CN116463252A - Weissella multocida M1 and application thereof - Google Patents
Weissella multocida M1 and application thereof Download PDFInfo
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- CN116463252A CN116463252A CN202310277091.2A CN202310277091A CN116463252A CN 116463252 A CN116463252 A CN 116463252A CN 202310277091 A CN202310277091 A CN 202310277091A CN 116463252 A CN116463252 A CN 116463252A
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- weissella
- strain
- preservative
- enteroid
- multocida
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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
- 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
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Life Sciences & Earth Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
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- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to an enterokinesia-like Weissella M1 and application thereof. The invention relates to an enteroid Weissella, latin is named Weissella Paramesenteroides; the preservation number is CGMCCNO:26182. the Weissella multocida is negative in amino acid decarboxylase, negative in nitrate reductase, sensitive to chloramphenicol, moderately resistant to penicillin and 2 antibiotics of tetracycline and cefoxitin, free of drug resistance transfer risk, excellent in antibacterial property, capable of being used as a preservative, and obviously superior to the conventional preservative in use effect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to an enterokinesia-like Weissella M1 and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus) is a gram-positive bacterium, a common food-borne pathogenic bacterium, and can cause a variety of acute and chronic infections. Among the most harmful of staphylococcus aureus is methicillin-resistant staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus aureu. Since the beginning of the 60 s of the 20 th century, MRSA strains have been isolated for the first time, and have spread worldwide and outbreak in countless hospitals, making MRSA one of the most common causes in nosocomial infections, which can lead to a series of problems ranging from skin infections to blood infections. MRSA infection has become a major threat to humans, with about 8 tens of thousands of infections and 11000 deaths occurring annually. Currently, MRSA can only be treated with antibiotics such as clindamycin and linezolid, but studies have reported that MRSA has developed resistance to these "last resort" antibiotics. For these reasons, the world health organization identified MRSA as one of six "highly preferred" pathogens that pose a great threat to public health. Thus, there is an urgent need to develop and find new therapies and antibacterial agents with new mechanisms of action to combat such high threat pathogens.
Lactic acid bacteria are a class of spore-free, gram-positive bacteria that can produce large amounts of lactic acid using glucose, and are recognized as safe (Generally recognized as safe, GRAS), widely used in the food, pharmaceutical and feed industries. The bacteriologists divide the lactic acid bacteria into 4 genera, lactobacillus, leuconostoc, pediococcus, and Streptomyces. The latest taxonomies among the lactic acid bacteria categories are the following: aerococcus, allophycoccus, carnobacter, dolosignaulum, enterococcus, globicaella, lactobacillus, oenococcus, tetragenococcus, vagococcus and Weissella. The lactobacillus can synthesize organic acid such as acetic acid, lactic acid, lactobacillus, and H 2 O 2 Substances such as hydroxy fatty acid and cyclic peptide can inhibit the growth and reproduction of various pathogenic bacteria such as bacteria and fungi and other putrefying bacteria. Lactobacillus rhamnosus SHA113 has been found to be effective against multiple resistant Staphylococcus aureus (MRST), and Lactiplantibacillus pantarum, L.acidophilus and L.casei have also been foundThe var combined fermentation has good inhibition effect on the clinical isolated MRST.
Therefore, how to provide a larger variety of lactobacillus strains and apply the lactobacillus strains to the preparation of food or the preservation or preservation of brewing is a problem to be solved by the person skilled in the art.
Disclosure of Invention
The invention takes lactobacillus separated from Tibetan mushroom as a research object, adopts oxford cup diffusion method to screen out a lactobacillus with MRSA inhibiting activity, identifies the strain and evaluates the safety of the strain, researches the property of antibacterial active substances of the strain, and provides a theoretical basis for developing novel biological bacteriostat.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an enterotype Weissella (Weissella Paramesenteroides) M1, which is preserved in China general microbiological culture Collection center (CGMCC), and the preservation date is 2023, 2 months and 14 days, and the preservation number is CGMCC NO:26182, address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The invention provides application of the enteroid Weissella in preparation of a preservative.
