CN116376746A - Lactobacillus plantarum ZJUIDS16 with high folic acid yield and application thereof - Google Patents
Lactobacillus plantarum ZJUIDS16 with high folic acid yield and application thereof Download PDFInfo
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- CN116376746A CN116376746A CN202310049947.0A CN202310049947A CN116376746A CN 116376746 A CN116376746 A CN 116376746A CN 202310049947 A CN202310049947 A CN 202310049947A CN 116376746 A CN116376746 A CN 116376746A
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- lactobacillus plantarum
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- folic acid
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A—HUMAN NECESSITIES
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
The invention provides a lactobacillus plantarum strain for high folic acid production（Lactobacillus plantarum) ZJUIDS16 and its application. The strain has the preservation number of: CGMCC No.26162. The strain has high folic acid metabolism capacity, can resist gastrointestinal tract environment, has no antibiotic resistance of culture solution, can inhibit intestinal harmful pathogenic bacteria, and has strong antioxidant capacity. Because ofThe lactobacillus plantarum ZJUIDS16 can be applied to preparation of functional foods, health-care products and pet foods. The food comprises: fermented fruit and vegetable juice, fermented dairy products, fungus powder and the like. The lactobacillus bacteriocin can also be prepared by fermentation and used as a food preservative, and is applied to food processing and storage. The lactobacillus plantarum ZJUIDS16 provided by the invention can be widely used for developing related products such as probiotics.
Description
Technical Field
The invention belongs to the technical field of food microorganisms, and particularly relates to lactobacillus plantarum ZJUIDS16 capable of producing folic acid in high yield and application thereof, and the lactobacillus plantarum ZJUIDS16 capable of producing 5-methyltetrahydrofolate and application thereof.
Background
Folic acid is a water-soluble B-group vitamin consisting of purine, para-aminobenzoic acid, polyglutamic acid tail and the like, and is widely distributed and commonly exists in animals, plants and traditional fermented foods. Folic acid has important physiological functions and can have adverse effects on the development of the nervous system of a fetus and an infant when lacking; the research shows that folic acid also has the functions of resisting tumor, and preventing and treating cardiovascular and cerebrovascular diseases and intestinal diseases. However, higher animals such as humans cannot synthesize their own folic acid due to the lack of the corresponding folic acid synthesis genes, and these folic acids can only be taken from the outside by eating or absorbing the intestinal flora to catabolize folic acid. In addition, the chemically synthesized folic acid has the defects of high absorptivity, potential side effects and the like, and the natural folic acid has the advantages of high safety, good absorption effect and no toxic or side effects when being taken into a human body. Lactic acid bacteria are widely applied to the food industry and the medical care industry, and meanwhile, researches prove that the lactic acid bacteria in intestinal microorganisms can metabolize and produce vitamins such as folic acid and the like, which are one of main sources of host vitamins, so that the lactic acid bacteria are the best choice for biosynthesis of folic acid.
So that the screening of the lactobacillus with high folic acid production capability has high application value. Meanwhile, compared with other lactic acid bacteria, the strain has obvious advantages in acid resistance and bile salt resistance, is suitable for growth in gastrointestinal environment, has multiplication capacity without antibiotic resistance, does not release hemolysin, has antibacterial activity and stronger antioxidant capacity, and has obvious advantages in determining folic acid content when being applied to fermented dairy products. Therefore, the lactobacillus plantarum can provide a foundation for developing probiotic products.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum ZJUIDS16 with high folic acid yield, which is lactobacillus plantarum ZJUIDS16 with 5-methyltetrahydrofolate yield. The invention provides a lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16 for producing 5-methyltetrahydrofolate, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 11 months and 21 days in 2022, and is classified and named: lactobacillus plantarum Lactobacillus plantarum, accession number: CGMCC No.26162, the preservation address is North Chen West Lu No.1, 3 of the Chaoyang district of Beijing, china academy of sciences of microorganisms.
Further, the full sequence of the 16SrDNA of the lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16 is shown in SEQ ID No. 1.
Furthermore, the lactobacillus plantarum ZJUIDS16 has obvious advantages in acid resistance and bile salt resistance compared with other lactobacillus, is suitable for gastrointestinal environment and has proliferation capacity; the culture solution of lactobacillus plantarum ZJUIDS16 (Lactobacillus plantarum) has no antibiotic resistance, which indicates that the strain does not carry a drug resistance gene; lactobacillus plantarum ZJUIDS16 (Lactobacillus plantarum) has antibacterial activity.
Another object of the invention is to provide the use of said lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16 for the preparation of functional foods, health products and pet foods.
The functional food comprises: fermented fruit and vegetable juice, fermented dairy products, fungus powder and other foods.
The health product comprises: high-yield folic acid bacterial agent or bacterial powder.
The pet food comprises: milk powder for pets and probiotic powder for pets.
