culture medium in an amount of 1%, and carrying out culture at 37 ℃ for 18 h. Centrifuging the culture solution at 4 deg.C at 8000r/min for 5min to collect bacteriaThe body was washed 2 times with PBS buffer (pH 6.8, 0.1 mol/L). The thallus is suspended in MRS liquid culture medium with pH adjusted to 3.0, and initial viable count is adjusted to about 108CFU/mL, cultured at 37 ℃ for 3 h. Counting viable bacteria in the samples of 0h and 3h by adopting a pouring plate method, culturing the poured plate at 37 ℃ for 48h, and determining the survival rate, wherein the survival rate calculation formula is as follows:
in the above formula, N0The number of viable bacteria (CFU/mL) of the test strain is 0 h; n is a radical oftTo test the viable count of the strain for 3h (CFU/mL).
2. Bile salt resistance test
Inoculating 1% of activated and expanded Lactobacillus plantarum ZJUIDS04 bacterial suspension into MRS liquid culture medium, culturing at 37 deg.C for 18h, mixing by vortex, and correcting initial viable count to about 109CFU/mL. The culture was inoculated in an amount of 10% to MRS liquid medium containing 0.3% (m/v) of bovine bile salt (control was MRS liquid medium containing no bovine bile salt), and cultured at 37 ℃ for 3 hours. The number of viable bacteria in the sample was then counted using the pour plate method. The poured plates were incubated at 37 ℃ for 48 h. The bile salt tolerance of the strain is expressed as the logarithm of the difference between the viable count per mL of medium containing bile salts and the viable count per mL of medium without bile salts at 3h (log CFU/mL).
3. The acid and bile salt resistance measurements described above were carried out with Lactobacillus rhamnosus ATCC53103 as a control.
4. As shown in Table 5, the acid and bile salt resistance of Lactobacillus plantarum ZJUIDS04 was significantly better than that of the control strain ATCC 53103. Its survival rate in MRS medium with pH 3.0 is up to 101.51%. The viable count in the environment containing 0.3% of ox bile salt still reaches 4 x 106CFU/mL or above indicates that the bile salt tolerance is better. Experiments prove that the lactobacillus plantarum ZJUIDS04 has higher gastrointestinal tract viability.
TABLE 5 results of the strains' tolerance to acids and bile salts
| Bacterial strains | Acid resistance (%) | Bile salt tolerance ability (Δ Log CFU/mL) |
| Lactobacillus plantarum ZJUIDS04 | 101.51±0.41 | 4.28±0.15 |
| Lactobacillus rhamnosus ATCC53103 | 62.12±0.18 | 1.03±0.23 |
5. Probiotics must be able to survive a range of adverse environments such as gastric acid and bile in the gastrointestinal tract to exert their probiotic effects. The lactobacillus plantarum ZJUIDS04 provided by the invention can grow and proliferate under the condition of pH 3.0, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. Meanwhile, the lactobacillus plantarum ZJUIDS04 can tolerate bile salt and can survive in intestinal tracts, so that the intestinal flora can be effectively improved, and the blood sugar is reduced.
Example 6 confirmation of the hydrophobic Capacity of Lactobacillus plantarum ZJuuds 04
1. Measurement of hydrophobicity
The lactobacillus pellet was washed twice with clean PBS buffer (0.1mol/L, pH 6.8) and resuspended to OD610The absorbance of the suspension is about 0.5, and the lactobacillus suspension is obtained.
Thoroughly mixing 2ml of lactobacillus suspension and 2ml of xylene, shaking in water bath at 37 deg.C for 5min, and measuring OD of water phase after 0h and 2h respectively610And (4) light absorption value.
A0Absorbance of 0h, AtTh absorbance.
The results obtained are shown in table 6 below.
TABLE 6 surface hydrophobicity of different strains (%)
2. Analysis of results
The hydrophobicity of lactobacillus plantarum ZJUIDS04 was measured to be 33.97%, significantly higher than the control standard strain. The strain is shown to have stronger adhesive capacity, can be adhered to the intestinal tract of a human body, and improves the health of intestinal flora.