The invention provides application of the enteroid Weissella to preparation of a preparation for preventing and treating diseases or diseases caused by staphylococcus aureus.
Preferably, the preparation is a health product or a cleaning product.
The invention also provides a preservative comprising the enteroid Weissella according to claim 1.
Preferably, the enteroid Weissella is a bacterial suspension of enteroid Weissella.
Preferably, the concentration of the bacterial suspension is 1X 10 6 ～1×10 10 CFU/mL。
The invention also provides application of the preservative in food preservation.
Preferably, the 16SrDNA sequence of the Weissella enteroides is shown as SEQ ID NO: 1.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention screens out a strain M1 with MRSA inhibiting activity from 9 strains of lactic acid bacteria separated from Tibetan mushroom, and identifies the strain M1 as enterokinesia-like Weissezia W.paramesenteroides M1 through morphology, physiological biochemistry and molecular biology. The dynamic detection results of the growth curve, pH and antibacterial activity show that the fermentation supernatant of the intestinal-membrane-like Weissella M1 after 8h fermentation shows the inhibition effect on MRSA, the pH value of the fermentation liquor is 4.62, and the diameter of the inhibition zone is 12.57+/-1.16 mm.
The antibacterial active substance produced by the Weissella multocida M1 is insensitive to protease and temperature, but is sensitive to pH, and when the pH is adjusted to 7.0, the antibacterial activity is completely lost. The ethyl acetate can effectively extract antibacterial substances in the fermentation liquor. Finally, the safety evaluation result shows that the Weissella multocida M1 is negative for amino acid decarboxylase, negative for nitrate reductase, sensitive to chloramphenicol, moderately resistant to penicillin, resistant to 2 antibiotics of tetracycline and cefoxitin, and free of drug resistance transfer risk. This study may provide a theoretical reference for the development of novel inhibitors of MRSA.
2. The data recorded by the invention show that the intestinal membrane-like Weissella M1 bacterial suspension can be used as a preservative, especially in the pickle fermentation process, the preservation time of pickle can be obviously prolonged, the shelf life of meat can be prolonged, and the use effect of the intestinal membrane-like Weissella M1 bacterial suspension is obviously better than that of the conventional preservative.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the inhibitory effect of strain M1 of example 1 on MRSA;
FIG. 2 is a graph showing colony characteristics and fungus shapes of the strain M1 of example 2;
FIG. 3 is a phylogenetic tree of example 2 constructed based on the rDNA sequence of strain M1 16S;
FIG. 4 is a graph of the bacteriostasis kinetics of Weissella multocida M1 of example 3;
FIG. 5 is an electrophoresis chart of plasmid extraction of example 6.
Description of biological preservation
The Weissella multocida (Weissella Paramesenteroides) M1 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2023, 2 and 14 days, and the preservation number is CGMCC NO:26182, address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Test materials used in the following examples
Lactobacillus to be screened is separated from Tibetan mushroom and stored in a food quality and safety laboratory of a Huainan academy of teachers; methicillin-resistant staphylococcus aureus BNCC326053, beijing north na-invasive joint biotechnology institute; bacterial genome DNA extraction kit (DP 302), plasmid extraction kit, marker2000, beijing Tian Gen Biotechnology Co., ltd; beyoRed DNA loading buffer, pekoe biotechnology limited, proteinase K, siro biotechnology; green streptomycin, chloramphenicol, and aletin; pepsin, catalase, cefoxitin, vancomycin, source foliar organisms; ethyl acetate, the company of the chemical industry, cyLong, inc.
Lactic acid bacteria medium (Mann Rogosa and Sharpe broth, MRS): 10g of beef extract, 10g of peptone, 2g of diammonium citrate, 2g of dipotassium hydrogen phosphate, 5g of yeast extract, 5g of anhydrous sodium acetate, 20g of glucose, 0.2g of magnesium sulfate, 80 1mL of tween-80, 0.05g of manganese sulfate, 1L of distilled water, sterilizing at 121 ℃ for 15min, and adding 18-20 g of agar to obtain the MRS solid culture medium.