It is a further object of the invention to provide the use of lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16 in the manufacture of a food preservative. Lactic acid bacteria bacteriocin
Furthermore, the lactobacillus bacteriocin prepared by lactobacillus plantarum fermentation can be used as a preservative for food preservation of meat products, milk products, alcoholic beverages and the like, or can be combined with other preservation technologies to be used as a barrier technology for food processing and storage.
Aiming at the lactobacillus which lacks high folic acid production capability at present and a more convenient and accurate measuring method and culture conditions, the invention provides the lactobacillus plantarum which produces the 5-methyltetrahydrofolate. Researches show that the strain has higher folic acid metabolism capability, can resist gastrointestinal tract environment, has no antibiotic resistance of culture solution, can inhibit harmful pathogenic bacteria in intestines, and has stronger antioxidant capability. Therefore, the lactobacillus plantarum ZJUIDS16 can be widely used for developing related products such as probiotics and the like.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum ZJUIDS16 according to the invention.
FIG. 2 is a diagram showing the morphology of gram-stained cells of Lactobacillus plantarum ZJUIDS16 according to the present invention.
FIG. 3 is an electrophoretically identified graph of 16SrDNA of Lactobacillus plantarum ZJUIDS16 according to the present invention.
FIG. 4 shows a Lactobacillus plantarum ZJUIDS16 phylogenetic tree.
Fig. 5 is a folic acid standard graph.
FIG. 6 shows the measurement result of the self-aggregation rate of Lactobacillus plantarum ZJUIDS16.
FIG. 7 shows the results of blood agar plates for detecting the type of hemolysis.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
EXAMPLE 1 isolation and characterization of Lactobacillus plantarum ZJUIDS16
1 isolation of Lactobacillus plantarum ZJUIDS16
1.1 sample Source
The strain used in the invention is derived from northeast pickle samples. Samples were collected in a total of 50 parts.
1.2 isolation and purification of Strain
About 5g of the sample was collected in a sterile tube and immediately sent to a laboratory for strain isolation and purification. 1g of sample is taken and placed into 9mL of MRS liquid culture medium during separation, and after vortex mixing, enrichment culture is carried out for 48 hours at 37 ℃; then 1mL of culture solution is sucked in an ultra-clean bench, ten-time gradient dilution is carried out by using sterile normal saline, and 10 is selected -5 、10 -6 、10 -7 Three dilution gradients, each of which was taken separately from the bacterial suspension 100, each of which was spread on MRS solid medium and incubated at 37℃for 48h. After the cultivation is finished, fromSelecting a flat plate growing with 30-300 single colonies in an MRS solid culture medium, picking typical colonies, carrying out repeated streak separation on the MRS agar flat plate until the colony morphology on the whole flat plate is consistent, and picking single colonies to be amplified and cultured in the MRS liquid culture medium. The resulting strain was stored in a-80℃refrigerator with 40% (w/v) glycerol in MRS liquid medium.
2. Identification of Lactobacillus plantarum ZJUIDS16
2.1 colony characterization
After lactobacillus plantarum ZJUIDS16 is cultured in MRS solid culture medium for 48 hours, the surface edges are smooth, the shape is regular and circular, and the lactobacillus plantarum ZJUIDS16 is milky white, as shown in figure 1.
2.2 morphology under microscope
Lactobacillus plantarum ZJUIDS16 colony smear: gram staining was positive, sporulation was absent, rod-like, see figure 2.
2.3 16SrDNA identification
Extracting 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 a PCR experiment of 16SrDNA by using bacterial universal primers 27F and 1492R, and taking a PCR product to carry out agarose gel detection and photographing after the PCR reaction amplification is finished, wherein the length of an amplified fragment is about 1500bp, as shown in figure 3. The PCR product was sent to Beijing Liuhua large gene technologies Inc. for sequencing, and after homology comparison, phylogenetic tree results are shown in FIG. 4, BLAST sequence alignment was performed on NCBI website, and the results show that the sequence has more than 99% homology with the identified 16SrDNA sequence of Lactobacillus plantarum.
According to the combination of the sequence comparison result and the physiological and biochemical result of the strain ZJUIDS16, the screened lactobacillus ZJUIDS16 is determined to be lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16.
Example 2 Lactobacillus plantarum ZJUIDS16 folate production assay
1 quantitative screening of high-yield folic acid bacterial strain and folic acid content determination thereof
1.1 sample pretreatment
Shaking and mixing lactobacillus fermentation broth, centrifuging (10000 rpm,10 min), collecting 10ml supernatant, adding methanol deproteinized (volume ratio of sample to be tested and methanol is 1:4 when methanol deproteinizing), blow-drying with nitrogen flow, redissolving with methanol (chromatographic grade), and filtering with 0.22 μm filter membrane.
