CN117264854A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
- Publication number
- CN117264854A CN117264854A CN202311539375.0A CN202311539375A CN117264854A CN 117264854 A CN117264854 A CN 117264854A CN 202311539375 A CN202311539375 A CN 202311539375A CN 117264854 A CN117264854 A CN 117264854A
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- China
- Prior art keywords
- lactobacillus plantarum
- plantarum
- lactiplantibacillus
- lactobacillus
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- 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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- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
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Abstract
The invention discloses a lactobacillus plantarum and application thereof, which is named as lactobacillus plantarum L3 Lactiplantibacillus plantarum L3, and the strain is registered and preserved in China center for type culture collection; the preservation number is CCTCC M2023863. The strain can produce phenyllactic acid with high yield and has strong antibacterial effect on pathogenic microorganisms such as mould, staphylococcus aureus and the like. The lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 provided by the invention can obviously inhibit the growth of harmful microorganisms in the storage and preservation of fermented milk and apples, ensure the quality of products and prolong the storage period of the products, and can provide theoretical basis for the development of food storage and preservation methods. In addition, the strain also has the capability of producing the probiotics functional factors such as conjugated linoleic acid, biomembrane and the like.
Description
Technical Field
The invention relates to the field of lactobacillus strains, in particular to lactobacillus plantarum and application thereof.
Background
Food safety is a worldwide challenge, and microbial contamination such as bacterial contamination, viral contamination, fungal contamination and the like is currently a major food safety issue. At present, chemical preservatives such as calcium propionate, sodium diacetate, potassium sorbate and the like are mainly used for microbial contamination, however, frequent or excessive use of the chemical preservatives can cause problems of microbial resistance, chemical preservative residues, food quality change and the like, and cannot meet the requirements of consumers on high-quality foods. It has been reported that lactic acid bacteria (Lactic acid bacteria, LAB) are recognized safety-class strains that produce phenyllactic acid and the like during metabolism to inhibit harmful microorganisms.
Phenyllactic acid (phenyllactic acid, PLA), 2-hydroxy-3-phenylpropionic acid, is a naturally occurring organic acid in honey and fermented foods. Molecular formula C 9 H 10 O 3 Molecular weight 166 g/moL, melting temperature of about 98 ℃, is generally considered safe, non-cytotoxic to animal and human cells, and free of any unpleasant odors. PLA has physiological functions of treating coronary heart disease, resisting myocardial anoxia, resisting HIV, improving immunity, improving meat quality, inhibiting bacteria, etc. Among these functions, most attention is paid to bacteriostasis, and PLA has better solubility in water than Nisin (Nisin) and other natural bacteriostasis substances, and can maintain good thermal stability (120 ℃ for 20 min) and acid adaptability, because of these excellent characteristics, PLA is more easily diffused in foods than Nisin and other bacteriostasis substances. The PLA has a broad antibacterial spectrum, and can inhibit gram-positive bacteria, gram-negative bacteria, fungi and the likeActing as a medicine. Therefore, the lactic acid bacteria phenyllactic acid has great prospect in the development and utilization of foods. Research and development of a starter with the characteristic of producing antibacterial substances, and substitution of chemical preservatives has important significance.
Disclosure of Invention
The invention aims to provide a lactobacillus for producing phenyllactic acid at high yield and application thereof. The invention separates a lactobacillus plantarum from fermented dough of the Daxizhou cake in Yunnan, and the lactobacillus plantarum can produce phenyllactic acid, conjugated linoleic acid and biological membrane with high yield, obviously inhibit the growth of pathogenic microorganisms and prolong the shelf life of food.
In a first aspect of the present invention, there is provided a lactobacillus plantarum designated lactobacillus plantarum L3 Lactiplantibacillus plantarum L, having a preservation number of CCTCC M2023863, and a preservation unit of: china center for type culture collection, with the preservation addresses: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: 2023-05-30.
Further, the lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 was isolated from yunnan da xizhou tsba fermented dough.
The bacterial colony morphology observed by naked eyes of the lactobacillus plantarum L3 Lactiplantibacillus plantarum L is as follows: round, semitransparent, convex, neat in edge and milky colony with moist surface; the strain cells are rod-shaped under an optical microscope, do not produce spores and are gram-positive.