Beef extract peptone agar medium: beef extract 3g, peptone 10g, sodium chloride 5g, agar 20g, distilled water 1L, and sterilizing at 121 ℃ for 15min.
Amino acid medium: 5g of peptone, 3g of yeast extract, 1g of glucose, 1mL of 1.6% bromocresol purple-ethanol solution with pH of 6.8, and 0.5% (mass fraction) of tyrosine, ornithine, lysine, histidine and arginine are added to prepare 5 groups of amino acid culture mediums.
Nitrate medium: adding 0.2g KNO into 1000mL MRS culture medium 4 Heating and stirring until the mixture is completely dissolved, and adjusting the pH value to 7.4 to prepare the nitrate culture medium.
Apparatus and device
SW-CJ-2F double-sided vertical purification workbench, suzhou West air conditioner purification Co., ltd; EX Zone2 electronic balance, shenzhen Anpule electronic technologies Co., ltd; BSC-250 constant temperature and humidity incubator, shanghai Boqing Xie medical order equipment factory; an LDZW-60 KCS-II vertical pressure steam sterilizing pot and Shanghai Shen An medical equipment; unique-R20+UV laboratory ultra-pure water System, xiamen Yue Jie Water purification technologies Co., ltd; h0347 electric heating constant temperature drying oven, shanghai Chengheng instruments and technology Co., ltd; BCD-152 Mitsubishi refrigerator, hefei Mitsubishi Co., ltd; HZQ-F160A constant temperature culture oscillator, shanghai Chengsu Intelligent science and technology Co., ltd; RE-5286A rotary evaporator, shanghai Asia Biochemical instruments factory; HH-420201 digital display heating water bath box, hangzhou you Ning instruments Co.
Example 1
1 screening of MRSA-inhibiting Strain
9 strains are obtained by screening and purifying Tibetan mushroom, the 9 strains to be screened after purification are respectively transferred into MRS liquid culture medium, and are subjected to stationary culture at 37 ℃ for 48 hours, 12000r/min and centrifugation at 4 ℃ for 10 minutes, and the supernatant is obtained by filtration through a 0.45 mu m sterile filter membrane. MRSA is used as indicator bacteria, and the oxford cup diffusion method is adopted to detect the antibacterial activity of the strain to be detected. OD is set to 600 A bacterial suspension of indicator bacteria MRSA (methicillin-resistant staphylococcus aureus) of 0.2 is uniformly coated on a beef extract peptone solid culture medium by using a sterile cotton swab, two oxford cups are gently placed in each dish, 100 mu L of fermentation supernatant and sterile MRS liquid culture medium are respectively injected into the oxford cups for comparison,standing at 4deg.C for 3 hr, culturing at 37deg.C for 18 hr, observing inhibition zone, and measuring inhibition zone diameter.
Results: the inhibitory effect of 9 strains to be tested on the indicator bacteria MRSA is measured by adopting an oxford cup diffusion method, and a strain M1 with obvious inhibitory effect on the MRSA is preferably selected, wherein the diameter of a bacteriostasis circle of the strain M1 can reach 18.42+/-0.33 mm (the inhibitory effect of the strain M1 on the MRSA in figure 1, the left circle is an M1 bacteriostasis circle, and the right circle is an MRS liquid culture medium contrast).
Example 2
Identification of strains
(1) Morphological observation and physiological biochemical reaction
Activating the strain M1 screened in the example 1 for 2 generations, streaking the bacterial liquid cultured to the logarithmic phase on an MRS solid culture medium, culturing at 37 ℃, observing colony morphology, color, smoothness and the like after colony formation, then picking single colony, carrying out gram staining, and observing cell morphology under a biological microscope; various physiological and biochemical experiments are carried out by referring to ' golden red star, yang Xiyin, cheng Wenyu ' the method for separating Weissella containing endogenous plasmid from spicy cabbage and identifying [ J ]. Chinese brewing, 2012,32 (1): 77-79 '.