1.2 preparation of Standard Folic acid stock solution
55-56mg (accurate to 0.1 mg) of standard substance is weighed, 50mL of distilled water is transferred into a 100mL volumetric flask, 2mL of ammonia water is added, after the solution is prepared, the volume of the solution is calculated according to the following formula, and the concentration of folic acid in stock solution is required to be 0.50mg/mL:
1.3 liquid chromatography determination
The liquid chromatography analysis is carried out after the pretreatment of the fermentation liquor sample to be tested and the folic acid standard sample of 0.50mg/mL by the folic acid production strain HPLC method, the operation steps are as follows, the retention time of folic acid between the two is compared, and the bacterial strain obtained by screening by using FACM is verified to have the folic acid production capability. The liquid phase selection stationary phase is C18 column, ultraviolet detector (280 nm); the mobile phase is methanol: water (containing Na 2 HPO 4 And Na H 2 PO 4 pH 7.6) =10: 90, filtering the mobile phase by using 0.456) organic film, and then performing ultrasonic degassing for 15min; retention time 20min; the sample was introduced in an amount of 20. Mu.L, and the results were expressed in micrograms of folic acid produced per milliliter of fermentation broth. The leaf acid content of the fermentation supernatant after 24h incubation at 30℃was determined using the medium without inoculation as a blank. Results are expressed as the mass of folic acid per milliliter of fermentation broth. The folic acid yield of the lactobacillus is calculated according to the following formula:
A＝B－A 0 +C
wherein: a is extracellular folic acid yield of lactobacillus; b is the yield of the leaf acid in the fermentation liquor; a0 is the yield of leaf acid in the blank group; c is folic acid loss in the measuring process. The amount of loss was determined by the same procedure as described above for pretreatment of the sample using folic acid standard (0.05 mg/mL) and the difference between the measured folic acid and the measured stock standard.
1.4 drawing of Standard Curve
The folic acid standard is dissolved in folic acid buffer solution to prepare standard solutions with the concentration of 1 mug/mL, 1.5 mug/mL, 2 mug/mL, 5 mug/mL and 10 mug/mLL respectively, and the folic acid yield in the standard solution is measured. The standard curve is shown in fig. 5, and the formula is as follows: y=64.367x+3.09679r 2 =0.9987; content unit: μg/mL.
The folic acid yield of different strains is compared with that of the different strains shown in Table 1, and lactic acid bacteria with better folic acid production capacity are screened out by measuring the folic acid yield of 50 strains through liquid chromatography.
TABLE 1 comparison of folate production by different strains
EXAMPLE 3 confirmation of acid resistance and bile salt resistance of Lactobacillus plantarum ZJUIDS16
1. Acid resistance test
Selecting Lactobacillus plantarum ZJUIDS16 single colony, performing amplification culture in MRS liquid culture medium for 18 hr, inoculating the amplified bacterial suspension into MRS broth culture medium at 1% (v/v), and culturing for 18 hr until the bacterial solution concentration reaches 10 8 CFU/mi or so. The culture solution was centrifuged at 5000r/min at 4℃for 5min to collect the cells, which were washed 2 times with Phosphate Buffer (PBS), and then suspended in MRS liquid medium at pH=3.0 for 3 hours, followed by 37 culture. The viable bacteria in the 0h and 3h samples were counted using a pour plate method against MRS without pH adjustment, and the pour plate was incubated for 48h in 37 to determine the viability, calculated as follows:
2. Experiment of bile salt resistance
Implantation after expansion of activationInoculating lactobacillus ZJUIDS16 bacterial suspension into MRS broth culture medium at 1% (v/v), culturing at 37deg.C for 18 hr, and culturing to obtain bacterial solution with concentration of 10 8 CFU/mi or so. Vortex mixed and inoculated into MRS broth containing 0.3% (m/v) ox gall salt (control is MRS broth without ox gall salt) at 2% and cultured for 3h at 37 ℃. The number of viable bacteria in the sample was then counted using the pour plate method. The poured plate was incubated at 37℃for 48h. The bile salt tolerance of the strain is represented by the following formula:
TABLE 2 acid and bile salt resistant conditions of Lactobacillus plantarum ZJUIDS16
| Strain | Survival in ph=3.0, 3h, MRS | Survival in 0.3% bile salts, 3h, MRS |
| Lactobacillus plantarum ZJUIDS16 | 68.31％ | 41.60％ |
As shown in Table 2, the pH of gastric juice is generally about 2-3, and lactic acid bacteria with acid tolerance can enter the gastrointestinal tract to play a role, and as shown in the table, the test results show that the acid tolerance rates of ZJUIDS16 are 68.31% respectively, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. The content of bile salt in the small intestine of a human body is about 0.30%, and the lactobacillus with bile salt resistance can maintain activity in the small intestine and exert the function of the lactobacillus, so that the result shows that the bile salt tolerance rate of the lactobacillus plantarum ZJUIDS16 is 41.60%, which is obviously superior to that of a control strain, and has stronger bile salt resistance. Probiotics must be able to withstand a range of adverse conditions in the gastrointestinal tract such as gastric acid and bile to survive their probiotic action. The lactobacillus plantarum ZJUIDS16 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.