Further, the 16S rDNA sequence of the lactobacillus plantarum L3 Lactiplantibacillus plantarum L is shown in SEQ ID NO. 1.
In a second aspect of the invention, a microbial agent is provided, said agent comprising said lactobacillus plantarum.
In a third aspect, the invention provides an application of the lactobacillus plantarum or the microbial agent in preparing phenyllactic acid, conjugated linoleic acid or a biological membrane.
In a fourth aspect, the invention provides the use of said lactobacillus plantarum or said microbial agent for preserving food or inhibiting spoilage and pathogenic microorganisms. The spoilage and pathogenic microorganisms are mold, staphylococcus aureus, vibrio parahaemolyticus, and the like.
In a fifth aspect, the invention provides the use of said lactobacillus plantarum or said microbial agent in fermented food products.
Preferably, the fermented food is a flour product or a fermented milk.
Preferably, the lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is prepared to have a viable count of fermented milk of no less than 1×10 6 CFU/mL。
The strain provided by the invention can be used for high-yield phenyllactic acid, the phenyllactic acid content can reach more than 250mg/L, conjugated linoleic acid and a biological film can be also high-yield, and the conjugated linoleic acid with the concentration of more than 330ug/mL can be produced after 24 hours of culture.
In the storage and preservation of fermented milk, lactobacillus bulgaricus: streptococcus thermophilus: lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is inoculated into milk, fermented milk (test group) is obtained by fermentation, and lactobacillus plantarum L3 Lactiplantibacillus plantarum L is not inoculated as a control group. The results showed that the mould count in the control fermented milk was essentially unchanged during 21d storage; the test group had a reduced number of moulds in the fermented milk. The lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 effectively inhibits the growth of mould in the storage of fermented milk, and has important significance for developing a starter with the characteristic of producing antibacterial substances and replacing the addition of chemical preservatives.
In the food preservation experiment taking apples as an example, lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 shows strong antibacterial capability, can effectively control the growth of mould in apples, and can inhibit the expansion of lesion areas; can prolong the storage period of apples.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention separates and screens lactic acid bacteria with high phenyllactic acid yield from fermented dough samples of Yunnan Daxizhou cake. Identification of the lactic acid bacteria by morphological identification, 16S rDNA Gene sequencing and Whole genome sequencing, genus on classificationLactiplantibacillus plantarumIs Lactobacillus plantarum and is named Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3。
(2) The lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 provided by the invention produces phenyllactic acid, conjugated linoleic acid and a biological film, the phenyllactic acid can inhibit bacteria, the conjugated linoleic acid has a plurality of physiological functions of resisting cancer, reducing lipid, resisting oxidization and the like, and the biological film is used for resisting bad environments and keeping the activity of the bacterial strain.
(3) The lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 provided by the invention can obviously inhibit the growth of pathogenic microorganisms such as mould, staphylococcus aureus, vibrio parahaemolyticus and the like, and can inhibit the growth of the pathogenic microorganisms in the food storage process, so that the strain can be used as an antiseptic microbial agent, the quality guarantee period of the food is prolonged, and the quality of the food are maintained.