(2) 16S rDNA sequence analysis
Inoculating the activated strain M1 into MRS culture medium, standing at 37 ℃ for 16 hours, centrifuging to collect precipitate, and extracting total DNA of the strain M1 according to a bacterial genome DNA extraction kit method. The extracted DNA template is subjected to PCR amplification, and forward primers are adopted for the following steps: 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO: 2), reverse primer is 1492R:5'-ACGGCTACCTTGTTACGACTT-3' (as shown in sequence 3). The PCR products were checked by agarose gel electrophoresis and then sent to the general biosystems (Anhui) Limited for sequencing (PCR reaction (50. Mu.L) where 3. Mu.L of template DNA, 1. Mu.L of forward and reverse primers, 25. Mu.L of Premix, and the balance of ultrapure water were used to 50. Mu.L. PCR reaction conditions of pre-denaturation 94℃for 5min, denaturation 94℃for 1min, annealing 52℃for 1min, extension 72℃for 1.5min,30 cycles, and extension 5min at 72 ℃). The sequence obtained is subjected to sequence correlation analysis by BLAST (http:// www.ncbi.nlm.nih.gov/BLAST), a sequence with high similarity to the 16S rDNA sequence is obtained, homology analysis is performed, and a phylogenetic tree is constructed by adopting MEGA4.0 software through a Neighbor-joining method.
Results: strain M1 was streaked on MRS solid medium at 37℃for 24 hours, and the morphological characteristics of the strain were observed, and the results are shown in FIG. 2. As can be seen from FIG. 2, strain M1 forms white colonies, circles, edges are regular, the colonies are micro-raised, smooth and opaque, and the morphology is gram positive, rod-like, spore-free, flagellum-free and motionless under microscopic examination on MRS solid medium. The physiological and biochemical characteristics of strain M1 are shown in Table 1. As is clear from Table 1, the strain M1 was unable to use hydrogen peroxide, lactose, raffinose, mannitol, sorbitol, sucrose, salicin, inulin, maltose, etc., and the physiological and biochemical characteristics of the strain M1 and Weissella enterica (Weissella Paramesenteroides) were highly similar to those of the strain M1 according to the "classification and identification of lactic acid bacteria" and the "Burjie's bacteria identification handbook". FIG. 2 shows colony characteristics and cell morphology of strain M1.
Further PCR amplification and sequence determination to obtain 16S rDNA sequence fragment of strain M1, BLAST on-line comparison, similarity with W.parameteroides strain 3151 (MT 515963.1) up to 99.73%, and construction of phylogenetic tree (phylogenetic tree constructed based on strain M116S rDNA sequence in FIG. 3) by MEGA software, the strain M1 was determined to be Weissella enteroides, named W.parameteroides M1. The intestinal membrane-like Weissella is an important lactic acid bacterium, and is freshly isolated in Tibetan mushroom or kefir, but is distributed in other fermented foods such as fermented vegetables, cheeses, sausages, fermented soybeans and the like, and part of strains can produce bacteriocin and bacteriostasis substances other than bacteriocin in the fermentation process, so that various pathogenic bacteria such as gram-negative bacteria, gram-positive bacteria, fungi and the like can be inhibited.
TABLE 1 physiological and biochemical characterization of Strain M1
Note that: the "+" test result is positive; the "-" test result was negative
Example 3
1 bacterial strain M1 bacteriostasis kinetic curve
The activated strain M1 of example 2 was inoculated into MRS liquid medium at an inoculum size of 3%, and cultured at 37℃for 48 hours with OD measured every 4 hours 600 Values and pH. Meanwhile, collecting fermentation liquor, centrifuging at 12000r/min and 4 ℃ for 10min, and filtering by a sterile filter membrane with the diameter of 0.45 mu m to obtain supernatant. The inhibition of MRSA by supernatants at different fermentation times was determined by oxford cup diffusion.