EXAMPLE 4 confirmation of the self-agglutination ability of Lactobacillus plantarum ZJUIDS16
Washing lactobacillus precipitate twice with PBS liquid culture medium subjected to high-temperature sterilization treatment, and re-suspending to make OD610 absorbance be 0.5 to obtain lactobacillus suspension; placing proper amount of bacterial liquid into a sterilized clean test tube, standing at 37deg.C for culturing, and measuring OD at different time points within 24 hr 610 Is used for the light absorption value of (a).
The results are shown in FIG. 6. Compared with the control strain GQ1701 strain, ZJUIDS16 has better self-agglutination performance. The higher the self-agglutination rate, the higher the agglutination rate of the strain itself. From the data we can see that the self-clotting rate of all strains was enhanced with time increase over 24h, and that ZJUIDS14 self-clotting rate reached about 80% at 24h.
The rate of self-clotting of the strain is related to its adhesion in the intestinal tract, the higher the rate of clotting, indicating a higher adhesion in the gastrointestinal tract. The bacterial strain has high rate of aggregation to a certain level, and the bacterial strain can form biological protection film to protect gastrointestinal tract mucous membrane. In addition, the agglutination of the strain can keep the bacterial body active, improve the upper digestive tract of the human body against pathogenic bacteria, and form steric hindrance to prevent the pathogenic bacteria from colonizing the intestinal tract.
Example 5 antioxidant Capacity verification of Lactobacillus plantarum ZJUIDS16
Antioxidant capacity assay of Lactobacillus plantarum ZJUIDS16
5.1 sample preparation
Will be stored in glycerol tubesThe strain was streaked on MRS solid medium and cultured upside down at 37℃for 48 hours. And (3) picking single colonies by using an inoculating loop, inoculating the single colonies into a sterilized MRS liquid culture medium, and standing and culturing for 18-24 hours at 37 ℃ to obtain a culture solution. The culture solution was adjusted to a lactic acid bacteria concentration of 10 with distilled water 10 CFU/mL,4 ℃, 8000 Xg centrifugal 20min, collecting the supernatant, namely fermentation supernatant. The centrifuged cell pellet was resuspended and washed with 0.02M PBS buffer (ph=7.4), and centrifuged at 8000×g for 20min at 4 ℃ for 3 replicates. Resuspending the washed cells in PBS buffer and adjusting the cell concentration to 10 10 CFU/mL to obtain the bacterial suspension.
5.2 determination of Total antioxidant Capacity
The total antioxidant capacity (FRAP method) was determined by adding 150. Mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, and V: V=10:1:1, as-is) and 20. Mu.L of sample to the ELISA plate, shaking and mixing, reacting at 37℃for 10min, and measuring the absorbance of the solution at 593 nm. Substituting the absorbance measured by the sample into a ferrous sulfate standard curve, wherein the antioxidant capacity of the sample is expressed as ferrous sulfate equivalent (mu mol FeSO) 4 /mL sample). Each sample was run in 3 replicates and the average was calculated.
Ferrous sulfate standard curve: ferrous sulfate solutions were prepared at different mass concentrations (0. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 400. Mu.M, 600. Mu.M, 800. Mu.M), and different molar concentrations of ferrous sulfate solution, 10mM TPTZ buffer, 0.3M acetate buffer at V: v: v=1: 1:10, adding 170 mu L of the mixed solution into an ELISA plate, reacting for 10min at 37 ℃, and measuring the absorbance of the solution at 593 nm. And drawing a standard curve by taking the mass concentration of ferrous sulfate as an abscissa and the absorbance as an ordinate.
5.3 determination of reducing Capacity
1mL of the sample was placed in a centrifuge tube, and 0.2M PBS solution (pH 6.6) and 1mL of each of 1% (w/v) potassium ferricyanide solution were added and mixed well. Water bath at 50 ℃ for 20min, and cooling in ice bath. Then 10% (w/v) trichloroacetic acid 1mL is added, 6000 Xg is centrifugated for 5min, 1mL of supernatant is taken, 0.1% (w/v) ferric trichloride 1mL and distilled water 1mL are added, the mixture is fully and uniformly mixed, the mixture is stood for reaction for 10min, and the absorbance of the sample at 700nm is measured. The samples were replaced with PBS buffer or MRS broth as blank. Each sample was run in 3 replicates and the average was calculated.