Drawings
FIG. 1 colony morphology of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3;
FIG. 2 is a gene circle diagram of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3;
FIG. 3 growth curve of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3;
FIG. 4 shows the antibacterial ability of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L, wherein A is the antibacterial ability of mould, B is the antibacterial ability of Staphylococcus aureus, and C is the antibacterial ability of Vibrio parahaemolyticus;
FIG. 5 Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 phenyllactic acid producing ability;
FIG. 6 use of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 in fermented milk;
FIG. 7 shows the application of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 in apple preservation, wherein A is the influence of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 on the apple on mould, and B is the lesion diameter of the apple;
FIG. 8 Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 biofilm producing ability.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the following description of the technical solution of the present invention is given by way of example and illustration only, and should not be construed as limiting the scope of the present invention in any way. The mould used in the invention is separated from metamorphic yoghurt, after 18s rDNA sequencing, the sequencing result is compared with a GenBank database, the result shows that the homology with penicillium roqueforti is 100%, and the penicillium roqueforti is determined to be penicillium roqueforti. The staphylococcus aureus is separated from the milk cake, and the sequencing result is compared with a GenBank database through biochemical detection and whole genome sequencing, so that the homology with the staphylococcus aureus is 100%, and the staphylococcus aureus is determined to be staphylococcus aureus; vibrio parahaemolyticus is separated from freshwater fish, and is subjected to biochemical detection and whole genome sequencing, and the sequencing result is compared with a GenBank database, so that the homology with Vibrio parahaemolyticus is 100%, and the Vibrio parahaemolyticus is determined. Lactobacillus plantarum L2 is separated from thick broad-bean paste, lactobacillus fermentum A1 is separated from schisandra chinensis wine, lactobacillus fermentum A3 is separated from garlic, lactobacillus fermentum A5 is separated from dried yak, lactobacillus fermentum A10 is separated from milk residue, lactobacillus plantarum C67 is separated from goat milk, lactobacillus plantarum C6 is separated from milk cake, lactobacillus plantarum E8 is separated from milk residue, lactobacillus plantarum E64 is separated from sweet white wine, and after the above 9 lactobacillus strains are separated, the lactobacillus plantarum is subjected to sequencing through 16s rDNA, and the sequencing result is compared with a GenBank database, so that the corresponding lactobacillus variety is identified.
EXAMPLE 1 isolation and purification of seed
Taking 1g of a fermentation dough sample of Xizhou cake obtained from Yunnan China, inoculating the fermentation dough sample into sterilized normal saline, and sequentially carrying out 10-time gradient dilution. Respectively take 10 -4 、10 -5 、10 -6 The diluted solution was coated on a plate containing 0.5% CaCO by the plate coating method 3 On MRS solid medium, placing into a constant temperature incubator, and standing and culturing at 37 ℃ for 48h. And (3) observing and recording colony characteristics, picking out colonies with different colony morphologies and containing a calcium dissolving ring, and further carrying out streak separation and purification for 3-4 times until the final plate is a colony with the same morphology. Single colony inoculation is selected and cultured in MRS liquid culture medium at 37 ℃ for 24 hours for preservation and identification.
Identification of species
(1) Morphological characterization of the species
As shown in FIG. 1, after lactobacillus L3 is cultured in MRS agar medium for 48 hours, the colony form is milky white, semitransparent, moist, smooth, and regular in edge, and has obvious bulges. The strain cells are rod-shaped under the lactobacillus L3 optical microscope, do not produce spores, and are gram-positive.
(2) Molecular genetics identification of species
(1) 16S rDNA Gene sequencing
Bacterial genomes were extracted using a DNA extraction kit (universal) for TSINGKE, and bacterial universal primers were used with the genome of the extracted strain as a template: 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were subjected to PCR experiments on 16S rDNA. The PCR product was sent to Beijing engine biotechnology Co. The nucleic acid sequence of the 16S rDNA is shown as SEQ ID NO.1, and the sequencing result is compared with the existing sequence of the GenBank database in NCBI database (www.ncbi.nlm.gov/BLAST /) by using a BLAST tool to analyze the homology of the strain to be tested with the corresponding sequence of the known strain. The comparison result of the GenBank database shows that the homology with lactobacillus plantarum is 100 percent, and the screened lactobacillus L3 is determined to be lactobacillus plantarum.
(2) Whole gene sequencing
Taking 2.5 mug of DNA sample qualified in lactobacillus L3 quality inspection, adding magnetic beads for purification, and taking 1 mug of sample Qubit for quantification. Fragmenting DNA by using a mechanical breaking method (ultrasonic), then carrying out fragment purification, terminal repair, adding A at the 3 terminal, connecting a sequencing joint on the fragmented DNA, carrying out fragment size selection by using agarose gel electrophoresis, carrying out PCR amplification to form a sequencing library (NEBNExt Ultra ™ DNA Library Prep Kit for Illumina), carrying out library quality inspection on the built library, and sequencing the library qualified in quality inspection by using IlluminaNovaSeq. The results in FIG. 2 show that the total length of the L3 gene of lactic acid bacteria is 3167484 bp and the average G+C content is 44.65%.
Finally, the screened strain is determined to be lactobacillus plantarum by morphology, 16S rDNA sequencing and whole genome sequencing, and is named as lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 and is preserved in China center for type culture Collection, and the preservation number is: cctccc M2023863.