Results: the growth curve, pH and bacteriostatic activity results of the intestinal membrane-like Weissella M1 are shown in FIG. 4. As can be seen from FIG. 4, during the 48h cultivation, the biomass of Weissella multocida M1 increased from 0.02 to 2.43 (OD 600 ). The pH of the broth was reduced from 5.36 to 3.52. The intestinal membrane-like Weissella M1 starts to show the inhibition effect on MRSA after 8 hours of fermentation, the pH value of the fermentation liquor is 4.62, and the diameter of a bacteriostasis ring is 12.57+/-1.16 mm. After fermentation for 36h and entering a stabilization period, the fermentation supernatant has the strongest inhibitory effect on MRSA, and the diameter of a inhibition zone reaches 24.41+/-0.60 mm, and then the fermentation supernatant is maintained stable. FIG. 4 is a graph of the bacteriostasis kinetics of Weissella multocida M1.
Example 4
Analysis of the Properties of bacteriostatic substances
(1) Influence of enzymes on bacteriostatic substances
The fermentation supernatant obtained in example 3 of strain M1 was treated with bromelain, catalase, pepsin and proteinase K to a final enzyme concentration of 2mg/mL and adjusted to the optimal pH of the enzyme: bromelain pH 7.0, catalase pH 7.0, pepsin pH 2.0 and proteinase K pH 7.5. And (3) after enzymolysis for 2 hours in a water bath at 37 ℃, the pH value of the fermentation supernatant is readjusted back to the original pH value, the influence of enzyme on the activity of antibacterial substances generated by the strain M1 is detected, the fermentation supernatant of the strain which is not subjected to enzyme treatment is used as a blank control group, and MRSA is used as an indicator bacterium.
(2) Influence of temperature on bacteriostatic substances
The fermentation supernatants obtained in example 3 of strain M1 were treated at 65℃at 85℃at 100℃at 121℃for 40 minutes, respectively, and the inhibitory effect of temperature on MRSA by the fermentation supernatants was measured.
(3) Influence of pH on bacteriostatic substances
The fermentation supernatant obtained in example 3 was adjusted to pH 7.0 (fermentation supernatant original pH 3.52) with 1mol/L NaOH, and the supernatant to which an equal amount of sterile water was added was used as a control. The inhibitory effect of the alkali-treated fermentation supernatant on MRSA was measured.
Results: the inhibitory effect of the fermentation supernatant of the Weissella multocida M1 after being treated by different enzymes is shown in Table 2, and the inhibitory effect of the fermentation supernatant of the Weissella multocida M1 after being treated by proteinase K, pepsin and catalase on the MRSA is not significantly different from that of the control (P < 0.05), so that the antibacterial active substances produced by the Weissella multocida M1 are insensitive to proteinase and are insensitive to catalase. The temperature sensitivity experiment result shows that after the fermentation supernatant is treated for 40min at 65-121 ℃, no significant difference (P > 0.05) exists between the inhibitory activities of the fermentation supernatant to MRSA (Table 3), and the antibacterial active substances are stable to heat. When the pH was adjusted to 7.0, the inhibitory activity of the fermentation supernatant against MRSA was completely lost (Table 4).
TABLE 2 Effect of different enzymes on bacteriostatic Activity
| Enzyme species | Antibacterial diameter (mm) |
| Catalase enzyme | 25.48±2.40a |
| Proteinase K | 24.08±0.24a |
| Pepsin | 25.01±1.17a |
| Control | 25.08±2.22a |
Note that: the same column of shoulder letters indicates significant differences (P < 0.05).
TABLE 3 influence of different temperatures on bacteriostatic Activity
| Treatment mode | Antibacterial diameter (mm) |
| 65℃ | 24.08±0.23a |
| 85℃ | 23.96±0.30a |
| 100℃ | 24.21±0.08a |
| 121℃ | 24.11±0.20a |
| Control | 24.15±0.15a |
TABLE 4 influence of pH on bacteriostatic substances
Note that: "-" means no bacteriostasis.