Reduction ability (%) = [ (a) s -A b )/A b ]*100% of the formula: a is that s -sample group absorbance; a is that b Blank absorbance
5.4DPPH free radical scavenging Capacity determination
Preparing V with different concentration gradients C Solution (0-30. Mu.g/ml). 100 mu L of the sample to be tested (or V) is added into the ELISA plate C Standard solution) and 100 mu L of 0.2M DPPH ethanol solution (prepared by absolute ethanol, stored at 4 ℃ in dark place and used at present) are shaken uniformly and kept away from light for 30min at room temperature, and the absorbance of the solution at 517nm is measured; 100 mu L of absolute ethyl alcohol is used to replace 100 mu L of DPPH ethanol solution to form a blank group; 100. Mu.L of PBS buffer (or MRS liquid culture medium) is used for replacing 100. Mu.L of the sample to be tested as a control group, and 100. Mu.L of PBS buffer (or MRS liquid culture medium) and absolute ethanol mixed solution are used for blank zeroing. The average was calculated by repeating 3 replicates for each sample. DPPH radical scavenging ability (%) = [1- (a) s -A b )/A c ]*100% of the formula: a is that s -sample group absorbance; a is that b -blank absorbance; a is that c Control absorbance.
TABLE 3 in vitro antioxidant properties of Lactobacillus plantarum ZJUIDS16
EXAMPLE 6 confirmation of the hydrophobic Capacity of Lactobacillus plantarum ZJUIDS16
Washing lactobacillus precipitate twice with PBS liquid medium subjected to high temperature sterilization, and re-suspending to obtain OD 610 The absorbance of the solution is about 0.5, and the lactobacillus suspension is obtained; thoroughly mixing 2ml lactobacillus suspension and 2ml xylene, shaking in 37 deg.C water bath for 5min, and measuring OD of water phase after 0 hr and 2 hr respectively 610 Absorbance values.
TABLE 4 surface hydrophobicity of different strains (%)
| Bacterial strain | Hydrophobicity of |
| ZJUIDS16 | 56.74％ |
The hydrophobicity of ZJUIDS16 was measured to be 56.74% significantly higher than the control strain. The strain has strong adhesion capability, can adhere to human intestinal tracts, and improves the health of intestinal flora.
EXAMPLE 7 confirmation of the anti-thawing Capacity of Lactobacillus plantarum ZJUIDS16
The test adopts a blood agar plate culture method to detect the hemolysis type of lactobacillus plantarum ZJUIDS16, and the method is as follows:
(1) Activating and culturing ZJUIDS16 in liquid MRS culture medium at 37 ℃;
(2) Streaking on blood agar plates (available from Beijing land bridge technologies Co., ltd.) using sterile inoculating loop to pick up the third generation of culture fluid;
(3) Placing the flat plate at 37 ℃ for culturing for 48 hours;
(4) The blood agar plates were observed for color change and for the presence of a hemolytic loop. Three types of hemolysis and judging methods: alpha (form a) hemolysis: the hemolysin is generated, the hemolysis is incomplete, and a grass green hemolysis ring with the thickness of 1-2 mm and narrower translucency is arranged around a colony on a blood agar plate. Beta (b) hemolysis: is completely hemolyzed, and has a hemolysis ring which is 2-4 mm wide, well-defined, colorless and transparent around the colony on the blood plate. (type c) hemolysis: no hemolysin was produced and no hemolytic ring was present around colonies on blood agar plates. As shown in FIG. 7, the Lactobacillus plantarum ZJUIDS16 grows well in blood agar plates, and the color of the culture medium near the colony does not change obviously, so that the Lactobacillus plantarum is judged to have no hemolysis.
Example 8 confirmation of antibiotic susceptibility of Lactobacillus plantarum ZJUIDS16
The antibiotic susceptibility of lactic acid bacteria strains was measured by the paper diffusion method, which is referred to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI). Preparing an MRS solid culture medium by using a wide-mouth conical flask, placing the culture medium in a 55 ℃ water bath for heat preservation after the culture medium is sterilized, taking out the culture medium after the temperature is reduced, placing the culture medium into a sterilized super clean bench, inoculating lactobacillus suspension (108 CFU/mL) into the conical flask according to the amount of 1%, shaking the culture medium until the culture medium and the lactic acid bacteria suspension are uniformly mixed, and pouring the culture medium and the culture medium into a sterile plate to prepare an LB plate of 15 mL/plate. After the MRS plates were solidified, two antibiotic paper sheets were gently attached to each plate with forceps uniformly. Plates with antibiotic paper were incubated at 37℃for 24h. After the cultivation is finished, the diameter of the inhibition zone is measured by a vernier caliper and recorded.
The diameter of the antibiotic susceptibility zone of lactobacillus plantarum ZJUIDS16 is shown in table 5. The strain was evaluated for susceptibility to the common 9 antibiotics according to the drug susceptibility test criteria of CLSI, and the results are shown in table 5.
TABLE 5 diameter of zone of bacteriostasis of Lactobacillus plantarum ZJUIDS16 on antibiotic susceptibility
Lactobacillus plantarum ZJUIDS16 is sensitive to 3 antibiotics (erythromycin, penicillin and ceftriaxone), wherein the sensitivity to penicillin is the most, and the inhibition zone reaches 32.19mm. Experimental results indicate that Lactobacillus plantarum ZJUIDS16 is sensitive to common antibiotics.
