CN112410251B - Lactobacillus plantarum with quick acid production and high acid production capacity and application thereof - Google Patents
Lactobacillus plantarum with quick acid production and high acid production capacity and application thereof Download PDFInfo
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- CN112410251B CN112410251B CN202011266087.9A CN202011266087A CN112410251B CN 112410251 B CN112410251 B CN 112410251B CN 202011266087 A CN202011266087 A CN 202011266087A CN 112410251 B CN112410251 B CN 112410251B
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Abstract
The invention provides lactobacillus plantarum with quick acid production and high acid production and application thereof, wherein the lactobacillus plantarum is lactobacillus plantarum (A)Lactobacillus plantarum) WG03, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC 20876, and the preservation date is 10 months and 12 days in 2020. The invention provides a microbial agent containing the lactobacillus plantarum. The lactobacillus plantarum or the microbial inoculum can be applied to silage fermentation. The lactobacillus plantarum with high acid production speed and high acid production quantity is obtained by combining the protoplast fusion technology with the traditional mutagenesis method, and the lactobacillus plantarum is used as the silage additive, so that the growth of putrefying bacteria can be well inhibited, the anaerobic fermentation process can be quickly started, and the quality of silage is improved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum with quick acid production and high acid production and application thereof.
Background
Lactic acid bacteria are a general term for a large group of bacteria which produce a large amount of lactic acid by fermenting sugar, and mainly comprise lactobacillus, bifidobacterium, lactococcus, streptococcus, enterococcus, leuconostoc, pediococcus, bacillus and other genera. The lactobacillus has high application value in the important fields closely related to human life, such as medicine, industry, agriculture and the like, such as yoghourt fermented by lactobacillus, bifidobacterium and streptococcus hygrothermal, and is applied to health-care food; the lactobacillus, the bifidobacterium and the like are applied to clinical medical treatment; the lactobacillus plantarum is applied to silage. Microorganisms such as lactic acid bacteria attached to the raw materials in the ensiling fermentation process form organic acids such as lactic acid by using carbohydrates and generate an anaerobic acidic environment, so that the growth of putrefying bacteria such as yeast and mould is inhibited, and the nutritional ingredients of the raw materials are further preserved in a limited way. Since the fermentation raw material is generally attached with less lactic acid bacteria, the addition of homofermentation lactic acid bacteria capable of rapidly reducing the pH value is beneficial to the rapid start of the silage fermentation. Lactobacillus plantarum is the most common lactic acid bacteria additive. However, the acid production rate and the acid production amount of the lactic acid bacteria additive sold in the market at present cannot achieve ideal effects, and ensilage substances are easy to rot, so that the ensilage quality is influenced.
For example, patent document CN202010612259.7 discloses a lactobacillus plantarum strain with an acid production amount of 12.3g/L, which is relatively low, and a method for producing fermented feed using the same. The existing method for accelerating acid production rate and improving acid yield mainly focuses on the research of natural breeding and mutation breeding, but the mutation direction is difficult to master, and mutants are difficult to concentrate on a plurality of ideal states.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum with fast acid production and high acid production and application thereof, so as to solve the problems of non-ideal acid production rate and acid production of the existing lactobacillus plantarum.
The technical scheme adopted by the invention is as follows: a lactobacillus plantarum with fast and high yield is lactobacillus plantarum (L.) (L.)Lactobacillus plantarum) WG03, the preservation unit is China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No. 1 of Beijing republic of south China, the preservation number is CGMCC 20876, and the preservation date is 10 months and 12 days in 2020.
A microbial agent containing the lactobacillus plantarum.
The viable count of the lactobacillus plantarum in the microbial inoculum is not less than 109CFU/g。
The application of the lactobacillus plantarum in silage fermentation.
The application of the microbial agent in silage fermentation.
The invention utilizes the protoplast fusion technology to improve the acid production rate and the acid production amount of the lactobacillus plantarum, and has the advantages of rapidness, high efficiency, high positive mutation rate and the like compared with the traditional breeding technology. The protoplast fusion technology carries out multiple recursion fusion on strains with positive mutation so as to mutually superpose the advantages of the strains and obtain a target strain more easily, and the protoplast fusion technology is usually combined with the traditional mutagenesis method to carry out screening on the target strain. The lactobacillus plantarum obtained by the technology has high acid production speed and high acid production capacity, and the strain serving as the silage additive can well inhibit the growth of putrefying bacteria, quickly start an anaerobic fermentation process and improve the quality of silage.