Example 5
Identification of bacteriostatic active substances
(1) Extraction of bacteriostatic active substances
Reference is made to "Li Weina, huang Wenyu, liu Chenjian, et al," method of primary organic acids in lactobacillus casei fermentation broth and their bacteriostasis [ J ]. Food industry technology, 2019,40 (3): 66-70+6. Culturing for 48h, centrifuging the fermentation broth 12000r/min obtained in example 3 at 4deg.C for 15min to obtain supernatant, and mixing with 1: mixing the mixture with ethyl acetate in a volume ratio of 1, and extracting the water phase for 3 times to obtain an upper ethyl acetate phase, an intermediate flocculent phase and a lower water phase. Concentrating ethyl acetate layer phase and middle white flocculent layer phase at 45deg.C under 150r/min, concentrating by rotary evaporation at 60deg.C under 150r/min, concentrating by 10 times volume to obtain water layer rotary evaporation phase and water layer rotary evaporation residual phase, middle layer rotary evaporation residual phase, ethyl acetate layer rotary evaporation phase, and measuring antibacterial effect of each phase by oxford cup method with MRSA as indicator bacteria.
Table 5 inhibition effect (mm) of Weissella M1 extraction on MRSA
| Extracting each phase | Diameter of inhibition zone |
| Ethyl acetate phase rotary steaming phase | -- |
| Ethyl acetate phase rotary steaming residual phase | 35.56±0.30 |
| Intermediate phase rotary steaming phase | -- |
| Intermediate phase rotary steaming residual phase | -- |
| Aqueous phase rotary steaming phase | -- |
| Steaming the water layer phase to form a residual phase | 23.10±0.18 |
Note that: "-" means no bacteriostasis.
Example 6
Safety evaluation of strains
(1) Amino acid decarboxylase assay
The strain M1 was inoculated in an inoculum size of 3% into 4 amino acid decarboxylase detection media, and was subjected to stationary culture at 37℃for 48 hours, and the color change was observed, with MRS medium as a control group.
(2) Nitrate reductase assay
The strain M1 is inoculated into a nitrate culture medium according to the inoculation amount of 3 percent, after standing culture is carried out for 48 hours at 37 ℃, 10 drops of 5 percent (mass fraction) potassium iodide solution are firstly dripped, then 10 drops of 5 percent (mass fraction) starch solution are dripped, after fully and uniformly mixing, the color change of the culture medium is observed, and the culture medium without bacteria is used as a control group.
(3) Drug sensitivity test
The tetracycline, head, was determined by the paper diffusion method (Kirby-Bauer, K-B method) recommended by the American society for clinical and laboratory standards (Clinical laboratory standard institute, CLSI)5 antibiotics of the sporisotin, the green streptomycin, the chloramphenicol and the vancomycin are used for judging the diameter of a bacteriostasis circle of the strain M1 and judging the drug resistance. After overnight activation of strain M1, the bacterial suspension of strain M1 was diluted with sterile MRS broth to an OD 625 The bacterial suspension with the value of 0.08-0.13 (using sterile MRS liquid culture medium as blank for zeroing) is uniformly coated on MRS solid culture medium by using sterile cotton stick. Taking the drug sensitive paper sheet by using sterile forceps, sticking the drug sensitive paper sheet on the surface of a culture medium, culturing at 37 ℃ for 21 hours, taking out the drug sensitive paper sheet, and measuring the diameter of a bacteriostasis ring by using a vernier caliper. Each test was repeated three times.
(4) Plasmid extraction test
3-5 mL of activated bacterial suspension of the bacterial strain M1 is taken, and plasmids of the target bacterial strain are extracted by using a plasmid extraction kit and lysozyme according to instructions. Detecting with 1% agarose gel, imaging with full-automatic gel imaging system, and observing whether electrophoresis band appears.
Results: nitrate reductase can catalyze nitrate to reduce into nitrite, nitrite can produce nitrosamine, and nitrosamine can induce gastric cancer, liver cancer and other diseases. The results of the invention show that the bacterial solutions are all not blue and are negative (see Table 6), which shows that nitrate reductase in the metabolites of Weissella multocida M1 is inactive or does not contain nitrate reductase.