Example 7 confirmation of antibiotic susceptibility of Lactobacillus plantarum ZJuuds 04
Culturing for 18h at a concentration of about 107The suspension of the Lactobacillus plantarum ZJUIDS04 strain CFU/mL was added in an amount of 1% to the sterilized MRS agar medium cooled to about 45 deg.C, mixed well, and added in a fixed amount to 15 mL/dish. After coagulation, the drug sensitive paper is taken out with tweezers and placed on the culture medium. The plate was placed right side up in a 37 ℃ incubator for 24 h. Paper without antibiotic was used as a blank control. And measuring the diameter of the inhibition zone. Each was repeated three times. FIG. 4 is an example of a plate susceptible to antibiotics of Lactobacillus plantarum ZJUIDS 04.
The diameters of the antibiotic-sensitive zone of inhibition of Lactobacillus plantarum ZJUIDS04 are shown in Table 7. With reference to CLSI (2017) drug susceptibility test standards, lactobacillus plantarum ZJUIDS04 showed sensitivity to penicillin G, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, sulfamethoxazole, and chloramphenicol. Present as intermediaries for ciprofloxacin, norfloxacin. The experimental result shows that the lactobacillus plantarum ZJUIDS04 is sensitive to common antibiotics.
TABLE 7 results of the sensitivity of Lactobacillus plantarum ZJUIDS04 to antibiotics
Note: s, sensitivity; i, an intermediary; r, drug resistance
With the wide application of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is more and more serious, and the intake of drug-resistant lactic acid bacteria for a long time brings great difficulty to clinical treatment. The lactobacillus plantarum ZJUIDS04 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 8 confirmation of the inhibitory Activity against pathogenic bacteria of Lactobacillus plantarum ZJuuds 04
The antibacterial activity of the lactic acid bacteria is determined by adopting an international universal agar diffusion method. 10mL of LB agar medium was poured into a sterile petri dish, and cooled to prepare a lower layer medium. The concentration of the bacteria is about 10 after 18h of culture7CFU/mL indicator suspension was added at 1% to sterilized LB agar medium cooled to about 45 deg.C, mixed well and dosed to 10 mL/dish. Placing the sterilized oxford cup on the upper surface. After the upper medium was condensed, the Oxford cup was gently pulled out. A sample of fermentation supernatant of Lactobacillus plantarum ZJUIDS04 was added at 100. mu.L/well and PBS buffer (0.1mol/L, pH 6.8) was used as a control. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times. FIG. 5 is an example of a plate of Lactobacillus plantarum ZJUIDS04 for its inhibitory effect on four pathogenic bacteria.
As shown in Table 8, the metabolite of Lactobacillus plantarum ZJUIDS04 has certain inhibitory effect on pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Listeria monocytogenes and the like, and is superior to the bacteriostatic effect of ATCC 53103. The metabolite of the strain can be seen to have bacteriostatic properties.
TABLE 8 results of the inhibitory potency of the strains on pathogenic bacteria
Staphylococcus aureus is the most common pathogen in human pyogenic infections, some escherichia coli can cause severe diarrhea and septicemia, and some salmonella species can also cause food poisoning in humans. Bacteriocin, organic acid, hydrogen peroxide and other bacteriostatic substances generated by the metabolism of the lactic acid bacteria can inhibit the growth of the pathogenic bacteria individually or jointly. The metabolite of the lactobacillus plantarum ZJUIDS04 provided by the invention has certain antagonistic action on the three pathogenic bacteria, plays an important role in maintaining intestinal microecological balance and has a health promotion effect.
Comparative example 1, the remaining lactobacillus plantarum (lactobacillus plantarum 01-04) obtained in the inventive process described in table 9 below were examined with reference to the above examples, and the blood glucose lowering ability, acid resistance, bile salt tolerance were compared with those of the present invention as described in table 9 below.
TABLE 9 comparison of probiotic Properties of different Lactobacillus plantarum
Comparative example 2, lactobacillus plantarum described in table 9 and lactobacillus plantarum having hypoglycemic activity reported in the background art were determined with reference to example 3 above, and the bile salt hydrolase activities were all 1U/mg or less.