Drawings
FIG. 1 is a colony diagram of a Lactobacillus plantarum MRS medium plate of the present invention.
FIG. 2 is a diagram showing the effect of the first set of fermentation bags on the natural ensiling of alfalfa fermentation products.
FIG. 3 is a graph showing the effect of adding Lactobacillus plantarum HBU03 to alfalfa in the second set of fermentation bags on ensiling the fermented product.
FIG. 4 is the effect diagram of the third fermentation bag alfalfa added with Lactobacillus plantarum CGMCC 20876 for ensiling fermentation product.
Detailed Description
The present invention is described in detail below with reference to specific examples, wherein reagents and procedures not mentioned in the examples are all performed according to the routine procedures in the art.
The method for measuring the lactic acid content of lactobacillus plantarum adopted in the examples comprises the following steps: adding excess CaCO to the fermentation broth3And after the reaction is completed, taking 10mL of fermentation liquor, centrifuging for 10min at 8000r/min, sucking 2mL of supernatant by using a liquid transfer gun, adding 25mL of water, adjusting the pH value to 13 by using a 10% NaOH solution, finally adding 200mg of calcein (an indicator) into a triangular flask, titrating by using 0.05mol/L of EDTA standard solution after the calcein is completely dissolved, observing in a backlight mode, and obtaining the end point when the color of the solution is changed from yellow green fluorescent color to orange and the color is not changed any more.
Calculating the formula: w (lactic acid) ═ 90VC (g/L)
V: titration of the amount of EDTA consumed (mL)
C: EDTA concentration (mol/L)
Test materials and reagents
MRS culture medium: 10.0g of beef extract, 10.0g of peptone, 5.0g of yeast extract, 20.0g of glucose, 2.0g of dipotassium phosphate, 2.0g of ammonium citrate, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate and 801.0 mL of tween-sodium, adding distilled water to 1.0L, adjusting the pH value to 6.2 +/-0.2, and sterilizing at 115 ℃ for 20 min.
Regeneration culture medium: based on MRS liquid culture medium, no Tween-80 is added, and 2.5% of gelatin, 25mmol/L of calcium chloride, 25mmol/L of magnesium chloride hexahydrate, 0.5mol/L of sucrose, 1.6-1.8% of agar powder are added, and sterilization is carried out for 20min at 115 ℃.
Protoplast buffer solution (LPB): tris1.21g, sucrose 171.14g, magnesium chloride hexahydrate 4.07g, hydrochloric acid pH6.8, 115 ℃ sterilization for 20 min.
Lysozyme solution (100 mg/mL): 1g of lysozyme was dissolved in 10mL of sterilized LPB, sterilized by filtration through a 0.22 μm aqueous membrane filter, and stored at-20 ℃.
0.85% physiological saline: weighing 8.5g solid NaCl, adding distilled water to constant volume to 1L, and sterilizing at 115 deg.C for 15 min.
Example 1 Complex mutagenesis
1. DES mutagenesis
Lactobacillus plantarum HBU03 is used as an original strain, is separated from sediment of the white lake basin in New zone of Xiongan, and is stored in a laboratory for researching and applying the diversity of microorganisms in Hebei province. Placing 9.4mL of bacterial suspension of the strain into a sterile triangular flask, adding 10mL of sterile phosphate buffer solution with pH7.0 and 0.6mL of DES solution, shaking uniformly, placing in a constant temperature shaking table at 37 deg.C, shaking for 35min from the beginning of DES solution addition, adding 10mL of 25% Na2S2O3The reaction is stopped. Diluting the mutagenized bacterial liquid to a proper gradient, coating the diluted bacterial liquid on a bromocresol purple culture medium, inverting the culture dish, placing the culture dish in a dark paper box, placing the culture dish in an incubator at 39 ℃ for culture, taking the bacterial liquid out after 4-6 days, measuring the diameters of bacterial colonies and a color-changing ring by using a ruler, calculating the ratio of the former to the latter, namely the HC value, selecting the bacterial colonies with a large HC value, storing the bacterial colonies on a slope, and placing the bacterial colonies in a refrigerator at 4 ℃. And (4) performing activation test on the preserved plate, primarily screening the strain, performing fermentation culture, and detecting the acid production rate and the acid production amount after 72 hours. One of the mutant strains is named as HBU03D, and has the acid yield of 54.26g/LThe acid yield (51.65 g/L) of the fermentation strain HBU03 is improved by 5.05%, and the time for reaching the highest acid yield is basically consistent with that of the starting strain.