Example 2 bacteriostatic ability of Strain
(1) Growth curve of lactobacillus plantarum L3 Lactiplantibacillus plantarum L3
Seed liquid of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L which is activated for three generations in MRS liquid culture medium is inoculated into the MRS liquid culture medium according to the inoculum size (V/V) of 1%, 200 mu L of the seed liquid is taken and placed into a 96-well plate, the absorbance at 600nm is measured, the absorbance is measured once every 2h, 24h is measured in total, each time point is repeated for 3 times, the average value is taken as the abscissa, the absorbance is taken as the ordinate, and a growth curve is drawn.
As shown in FIG. 3, the OD600nm value of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 was increased with the increase of fermentation time, and the growth was in accordance with an S-shaped curve. The strain is in a growth delay period for 0-2 hours, and the growth rate of the strain is slower; 2-14 h in the growth log phase, and the growth rate of the strain is rapidly increased; the strain is in a stable growth period for 14-18 hours, and the OD600nm value of the strain is basically stable; and the bacterial OD600nm value is slightly reduced in the growth decay period of 18-24 h.
(2) Antibacterial ability measurement of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3
The sterilized oxford cup was placed on a plate, and the mold (penicillium roquefortii) suspension was inoculated into an unset wort agar medium in a volume fraction of 3% (v/v), and poured into the plate after mixing well. Placing the plate in a super clean bench for 30 min, extracting oxford cup (10 mm deep, 8mm wide) with forceps, and adding 200 μl lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 fermentation supernatant (CFS) into the hole, lactobacillus plantarum L2Lactiplantibacillus plantarumL2 fermentation supernatant (CFS), control group added 200. Mu.L sterilized sterile MRS broth, malt extract agar plate placed at 28℃for 3 d culture, observation and measurement of the size of inhibition zone, and repeating for three times.
Staphylococcus aureus suspension was inoculated into an uncured LB agar medium in a volume fraction of 3% (v/v), and poured into a plate after mixing well. Flat platePlacing the plate in a super clean bench for 30 min, extracting oxford cup (10 mm deep, 8mm wide) with forceps, and adding 200 μl lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 fermentation supernatant (CFS) into the hole, lactobacillus plantarum L2Lactiplantibacillus plantarumL2 fermentation supernatant (CFS), 200. Mu.L of sterilized sterile MRS broth and sterile water were added to the control group, and LB agar plates were incubated at 37℃for 24 hours, and the size of the inhibition zone was observed and measured, and repeated three times.
Inoculating Vibrio parahaemolyticus suspension at 3% (v/v) volume fraction onto 3% sodium chloride tryptone soybean agar plate, coating with coating rod, placing sterilized oxford cup on the plate, and adding 200 μl lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 fermentation supernatant (CFS), lactobacillus plantarum L2 into the holeLactiplantibacillus plantarumL2 fermentation supernatant (CFS), control group was added with 200. Mu.L sterilized sterile MRS broth and sterile water. The 3% sodium chloride tryptone soybean agar plate is placed at 37 ℃ for culture for 24 hours, and the size of the inhibition zone is observed and measured for three times.
As shown in ABC in fig. 4, a is a mold bacteriostatic ability assay, B is a staphylococcus aureus bacteriostatic ability assay, and C is a vibrio parahaemolyticus bacteriostatic ability assay; compared with a control group, the Lactobacillus plantarum L3 Lactiplantibacillus plantarum L CFS has obvious inhibition zones on the mould, staphylococcus aureus and vibrio parahaemolyticus plates, the diameters of the inhibition zones respectively reach 21.8+/-1.1 mm, 20.3+/-1.6 mm and 13.7+/-0.9 mm, so that the Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 has higher inhibition capability and can inhibit the growth of mould, staphylococcus aureus and vibrio parahaemolyticus.