Comparative example 3, lactobacillus plantarum having hypoglycemic activity informed in the background art was determined with reference to the above example 2, and both α -glucosidase inhibitory rate and DPP-IV inhibitory rate ability were significantly less than ZJUIDS04 of the present invention, taking the fermentation supernatant as an example.
Comparative example 4, lactobacillus plantarum described in table 9 and lactobacillus plantarum having hypoglycemic ability informed in the background art were examined with reference to the method of example 3 above, and taking the fermentation supernatant as an example, the total antioxidant ability was significantly less than ZJUIDS04 of the present invention.
Example 9 preparation of functional fermented yogurt Using Lactobacillus plantarum ZJuIDS04
1. The processing process flow of the yoghourt comprises the following steps:
raw material → preheating → homogenizing → blending → sterilizing → cooling → preparation inoculation → fermentation → after-ripening → refrigeration
2. The key points of the operation
(1) Raw materials: 2L of whole UHT sterilized milk or fresh cow milk;
(2) preheating: putting into a container and heating to 63 ℃;
(3) homogenizing: homogenizing in homogenizer under pressure of 15-25MPa), pouring the mixed solution into iron tank, adding 100g white sugar, and sterilizing in 90 deg.C water bath environment for 10 min;
(4) blending: adding ingredients into cow milk, and dissolving;
(5) and (3) sterilization: sterilizing the sweetened cow milk in water bath at 90 deg.C for 10 min;
(6) and (3) cooling: cooling the sterilized milk to 40-50 ℃ for later use;
(7) preparing a leaven: lactobacillus plantarum ZJUIDS04 strain was inoculated in a test tube containing sterilized skim milk (12%, w/v) under aseptic conditions and cultured at 37 ℃ for 20 hours. The inoculation amount of each passage is 2-4% (v/v), the vitality is restored after 2-3 passages, and the mixture is placed in a refrigerator at 4 ℃ for preservation;
(8) inoculation and fermentation: inoculating activated Lactobacillus plantarum ZJUIDS04 with the inoculation amount of 2-4% (v/v) under aseptic condition. Fermenting at 42 deg.C for 6-10 h;
(9) after-ripening: after fermentation, putting the mixture into a refrigerator with the temperature of 4 ℃ for after-ripening for 12-24 h;
(10) filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
Example 10 preparation of functional fermented fruit and vegetable juice Using Lactobacillus plantarum ZJuuds 04
1. Processing technological process of fermented fruit and vegetable juice
Raw material → washing → flash evaporation → pulping → blending → homogenizing → sterilizing → cooling → inoculating → closed fermentation → after-ripening → filling → refrigeration
2. The key points of the operation
(1) Raw materials: selecting fresh pumpkin and dragon fruit.
(2) Cleaning and cutting: cleaning, peeling (removing pulp from fructus Cucurbitae Moschatae), and cutting into small pieces.
(3) Flash evaporation: and (3) inactivating enzyme by adopting a flash evaporation method, treating for 0.5-1 min at 121 ℃, and quickly exhausting gas.
(4) Pulping: according to the weight ratio of 1:1, the pumpkin and water are gradually put into a colloid mill to be ground, and coarse grinding and fine grinding are carried out once respectively. Pulping the dragon fruit by a pulping machine until the pulp is uniform and has no blocks.
(5) Blending and homogenizing: according to 15 percent of pumpkin juice and 30 percent of dragon fruit juice, the content of soluble solids is adjusted to 10 DEG Brix by using cane sugar, 0.2 percent of stabilizer CMC is added for uniform mixing, and a two-stage homogenization method is adopted, wherein low pressure (15MPa) is firstly carried out, and then high pressure (25MPa) is carried out, so that the diameter of the melon pulp particles is 2-3 mu m.
(6) And (3) sterilization and cooling: keeping the temperature of the blended composite fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃.