2. Ultraviolet mutagenesis
Taking the mutant strain HBU03D as an original strain, sucking 10mL of the bacterial liquid diluted to a proper dilution degree to a sterile culture dish by using a pipette gun, and placing the sterile culture dish on a magnetic stirrer at a position 20cm below an ultraviolet lamp. And (3) turning on a power supply of the magnetic stirrer to uniformly mix the bacteria liquid, opening a vessel cover, turning on an ultraviolet lamp, timing by using a stopwatch, and setting the irradiation time to be 40 s. After irradiation, the culture dish is placed in a sterile room and kept stand for 1h, then 200 mu L of bacteria liquid is sucked into a bromocresol purple culture medium, the culture dish is inverted and placed in a dark color carton, the culture dish is placed in an incubator at 39 ℃ for culture, the culture dish is taken out and observed after 4-6 days, bacterial colonies growing on a flat plate become orange red, lemon yellow color-changing rings with different sizes are arranged around the bacterial colonies, the diameters of the bacterial colonies and the color-changing rings are measured by using a ruler, the ratio of the latter to the former, namely the HC value, the bacterial colonies with large HC values are selected and stored on an inclined plane, and the bacterial colonies are placed in a refrigerator at 4 ℃. Activating the ultraviolet mutagenic plate preserved in the test, primarily screening the strain, fermenting and culturing, and detecting the acid production rate and acid production amount after 72 hours. The acid yield of the mutant strain named as HBU03DV is 68.24g/L, which is 32.12% higher than that of the original strain HBU03 (51.65 g/L), and the time for reaching the highest acid yield is advanced by about 1 h.
3. UV-lithium chloride mutagenesis
Activating mutant strain HBU03DV obtained by ultraviolet mutagenesis, inoculating the mutant strain HBU03DV into a seed culture medium, diluting to obtain bacterial suspension, and treating the bacterial suspension by ultraviolet lamp irradiation for optimal mutagenesis time. And (3) absorbing 200 mu L of bacterial suspension obtained in the ultraviolet irradiation test into a lithium chloride mutagenesis culture medium by using a pipette, inverting the culture dish, placing the culture dish into a dark-colored carton, placing the culture dish into an incubator at 39 ℃ for culture, taking out the culture dish after 4-6 days, measuring the diameters of bacterial colonies and color-changing rings by using a ruler, calculating the ratio of the latter to the former, namely the HC value, selecting the bacterial colonies with large HC value, storing the bacterial colonies on an inclined plane, and placing the bacterial colonies in a refrigerator at 4 ℃. And (4) performing activation test on the preserved plate, primarily screening the strain, performing fermentation culture, and detecting the acid production rate and the acid production amount after 72 hours. One mutant strain is named as HBU03DV15, the acid yield is 67.72g/L, the acid yield is 31.11% higher than that of the original strain HBU03 (51.65 g/L), and the time for reaching the highest acid yield is advanced by 1.67 h.
EXAMPLE 2 preparation of protoplasts
The mutant strain HBU03DV with high acid yield and the mutant strain HBU03DV15 with the shortest time of reaching the highest acid yield are selected as two parent strains to carry out protoplast fusion, so that the characters of large acid yield and short acid yield time are realized in the same mutant strain.
1. Collecting mutant strains HBU03DV and HBU03DV15 cultured for 8h (late log phase), centrifuging (4000 r/min, 5 min), collecting thallus, washing with LPB buffer solution for 2 times, and suspending in LPB to obtain thallus concentration of about 107-108CFU/mL, adding lysozyme with a final concentration of 100mg/mL, and carrying out water bath at 37 ℃ for 30 min.