Example 3 determination of the phenyllactic acid production Capacity of Strain
Lactobacillus plantarum L3 Lactiplantibacillus plantarum L seed liquid, which was activated three times in MRS liquid medium, was inoculated in an inoculum size (V/V) of 1% into an MRS liquid medium containing 0,0.2,0.4,0.6,0.8,1 g/L phenylalanine, cultured at 37℃for 24h, centrifuged at 8000 rpm at 4℃for 5min, filtered with a sterile 0.22 μm filter to obtain a cell-free supernatant, and analyzed using reverse phase high performance liquid chromatography (RP-HPLC) equipped with an Agilent Zorbax SB-C18 column (150 mm X4.6 mm,5 μm), with 0.05% acetonitrile in phase A and 0.05% aqueous trifluoroacetic acid in phase B, as determined at 210 nm.
As shown in FIG. 5, with increasing phenylalanine addition amount (0-1 g/L), the PLA (phenyllactic acid) content tended to increase first and then decrease. The highest PLA content can reach 258.1+/-1.39 mg/L when the phenylalanine adding amount is 0.6 g/L.
EXAMPLE 4 application of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 in food
(1) Application of lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 in fermented milk
The activated strain was inoculated into MRS broth at an inoculum size of 1% (37 ℃ C., 24 h) to third generation, and Lactobacillus bulgaricus was inoculated: streptococcus thermophilus: lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is inoculated to milk in a sterile environment according to a ratio of 1:1:1, and then cultured at 36 ℃ to obtain fermented milk. The inoculation amount of the lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is 0.5-2% (v/v).
Antibacterial effect of fermented milk
The ready-to-use mould suspension was washed with PBS and resuspended to about 10 in fresh pasteurized milk 3 CFU/mL, lactobacillus bulgaricus at a ratio of 1:1:1: streptococcus thermophilus: lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is inoculated into milk. Mold numbers were measured using eosin blue plates at 0 d, 4 d, 8 d, 12 d, 16 d, 21d, respectively. Lactobacillus bulgaricus in the same ratio: milk from streptococcus thermophilus served as a control.
As shown in FIG. 6, the mold count in untreated fermented milk was substantially unchanged (7.30~7.29 log CFU/mL) during 21d (days) of storage; the amount of mold added to the lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 fermented milk was reduced from the original 7.31 log CFU/mL to 5.83 log CFU/mL. Clearly, lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 treatment effectively inhibited the growth of mold during storage of the fermented milk. The lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 can be used as a starter with a preservative effect in the production of fermented milk, so that the problem of exceeding standard of mould in the fermented milk is solved, and the economic loss is reduced.
(2) Application of lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 in food preservation
Fresh apples of the same size (each apple weighing about 200 g) were selected. Apples were soaked in 2% sodium hypochlorite solution (v/v) for 2 minutes, rinsed with tap water and dried at room temperature. Each apple surface was sterilized with 70% ethanol. One wound (10 mm deep, 7.8mm wide) was made on each apple with a sterile oxford cup. The mould suspension was then injected into 10 μl of each wound. The mould suspension was allowed to fully absorb within 1 hour, after which 20. Mu.L of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L CFS was inoculated, incubated with sterile MRS broth as control, at 30℃and observed for mould growth and apple decay.
As shown in fig. 7 a, at the early stage (0-2 d), lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 CFS treated group was not significantly different from the control group; however, in the 4 th d th, it can be observed that lactobacillus plantarum L3 Lactiplantibacillus plantarum L CFS shows strong antibacterial ability, can effectively control the growth of mould in apples, and can inhibit the expansion of lesion areas; in the final stage (7-9 d), the decay of mold and apple was observed in the sterile MRS broth treated group compared with the Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 CFS treated group, while as seen in FIG. 7B, the lesion area on the apples in the control group was significantly higher than that in the Lactobacillus plantarum L3 Lactiplantibacillus plantarum L CFS treated groupP<0.001). The plant milk stalks L3 Lactiplantibacillus plantarum L3 are shown to be effective in controlling fruit and vegetable spoilage, and can extend the shelf life of the food product.