(7) Inoculation and fermentation: inoculating activated Lactobacillus plantarum ZJuuds 04 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Fermenting at 37 deg.C for 24 h.
(8) After-ripening: after the fermentation is finished, putting the mixture into a refrigerator with the temperature of 4 ℃ for 3 hours.
(9) Filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
Example 11 preparation of functional fermented sour meat Using Lactobacillus plantarum ZJuuds 04
1. The processing process flow of the fermented sour meat comprises the following steps:
raw material → slice → mixed glutinous rice flour → inoculation → fermentation → pickling → packaging → finished product
2. The key points of the operation
(1) Slicing: commercially available fresh streaky pork was cut into 3cm square slices.
(2) Mixing glutinous rice flour: 1000g of raw meat is mixed with 250g of glutinous rice flour, and 1% of glucose is added.
(3) Inoculation and fermentation: inoculating activated Lactobacillus plantarum ZJuuds 04 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Fermenting at 37 deg.C for 18 h.
(4) Pickling: adding 2% salt, and pickling at 25 deg.C for 20 d.
Example 12 preparation of hypoglycemic powder Using Lactobacillus plantarum ZJuuds 04
1. Preparation of lactobacillus plantarum ZJUIDS04 bacterial sludge
A single colony of lactobacillus plantarum ZJUIDS04 is selected and inoculated in 50mL of MRS liquid culture medium, and the liquid culture medium is placed in an incubator at 37 ℃ for culturing for 18 h. Activated again in 250mL MRS liquid medium according to the inoculum size of 5%, and placed in an incubator at 37 ℃ for 24 h. Finally, the activated lactobacillus plantarum ZJUIDS04 was cultured in a 10L fermentor at 5% inoculum size for high-density anaerobic culture at 37 ℃ and pH6.8 for 18 h. Then centrifuging at 8000r/min and 4 deg.C for 15min, discarding supernatant, collecting thallus precipitate, and rinsing thallus with sterile phosphate buffer (pH 7.0) for 2 times. Thus obtaining the lactobacillus plantarum ZJUIDS04 bacterial sludge.
2. Preparation of the protective agent
The freeze-drying protective agent contains 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride. Water is used as the solvent. Sterilizing at 110 deg.C for use.
3. Preparation of lactobacillus plantarum ZJUIDS04 bacterial powder
And (3) fully and uniformly mixing the prepared lactobacillus plantarum ZJUIDS04 thallus precipitate with a protective agent solution according to the proportion of 1: 5. Pre-freezing for 5h at-40 ℃ to uniformly freeze the lactobacillus plantarum on the inner wall of the container, then carrying out vacuum freeze drying, and drying for 18-20 h to obtain lactobacillus plantarum ZJUIDS04 bacterial powder. Rehydrating with normal saline, washing twice, and determining viable count of Lactobacillus plantarum ZJUIDS04 powder to be 1.0 × 1011～1×1012CFU/g。
Example 13 preparation of silage Using Lactobacillus plantarum ZJuIDS04
The steps for preparing the alfalfa silage by the lactobacillus plantarum ZJUIDS04 are as follows:
(1) preparing raw materials: after the alfalfa is harvested, cleanness is guaranteed, no putrefaction and deterioration occur, and the alfalfa is cut into 1-2 cm.
(2) Preparation of silage: controlling the water content of the alfalfa grass to be 60-65% during ensiling, weighing 200g of alfalfa grass raw materials, filling the alfalfa grass raw materials into a vacuum packaging bag, inoculating lactobacillus plantarum ZJUIDS04 powder into the raw materials according to the inoculation amount of 0.02 per mill, carrying out vacuum packaging by using a vacuum packaging machine, and then placing the raw materials into a storage room for fermentation.
(3) Storage conditions were as follows: the temperature is 15-45 ℃ and the time is 30-60 days.