With the wide application of antibiotics in clinical treatment, the drug resistance of lactobacillus is also more and more serious, and long-term intake of the drug-resistant lactobacillus can bring great difficulty to clinical treatment. The lactobacillus plantarum ZJUIDS16 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 9 confirmation of pathogenic inhibitory Capacity of Lactobacillus plantarum ZJUIDS16
The antibacterial activity of the lactic acid bacteria is measured by an international agar diffusion method. The four frozen indicator strains (escherichia coli, salmonella, staphylococcus aureus and listeria monocytogenes) were activated 2-3 times on LB solid medium. The activated single colonies were picked up and cultured in LB medium, 37 for 18h. Collecting bacterial cells by centrifugation, and re-suspending in physiological saline to a concentration of 10 8 CFU/mL. The indicator fungus suspension is added into sterilized LB solid medium cooled to about 55 ℃ according to the amount of 1% (v/v), and the mixture is poured into a culture dish (15 mL/dish) after being uniformly mixed, and a sterile oxford cup placed in advance is pulled out after condensation. After the activated Lactobacillus plantarum ZJUIDS16 was grown in MRS medium for 18 hours, the supernatant was collected by centrifugation (8000 rpm,5min,4 m) and the bacterial pellet was discarded. The metabolic supernatant of lactobacillus plantarum ZJUIDS16 was added to the cup wells (200 wells) and non-inoculated MRS medium (ph=6.2) was used as a blank, the diameter of the zone of inhibition was measured after 24h incubation at 37 ℃, strains with distinct zones of inhibition around the wells were selected, the diameter of the zone of inhibition was measured, and each was repeated three times.
TABLE 6 diameter data for the coil sensitivity of Lactobacillus plantarum ZJUIDS16 to 9 common antibiotics
As shown in Table 6, the strain has a certain inhibition effect on 4 pathogenic bacteria, the strain has a remarkable inhibition effect (P < 0.05) on staphylococcus aureus and escherichia coli, wherein the inhibition effect of supernatant on listeria monocytogenes is the best, and the inhibition zone reaches 20.1mm. Therefore, the metabolic substances generated in the growth process of the lactobacillus plantarum ZJUIDS16 have certain antibacterial activity and have an effect of improving the intestinal health of a human body.
Staphylococcus aureus is the most common pathogen in suppurative infection of humans, 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 antibacterial products generated by lactic acid bacteria metabolism can inhibit the growth of pathogenic bacteria singly or jointly. The metabolite of the lactobacillus plantarum ZJUIDS16 provided by the invention has a certain antagonism to the four pathogenic bacteria, plays an important role in maintaining intestinal microecological balance and has a health promoting effect.
Example 10 preparation of functional fermented fruit and vegetable juice Using Lactobacillus plantarum ZJUIDS16
1. The processing technology of the fermented fruit and vegetable juice comprises the following steps:
raw material cleaning, flash evaporation, pulping, blending, homogenizing, sterilizing, cooling, inoculating, sealing fermentation, after-ripening, filling and refrigerating
2. Key points of operation
(1) Cleaning and cutting into blocks: firstly, cleaning pumpkin and dragon fruit, peeling, removing and cutting into small pieces;
(2) Flash evaporation: inactivating enzyme by flash evaporation, treating at 121 deg.C with 0.5-1min, and rapidly exhausting;
(3) Pulping: according to pumpkin: water (mass ratio) =1:1, and proper amount of pumpkin and water are gradually put into a colloid mill for grinding, and coarse grinding and fine grinding are performed once. Pulping the dragon fruits by a pulping machine until pulp is uniform and has no lumps;
(4) Blending and homogenizing: 15% (v/v) of pumpkin juice, 30% (v/v) of dragon fruit juice and regulating the content of soluble solids to 10 by using sucrose 。 Brix, adding 0.2% (w/v) stabilizer CMC-Na, mixing, and homogenizing under low pressure (15 MPa) and high pressure (25 MPa) to obtain pulp with particle diameter of 2-3 μm;
(5) Sterilizing and cooling: preserving the heat of the blended composite fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃;
(6) Inoculating and fermenting: under aseptic condition, inoculating activated Lactobacillus plantarum ZJUIDS16, and controlling initial bacterial count at 1×10 7 CFU/mL. Fermenting at 37 deg.C for 24 hr;
(7) Post-ripening: after fermentation, placing the mixture in a refrigerator at 4 ℃ for 3 hours;
(8) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.
Example 11 preparation of bacterial powder Using Lactobacillus plantarum ZJUIDS16 producing 5-methyltetrahydrofolate
Lactobacillus plantarum ZJUIDS16 was inoculated with an inoculating loop in 50mL MRS liquid medium and incubated in an incubator at 37℃for 18h. Then transferred to 250mL MRS broth after sterilization at an inoculum size of 5%. Placed in a bacterial incubator at 37℃and incubated for 24h. The resulting fermentation broth was centrifuged, after which the supernatant was discarded and the cell pellet was collected, and the cells were rinsed 2 times with sterile Phosphate Buffer (PBS) for 10min each at a centrifugation speed of 5000r/min. Thus obtaining lactobacillus plantarum ZJUIDS16 bacterial precipitate.