Some lactobacillus has amino acid decarboxylase activity, can decarboxylate and reduce amino acid into amine substances, if the amine substances are accumulated in a body too much, toxicological effects are caused, the culture environment becomes alkaline, and after bromocresol purple indicator is added dropwise, the culture medium shows yellow as negative and purple as positive. The experimental result is yellow, which shows that the metabolic product of the Weissella multocida M1 does not contain amino acid decarboxylase, i.e. is harmless to human health.
TABLE 6 detection results of harmful metabolites
Table 7 shows the results of drug sensitivity experiments, which show that chloramphenicol is highly sensitive to the diameter of the inhibition zone of Weissella multocida M1 reaching 27.12+ -0.29 mm, moderately sensitive to penicillin, but resistant to 2 antibiotics, tetracycline and cefoxitin. In recent years, research on drug resistance of lactic acid bacteria has attracted attention as to whether it carries a drug-resistant gene with transferability. Many studies have shown that resistance to lactic acid bacteria comes from two sources: and is self-contained and obtained from foreign DNA. It is thought that most lactic acid bacteria are resistant to metastasis, but there is a possibility that they will have metastasis after binding to plasmids. To further investigate whether the resistance of the enteroid Weissella M1 to antibiotics is at risk of metastasis, plasmids of the strains were extracted. As shown in the plasmid extraction electrophoresis chart 5, no band is found after the plasmid of the Weissella multocida M1 is detected, so that the condition that the Weissella multocida M1 does not contain drug resistant plasmid or drug resistant genes only exist on genome DNA can be presumed, and therefore, the risk of drug resistance transfer of the Weissella multocida M1 can be primarily judged. FIG. 5 plasmid extraction electrophoretogram; M-DL2000 Marker,1-W.Paramesenteroides M1 plasmid.
TABLE 7 results of drug sensitivity experiments
Note that: r (resistance) -drug resistance, I (Insensitive) -mediator, S (Sensitivity) -sensitivity, "-" indicates no zone of inhibition;
determination criteria: clinical & laboratory standards institute (Clinical & Laboratory Standards Instituts, CLSI) standard
Example 7
Comparison test:
(1) 9kg of pickled radish was prepared according to the method of example 1 in the preparation method of CN201510250096.1, and was equally divided into 9 parts, and each part was placed in a pickle jar for standby, and three jars were randomly selected for each test group.
(2) Experiment group 1 is the bacterial suspension of the Weissella multocida with the bacterial concentration of 1 multiplied by 10 8 CFU/mL; the addition ratio was 50g/kg.
The control group 1 was potassium sorbate with an addition ratio of 0.5g/kg.
Blank 1 was not subjected to any treatment.
And (3) respectively adding the preservatives of different test groups into the pickle jar, and uniformly mixing. The kimchi treated in each test group was then stored at 5 deg.c, 15 deg.c and 25 deg.c, respectively. The appearance, flavor and taste of kimchi were examined after 60d after storage, and the number of bacteria was measured to average. The results are shown in tables 8 to 10.
Note that: the bacteria are gram negative microorganisms such as Escherichia coli, acinetobacter, vibrio, proteus, etc. which are unfavorable for food fermentation, health or flavor generation. Cells were stained with crystal violet plus iodine, destained with 95% ethanol and re-stained with reddish solution. After such treatment, the microorganisms stained red are observed by a microscope, and are gram-negative bacteria. Counting was performed by a hemocytometer.
Table 85 ℃ stored survey results table
| Appearance of | Flavor of | Mouthfeel of the product | CFU/g of mixed bacteria count | |
| Experiment group 1 | The soup is clear | Unique flavor | Crisp and refreshing food | 1.2×10 2 |
| Control group 1 | The soup is clear | Unique flavor | Crisp and refreshing food | 1.5×10 2 |
| Blank control group 1 | Cloudy soup | Poor flavor | Soft and soft | 8.5×10 2 |
Table 9 table of investigation results stored at 15 c
| Appearance of | Flavor of | Mouthfeel of the product | CFU/g of mixed bacteria count | |
| Experiment group 1 | The soup is clear | Unique flavor | Crisp and refreshing food | 6.2×10 2 |
| Control group 1 | The soup is clear | Unique flavor | Crisp and refreshing food | 7.5×10 2 |
| Blank control group 1 | Cloudy soup | Poor flavor | Soft and soft | 8.6×10 3 |
Table 10 table of investigation results stored at 25 c
As can be seen from tables 8 to 10, the kimchi is stored under different conditions, which may be related to the inhibition of the activity of microorganisms at low temperature. However, the best results were obtained in the test group 1 and the control group 1 at any storage temperature. The intestinal membrane-like Weissella strain solution disclosed by the invention can be used as a preservative for pickle, and the use effect is obviously better than that of the preservative in the prior art.