Example 14 preparation of probiotic milk powder for pets Using Lactobacillus plantarum ZJUIDS04
1. Preparation of lactobacillus plantarum ZJUIDS04 bacterial powder
The freeze-dried powder of Lactobacillus plantarum ZJUIDS04 strain powder with viable count of 1.0 × 10 was prepared according to the above-mentioned cases11～1×1012CFU/g。
2. Preparation of pet formula powder
Primary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil and additives: vitamins, trace elements, functional factors, and others;
automatic batching: putting the obtained material raw materials into a material bin according to a formula;
crushing: crushing the weighed materials by a crusher;
mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniformly mixing;
puffing: the mixed materials are made into granular materials through a bulking machine;
drying: drying the mixed materials by a dryer, and controlling the temperature to be 65-70 ℃;
grading and screening: the material flow passes through a grading sieve, and the particle size is controlled between 2.5 and 5 mm.
3. Preparation of probiotic formula powder for pets
Mixing the bacterial powder prepared in the step 1 and the pet feed prepared in the step 2 according to the ratio of 1: 100, and the live bacteria leaving the factory in the final productAt 108CFU/g is higher than the standard. And (5) after filling, storing and selling the product.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang university
<120> hypoglycemic lactobacillus plantarum ZJUIDS04 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1248
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
aacctgccca gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt 60
ggaccgcatg gtccgagttt gaaagatggc ttcggctatc acttttggat ggtcccgcgg 120
cgtattagct agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag 180
agggtaatcg gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta 240
gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt 300
ttcggctcgt aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat 360
tgacggtatt taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 420
ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc 480
tgatgtgaaa gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc 540
agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac 600
cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc 660
aaacaggatt agataccctg gtagtccata ccgtaaacga tgaatgctaa gtgttggagg 720
gtttccgccc ttcagtgctg cagctaacgc attaagcatt ccgcctgggg agtacggccg 780
caaggctgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 840
attcgaagct acgcgaagaa ccttaccagg tcttgacata ctatgcaaat ctaagagatt 900
agacgttccc ttcggggaca tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 960
gagatgttgg gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattaag 1020
ttgggcactc tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1080
catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc 1140
gaactcgcga gagtaagcta atctcttaaa gccattctca gttcggattg taggctgcaa 1200
ctcgcctaca tgaagtcgga atcgctagta atcgcggatc agcatgcc 1248
Claims (5)
1. Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04, characterized by: the preservation number is CGMCC NO. 22609.
2. Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04, according to claim 1, characterized in that: the 16S rDNA complete sequence of Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 is shown as SEQ ID No: 1 is shown.
3. Use of Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 according to claim 1 or 2 for the preparation of a product with hypoglycemic effect.
4. Use according to claim 3, characterized in that: the product with blood sugar lowering function contains viable bacteria preparation of Lactobacillus plantarum ZJUIDS 04.
5. Use according to claim 4, characterized in that: in the viable bacteria preparation, the viable bacteria number of Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS04 is 1.0 × 1011～1×1012CFU/g。
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| CN116064285B (en) * | 2022-08-19 | 2023-08-11 | 浙江大学 | Lactobacillus rhamnosus ZJUIDS07 capable of reducing blood sugar and application thereof |
| CN116396890A (en) * | 2023-02-08 | 2023-07-07 | 浙江大学 | Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer and application thereof |
| CN116396890B (en) * | 2023-02-08 | 2024-03-12 | 浙江大学 | Lactobacillus plantarum ZJUIDS15 for preventing and treating colon cancer and application thereof |
| CN116656526A (en) * | 2023-03-13 | 2023-08-29 | 广东悦创生物科技有限公司 | Lactobacillus plantarum JF4 and application thereof in preparation of blood sugar and cholesterol reducing foods and medicines |
| CN116656526B (en) * | 2023-03-13 | 2023-11-17 | 广东悦创生物科技有限公司 | Lactobacillus plantarum JF4 and application thereof in preparation of blood sugar and cholesterol reducing foods and medicines |
| CN116555115A (en) * | 2023-05-17 | 2023-08-08 | 宁夏大学 | Lactobacillus plantarum NXU0011 and application thereof |
| CN116555115B (en) * | 2023-05-17 | 2024-04-12 | 宁夏大学 | Lactobacillus plantarum NXU0011 and application thereof |
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