Respectively weighing 4.1g of trehalose, 2.8g of sodium glutamate and 8g of sucrose, dissolving in 10mL of distilled water at 40 ℃, filtering and sterilizing by using a 0.22 mu m microporous filter membrane, and adding into 90mL of sterile water for later use. Dissolving 15g of skim milk powder in distilled water to obtain 100mL of 15% skim milk powder solution, and sterilizing at 110 ℃ for later use.
The lactobacillus plantarum ZJUIDS16 bacterial sediment prepared above is prepared according to the following ratio of 1: and 5, fully and uniformly mixing the mixture with the protective agent solution to obtain the bacterial suspension. And subpackaging 5mL of bacterial suspension into 10mL of sterile glass bottles with plugs, pre-freezing at-70 ℃ for 2 hours, taking out, freeze-drying in a vacuum freeze dryer under the conditions of vacuum degree of 5Pa, baffle heating temperature of 20 ℃ and cold trap temperature of-55 ℃, and crushing after freeze-drying for 30 hours to obtain lactobacillus plantarum ZJUIDS16 bacterial powder.
Example 12 preparation of solid beverages Using Lactobacillus plantarum ZJUIDS16 producing 5-methyltetrahydrofolate
3 parts of whole milk powder, 1-2 parts of theanine, 4-5 parts of gamma-aminobutyric acid, 0.20 part of vitamin C,2-5 parts of compound probiotics, 16.02-36.02 parts of inulin, 5.01-15.01 parts of fructo-oligosaccharide, 7.01-17.01 parts of erythritol, sweetener, thickener and essence are added; the compound probiotics comprise a mixture of streptococcus thermophilus, bifidobacterium lactis Bla08, lactobacillus acidophilus LA85, lactobacillus plantarum BL21, lactobacillus plantarum Lp90 and lactobacillus plantarum ZJUIDS16, all the substances are synergistic, and the finished product is obtained by mixing the substances according to the proportion, and packaging the mixture after uniform mixing.
Example 13 preparation of fruit and vegetable juice fermented with Lactobacillus plantarum ZJUIDS16 producing 5-methyltetrahydrofolate
Fresh pumpkin and dragon fruit are selected as raw materials, and the raw materials are cleaned, peeled (pulp removed) and cut into small pieces. The enzyme is deactivated by flash evaporation, 0.5 to 1min is treated at 121 ℃, and the gas is rapidly exhausted. According to pumpkin: water (weight ratio) =1:1, and proper amount of pumpkin and water are gradually put into a colloid mill for grinding, and coarse grinding and fine grinding are performed once. Pulping the dragon fruits by a pulping machine until pulp is uniform and has no lumps. Blending and homogenizing: 15% of pumpkin juice, 30% of dragon fruit juice, regulating the content of soluble solids to 10 degrees Brix by using sucrose, adding 0.2% of stabilizer CMC, uniformly mixing, and adopting a two-stage homogenization method, wherein the diameter and the grain size of the melon pulp are 2-3 mu m by adopting a low pressure (15 MPa) and a high pressure (25 MPa) firstly. Preserving the heat of the prepared compound fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃. Under aseptic condition, activated lactobacillus plantarum ZJUIDS16 is inoculated, and the initial bacterial count is controlled at 107CFU/mL. Fermenting at 37deg.C for 24 hr. After fermentation, the mixture is put into a refrigerator at 4 ℃ for 3 hours. After finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.
EXAMPLE 14 preparation of Lactobacillus plantarum ZJUIDS16 starter for 5-methyltetrahydrofolate production
The original strain of Lactobacillus plantarum ZJUIDS16 is inoculated into 11% skimmed milk (sterilized at 115 ℃ C. For 20 min), cultured at 37 ℃ C. For 18-24h to curd, and activated for two generations continuously to be used as mother starter. Inoculating the mother starter into 11% skimmed milk according to 3% -5% inoculum size, sterilizing for 20min, and culturing at 37deg.C for 18-24 hr to obtain curd, wherein viable count can reach 10 9 -10 10 CFU/mL to obtain the lactobacillus plantarum ZJUIDS16 starter.
EXAMPLE 15 preparation of functional fermented milk by fermentation of Lactobacillus plantarum ZJUIDS16
Heating the pretreated cow milk to about 60 ℃, adding 6% sucrose, fully dissolving, and homogenizing under 20 MPa. Then heat treated at 95℃for 5min and cooled to 38 ℃. Inoculating lactobacillus plantarum ZJUIDS16 starter prepared in example 10 according to 5% inoculum size, fermenting at 37deg.C for 14-18h to curd, cooling, and refrigerating at 4deg.C to obtain lactobacillus plantarum ZJUIDS16 functional fermented milk.
1. Preparation of fermented milk folic acid content determination
The folic acid content was measured by the method of 1.3 in example 2.