2. Inactivation of protoplasts
Ultraviolet inactivation: pouring the prepared mutant strain HBU03DV protoplasm body fluid into a glass culture dish with the diameter of 6cm, putting a magnetic needle serving as a rotor into the dish, putting the dish on a magnetic stirrer, putting the magnetic stirrer into a super clean bench, adjusting the irradiation distance to be 19.5cm, starting an ultraviolet lamp with the power of 18W for timing, and irradiating for 35min for inactivation.
Heat inactivation: transferring the prepared mutant strain HBU03DV15 protoplasm liquid into a centrifuge tube, and putting the centrifuge tube in a water bath at 60 ℃ for 40min for inactivation.
Example 3 protoplast fusion and selection
1. Protoplast fusion: respectively taking 500 mu L of the inactivated amphiphilic protoplasm body fluid, mixing, centrifuging at 3000r/min for 20min, and removing the supernatant. Resuspending in 500 μ LLPB, adding 9 times volume of 40% PEG6000, acting at room temperature for 20min, immediately adding 5mL LPB to dilute after the action time is over, removing the action of PEG6000, centrifuging at 3000r/min for 10min twice at low speed, and resuspending in 100 μ L buffer solution. After fusion, the cells were spread on a regeneration plate and cultured in an incubator at 37 ℃ for 48 hours. Protoplast fluid without inactivation was also plated as a control.
2. Screening of fusion strains: after bacterial colonies grow out, a single fusion strain is picked by a sterilized toothpick and put into an MRS liquid culture medium, standing and fermenting are carried out for 42-48h at 37 ℃, mutant strains HBU03DV, HBU03DV15 and an original strain HBU03 are used as controls, the acid production rate and the acid production amount are detected, a more ideal fusion strain is obtained, the acid production amount of the strain reaches 89.63g/L, is 71.38% higher than that of the original strain HBU03, and the time for reaching the highest acid production amount is advanced by 2.5 h. The fusion strain is preserved with the preservation number of CGMCC 20876 and the preservation unit is China general microbiological culture Collection center.
Activating the preserved Lactobacillus plantarum, performing amplification culture, performing liquid fermentation, and spray drying to obtain product of about 1010CFU/g of bacterial powder. The bacterial powder can be applied to silage fermentation.
Example 4 application of Lactobacillus plantarum of the invention in silage fermentation
1. Preparation of ensilaged alfalfa
Cutting the alfalfa dried to the required water content into small sections of about 1cm by a guillotine, putting the small sections into a sealed bag, arranging 3 treatment groups, inoculating lactobacillus plantarum in logarithmic phase, and naturally fermenting the first group without adding microbial inoculum; the second group is added with Lactobacillus plantarum HBU03 with content of 1 × 109CFU/g, the addition amount is1 g/kg; the third group was supplemented with the fusion strain obtained in example 3 at a content of 1X 109CFU/g, the addition amount is1 g/kg. And fully compacting, vacuumizing and sealing the sealing bag, and performing anaerobic fermentation for 30 days. Sensory evaluation analysis of silage was performed after 30 days.
2. Sensory evaluation analysis
And (3) taking out the silage sample after fermentation, and carrying out silage quality evaluation according to smell, structure and color by referring to a grading method of German DLG silage sensory grading standard (the specific value is shown in Table 1). The grades can be divided into four grades of excellence (20-16 grades), goodness (15-10 grades), medium (9-5 grades) and difference (4-0 grades) according to the scores. The quality sensory score and the silage quality evaluation result grade of the silage alfalfa produced by the experiment are shown in the table 2. The silage quality rating of the lactobacillus plantarum fermentation obtained by the experiment is a good rating.
Claims (5)
1. The lactobacillus plantarum with fast acid production and high acid production is characterized by comprising lactobacillus plantarum (A)Lactobacillus plantarum) WG03, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC 20876, and the preservation date is 10 months and 12 days in 2020.
2. A microbial inoculant comprising the lactobacillus plantarum of claim 1.
3. The microbial inoculum according to claim 2, wherein the viable count of lactobacillus plantarum in the microbial inoculum is not less than 109CFU/g。
4. Use of the lactobacillus plantarum of claim 1 in ensiled alfalfa fermentation.
5. Use of the microbial inoculant of claim 2 in ensiled alfalfa fermentation.
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