Example 8
Comparison test:
(1) One piece of chilled fresh pork was selected, divided into 9 pieces on average, and three pieces were randomly selected for each test group.
(2) Experiment group 2 is the bacterial suspension of the Weissella multocida with the bacterial concentration of 1 multiplied by 10 8 CFU/mL; the addition ratio was 50g/kg, and the bacterial suspension was sprayed onto the surface of chilled fresh meat using a small sprayer.
The control group 2 is YC9-6-1 chilled fresh meat preservative (WU Han fresh keeping biotechnology Co., ltd.) and the application method is referred to the instruction.
Blank group 2 was not subjected to any treatment.
The chilled fresh meat treated by each test group is wrapped by a preservative film and then stored in a refrigerator at 4 ℃. Meat quality was measured at 7d and 14d, and the average was taken three times. The results are shown in Table 11.
(3) Measured data:
1) The measurement of 2-thiobarbituric acid (TBA) was performed by a direct measurement method of the acid value of animal and vegetable oils and fats, 2-thiobarbituric acid.
The TBA value in the meat product reflects the extent of the final oxidation of fat in the muscle tissue, with a higher TBA value indicating a higher extent of fatty rancidity of the meat product and poorer meat quality. Evaluation criteria: TBA > 1mg/kg can be identified as spoiled meat.
2) Determination of volatile basic Nitrogen (TVB-N) Using semi-Trace Nitrogen determination method
Evaluation criteria: TVB-N value is less than or equal to 15mg/100g, primary freshness is 15mg/100 g-25 mg/100g, secondary freshness is more than 25mg/100g, and deteriorated meat is obtained.
3) The total number of colonies was determined by plate counting.
Evaluation criteria: the logarithmic value of the total number of the bacterial colonies in the meat product is smaller than 4.0 and is first-order freshness, the logarithmic value of the total number of the bacterial colonies is between 4.0 and 6.0 and is second-order freshness, and the logarithmic value of the total number of the bacterial colonies is larger than 6.0 and is spoiled meat.
4) The water holding capacity of the meat was determined using centrifugation.
Table 11 chilled meat assay results
As can be seen from the records in Table 11, the enteric-coated Weissella-like bacteria has excellent use effect on meat preservation, the total number of TBA, TVB-N and bacterial colony is obviously superior to that of a control group, and the water retention rate is obviously higher than that of other control groups. Therefore, the intestinal membrane-like Weissella can obviously prolong the shelf life of chilled meat and is obviously superior to the products in the prior art. This is sufficient to prove that the intestinal membrane-like Weissella provided by the invention can be used as a food preservative.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Weissella multocida (Weissella Paramesenteroides) M1, which is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation date of 2023, 2 and 14 days and a preservation number of CGMCCNO:26182, address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
2. Use of the enteroid weissella according to claim 1 for the preparation of a preservative.
3. The use of the enteroid Weissella according to claim 1 for the preparation of a preparation for controlling diseases or disorders caused by Staphylococcus aureus.
4. The use according to claim 3, wherein the formulation is a health product or a cleaning product.
5. A preservative comprising the enteroid weissella according to claim 1.
6. The preservative according to claim 5, wherein the enteroid Weissella is a bacterial suspension of enteroid Weissella.
7. The preservative according to claim 6, wherein the concentration of the bacterial suspension is 1X 10 6 ～1×10 10 CFU/mL。
8. Use of a preservative according to any one of claims 5 to 7 for preserving food.
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