EXAMPLE 5 Lactobacillus plantarum L3Lactiplantibacillus plantarumL3 produces other probiotic functional factors
(1) Determination of the ability of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 to produce Conjugated Linoleic Acid (CLA)
1 to be preserved in experiments0 strains of lactic acid bacteria were cultured in MRS liquid medium at 37℃for 24h with third generation of lactic acid bacteria activated. Centrifuging (8000 r,5min,4 ℃) to obtain fermentation supernatant after finishing, taking 10mL of fermentation supernatant after finishing, adding 30mL of normal hexane into a separating funnel, oscillating for 5min, standing for 20min, and then adding 20 mL normal hexane; the whole process was repeated three times, the lower liquid was discarded, the upper organic phase was collected in a clean beaker, and anhydrous sodium sulfate was added to absorb moisture, and then the impurities and bacteria were filtered using a 0.22 m filter. The filtered liquid was diluted 10 times and stored at-4℃for further examination. And (3) measuring the absorbance of the liquid to be detected at 232m by using an ultraviolet spectrophotometer, and then calculating the CLA content of the liquid to be detected according to a formula, wherein the obtained data is the CLA content in the fermentation liquid. The calculation formula of the colic acid in the fermentation liquid is that y=0.0781 x-0.0508 (R 2 =0.9993), x is absorbance.
As shown in Table 1, the CLA content 334.22.+ -. 1.26ug/mL of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 was much higher than that of the other strains among 10 strains. The result shows that the strain can produce other probiotics functional factors besides phenyllactic acid, and is beneficial to human health.
TABLE 1
(2) Determination of the ability of Lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 to produce a biofilm
The experimentally deposited 5 strains of lactic acid bacteria were incubated in MRS broth for three generations of lactic acid bacteria at 37℃for 24 h. After the incubation was completed, the contents of each tube were removed and each tube was washed 2 times with 5 mL sterile PBS (pH 7.2) to remove non-adherent bacteria. The remaining attached bacteria were mixed with 5 mL of 95% methanol for 15 min, the liquid was removed and dried in an oven at 60 ℃. Each tube was then stained with 5 ml0.1% (v/v) crystal violet for 15 min. Then washed with sterile PBS (pH 7.2) to remove any unbound dye. After the tube was dried in an oven at 60 ℃,5 mL of 33% glacial acetic acid was used to dissolve the bound crystal violet and the absorbance of the sample was measured with an ultraviolet spectrophotometer at 490nm wavelength.
The formation of the biological film can resist conditions such as high temperature, improper pH value, osmotic pressure, metal ions, antibiotics and the like, and the survival rate of bacteria is enhanced. In addition, the biological film can promote the adhesion of lactobacillus in intestinal tracts, and is beneficial to the colonization of lactobacillus. Crystal violet staining is a well-known method of measuring biofilms by which biofilms can be stained, the more biofilms the higher the crystal violet color and the higher the value measured at OD490 nm.
As shown in fig. 8, the amount of the biofilm produced by lactobacillus plantarum L3 was higher than that of other lactobacillus, indicating that the survival rate was higher in the adverse environment and gastrointestinal tract, and the function of probiotics was utilized.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explanation of the principles of the present invention and are in no way limiting of the invention. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.
Claims (9)
1. The lactobacillus plantarum is characterized by being named as lactobacillus plantarum L3 Lactiplantibacillus plantarum L3, and has a preservation number of CCTCC M2023863, wherein the preservation unit is as follows: china center for type culture collection, with the preservation addresses: in the Wuhan university of No. 299 in Wuhan district of Wuhan, hubei province.
2. The lactobacillus plantarum of claim 1, wherein the lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is isolated from a yunnan da xizhou cake fermented dough.
3. The lactobacillus plantarum according to claim 1, wherein the 16S rDNA sequence of lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is shown in SEQ ID No. 1.
4. A microbial agent comprising the lactobacillus plantarum of any of claims 1-3.
5. Use of the lactobacillus plantarum of any of claims 1-3 or the microbial agent of claim 4 for the preparation of phenyllactic acid, conjugated linoleic acid or a biofilm.
6. Use of the lactobacillus plantarum of any of claims 1-3 or the microbial agent of claim 4 for preserving food or inhibiting spoilage and pathogenic microorganisms.
7. Use of the lactobacillus plantarum of any of claims 1-3 or the microbial agent of claim 4 in fermented food products.
8. The use according to claim 7, wherein the fermented food product is a flour product or a fermented milk.
9. The use according to claim 8, wherein the viable count of the fermented milk prepared by lactobacillus plantarum L3 Lactiplantibacillus plantarum L3 is not less than 1x 10 6 CFU/mL。
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