2. Evaluation of flavor of fermented milk
And fermenting the strain to prepare fermented milk serving as an experimental material. The prepared samples are stored in a refrigerator at the temperature of 4 ℃ in a laboratory, and all indexes are measured in the shelf life.
3. Sensory evaluation
3.1 screening by raters: in view of the persistence of sensory experiments, the recruitment and screening of the raters was primarily directed to food professionals at school students. According to the method described in GB/T16291.1-2012, the basic taste recognition capacity, the observation threshold and the recognition threshold of the evaluator are respectively measured, and finally 6 sensory sensitive candidates are determined, wherein the ratio of men to women is 1:1.
3.2 yogurt sensory descriptors: finally determining that the yogurt smell sensory evaluation words are sweet taste, sour taste, milk flavor, milk fat taste and taste after sensory training; the taste sensory evaluation words are sweet taste, sour taste, milk flavor and milk fat taste. The descriptors are defined and a reference sample is determined, a food-grade plastic cup with a transparent cover is selected, 10g of yoghurt sample is weighed and put into 2 tasting cups, and the sensory evaluation of smell and taste is carried out respectively. The number of yoghurt samples evaluated in each test is controlled to be less than 5, and the sample number adopts 3-bit random numbers. The evaluator rinsed one sample with mineral water after each sample was tasted, and each sample was repeated twice. Sensory scoring criteria (0-9): 0 (none); 1 to 3 (weaker); 4 to 6 (medium strength); 7 to 9 (strong).
TABLE 7 sensory evaluation of different folate producing strains
| Bacterial strain | ZJUIDS16 | LZ217 |
| Sweet taste | 4.3±0.23 a | 5.1±0.14 a |
| Sour taste | 4.4±0.13 b | 4.1±0.39 c |
| Milk flavor | 4.4±0.62 a | 3.4±0.13 a |
| Sour and fatty taste | 6.2±0.31 a | 6.1±0.75 a |
| Cooking flavor | 2.2±0.3 a | 4.1±0.3 a |
| Smell intensity | 4.5±0.45 a | 2.2±0.53 a |
| Preference of | 6.2±0.23 a | 4.9±0.63 b |
The results of the sensory odor analysis of the prepared yogurt and the commercial yogurt are shown in table 3. The odor sensory analysis data showed flavor differences between different commercial yogurts. The flavor index score of the yoghurt milk is 3.7-5.6, the cooking taste score is 1.7-4.2, and the preference score is 2.8-6.7. The milk flavor in yogurt was determined to be at a moderate intensity, the milk flavor was weak, and there was also a weak cooking flavor. The odor sensory preference of the evaluator shows that the sample ZJUIDS16 has high preference; LZ217 favorites were moderate.
EXAMPLE 16 high folate production Strain yield and functional property to property comparison
In order to show the advantages of the invention in terms of folic acid production and other probiotic functions, the folic acid production capacity and the probiotic functions of the strain disclosed by the invention and other disclosed strains are particularly compared, and the comparison results are shown in Table 8.
TABLE 8 comparative examples of extracellular folate content of lactic acid bacteria compared with literature publications
As shown in Table 8, the Lactobacillus plantarum has higher folic acid yield in published documents, and has a synthetic gene sequence of high folic acid yield in Lactobacillus plantarum, so that subsequent experiments performed by using Lactobacillus plantarum have better theoretical basis and experimental significance, and have superiority of strain types for improving the folic acid yield of lactobacillus.
The prior art mainly adopts common bacterial strain fermentation to prepare the fermented dairy product, has the trend of product singleness, mainly uses the product function, lacks the product rich in high-content high-quality nutrients, does not have the fermented dairy product prepared by the high-yield folic acid bacterial strain at present, and does not have the fermented dairy product with higher folic acid content at the same time, so the invention has better originality and innovation in comparison with the commercial products.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (7)
1. Lactobacillus plantarum strain capable of producing folic acid in high yieldLactobacillus plantarum) ZJUIDS16, characterized by the taxonomic designation of the strain: lactobacillus plantarumLactobacillus plantarumThe microbial strain has been deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.26162.
2. Lactobacillus plantarum ZJUIDS16 according to claim 1, characterized in that its 16SrDNA complete sequence is shown in SEQ ID No. 1.
3. Use of lactobacillus plantarum ZJUIDS16 according to claim 1 for the preparation of functional foods, health products and pet foods.
4. The use according to claim 3, wherein the functional food comprises: fermented fruit and vegetable juice, fermented dairy products and fungus powder.
5. The use according to claim 3, wherein the health product comprises: high-yield folic acid bacterial agent or bacterial powder.
6. Use according to claim 3, wherein the pet food comprises: milk powder for pets and probiotic powder for pets.
7. Use of lactobacillus plantarum ZJUIDS16 according to claim 1 for the preparation of a food preservative, characterized in that lactobacillus plantarum ZJUIDS16 is prepared by fermentation for food preservation.
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