CN116731939A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
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- CN116731939A CN116731939A CN202311022692.5A CN202311022692A CN116731939A CN 116731939 A CN116731939 A CN 116731939A CN 202311022692 A CN202311022692 A CN 202311022692A CN 116731939 A CN116731939 A CN 116731939A
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- lactobacillus plantarum
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- fermented
- traditional chinese
- lactobacillus
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Abstract
The invention provides lactobacillus plantarum (Lactobacillus plantarum) named LBR-001 with a preservation number of CGMCC No.26774, which has strong inhibition effect on staphylococcus aureus, escherichia coli, salmonella and the like, has strong acid resistance and acid production capacity, and has extremely strong fermentation conversion capacity on Chinese medicines, and the lactobacillus plantarum (Lactobacillus plantarum) belongs to the technical field of functional microbial fermentation and application. Has definite application effect, safety and reliability, and is suitable for traditional Chinese medicine fermentation, feed additive and fermented feed development.
Description
Technical Field
The invention belongs to the technical field of functional microorganism fermentation and application, and relates to lactobacillus plantarum and application thereof.
Background
Along with the continuous expansion of the Chinese poultry cultivation scale and the continuous rise of the poultry cultivation density, the incidence rate of the respiratory diseases of the poultry is also increased, and serious economic loss is brought to the breeding industry. Upper respiratory tract infections are a common acute respiratory tract infectious disease, and if not treated in time, inflammatory reactions spread to other organs and cause corresponding symptoms. For the treatment of upper respiratory tract infection, targeted therapeutic measures such as antipyresis, fluid infusion, phlegm reduction, cough relief, anti-infection and the like are generally adopted clinically, and although clinical symptoms of poultry can be relieved, the treatment period is longer and the recovery of the poultry is slower. Bacteria are the primary causative agent of upper respiratory tract infections, with staphylococcus aureus, escherichia coli, and salmonella being common bacterial types. The winter is a high-rise season, and the prevention and the control are not timely and easily cause large-scale death, so that huge economic loss is caused.
Currently, the primary route for preventing and treating respiratory tract infections in poultry is the use of antibiotics. However, with the long-term use of antibiotics, the problems of quality safety and environmental pollution of livestock and poultry products have become key factors for restricting sustainable development of livestock industry in China. Furthermore, abuse of antibiotics is also a serious threat to food safety and human health. To address these problems, the search for suitable antibiotic replacement products has become an important point of industry research.
Lactobacillus plantarum (L.) KummerLactobacillus plantarum) The fermented feed additive is widely applied to vegetables, various fermented products and intestinal tracts, has the effects of resisting inflammation and oxidization, regulating balance of intestinal flora, improving immunity of organisms, promoting absorption of nutrient substances and the like, has good tolerance to the gastrointestinal tract environment of animals, and is beneficial to absorption and utilization of the animals. Has good inhibiting effect on staphylococcus aureus, escherichia coli and salmonella. However, the lactobacillus preparation in the market has complicated types and sources, the effective components are difficult to confirm, the lactobacillus plantarum strains are more, and the action effect and additive amount of different strains in different animal types are not clear. Therefore, the lactobacillus plantarum strain is researched and applied to livestock and poultry production aiming at different strains, a specific application scheme and a specific application method for being used in specific livestock and poultry are formed, and the application effect of the lactobacillus plantarum can be maximized.
The traditional Chinese medicine used as a medicine or feed additive has the advantages of high safety, no drug resistance, difficult residue and the like, can be used as an antibiotic substitute for preventing and treating the respiratory tract diseases of the poultry, and has positive effects on the healthy growth of the poultry. However, traditional Chinese medicines have slower effects, and the effect of directly utilizing traditional Chinese medicines by poultry is to be enhanced, so that great industry challenges exist for preventing and treating respiratory tract infection of poultry with fast morbidity, fast infection and high mortality rate.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum which has remarkable inhibition effect on staphylococcus aureus, escherichia coli, salmonella and the like, has strong acid resistance and acid production capacity, has extremely strong fermentation and conversion capacity on fermented traditional Chinese medicines, and can be used for preparing fermented traditional Chinese medicines, feed additives and fermented feeds.
In order to achieve the purpose of the invention, the lactobacillus plantarum (Lactobacillus plantarum) LBR-001 is obtained by screening 78 lactobacillus strains separated from Sichuan acid Chinese cabbage layer by layer, and the strain LBR-001 is lactobacillus plantarum (Lactobacillus plantarum). The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3 and 9 days of 2023, and is classified and named as Lactobacillus plantarum, and the preservation number is CGMCC No.26774.
The microbiological characteristics of the lactobacillus plantarum (Lactobacillus plantarum) LBR-001 provided by the invention are as follows: gram positive bacteria, the cell morphology is Brevibacterium, irregular shape. The colony is round, white, occasionally pale yellow, with a diameter of 1-2 μm, smooth and moist surface, slightly convex middle, opaque, orderly and serrated edge, and no halo.
The present invention identified the strain LBR-001 as Lactobacillus plantarum by 16S rDNA sequence analysis (Lactobacillus plantarum).
The invention provides lactobacillus plantarum (Lactobacillus plantarum) LBR-001, which is characterized in that the proper growth temperature is 28-40 ℃; the pH value is 5.5-7.5; the fermentation time is 24-72 hours.
Further, the invention provides lactobacillus plantarum (Lactobacillus plantarum) LBR-001, which is characterized in that the proper growth temperature is 37 ℃; the pH value is 6.5; fermentation time was 60h.
The invention provides a composite microbial inoculum containing the lactobacillus plantarum (Lactobacillus plantarum) LBR-001. The composite microbial inoculum is prepared from lactobacillus plantarum (Lactobacillus plantarum) LBR-001, clostridium butyricum, enterococcus faecium, enterococcus faecalis, lactobacillus thermophilus, lactobacillus acidophilus and saccharomyces cerevisiae in a mass ratio of 3:2:1:1:1:1:1 are compounded.
According to in vitro simulation identification, the lactobacillus plantarum (Lactobacillus plantarum) LBR-001 has the characteristics of acid resistance and resistance to the environment in the gastrointestinal tract, and has the potential of being developed into probiotics.
The invention also discovers that the survival rate of the lactobacillus plantarum (Lactobacillus plantarum) LBR-001 is still up to 80 percent when the pH value is 3.0, and has extremely strong fermentation and conversion capability on fermented traditional Chinese medicines; the invention also has strong inhibition effect on staphylococcus aureus, escherichia coli and salmonella, so the invention provides a fermented traditional Chinese medicine preparation containing lactobacillus plantarum LBR-001 for inhibiting staphylococcus aureus, escherichia coli and salmonella, and provides application of lactobacillus plantarum LBR-001 in reducing pollution of staphylococcus aureus, escherichia coli and salmonella in feed.
The invention provides a fermented traditional Chinese medicine preparation containing lactobacillus plantarum (Lactobacillus plantarum) LBR-001 or a fermentation product thereof.
The invention provides a fermented traditional Chinese medicine preparation, which is prepared from liquorice, dandelion, cordate houttuynia, common cold toad herb, radix isatidis, baical skullcap root, epimedium herb, subprostrate sophora and honeysuckle in a mass ratio of 2:2:2:1:1:1:1:1:1 is prepared by fermenting lactobacillus plantarum (Lactobacillus plantarum) LBR-001.
The invention provides a feed additive containing the lactobacillus plantarum (Lactobacillus plantarum) LBR-001.
The invention provides a fermented feed containing the lactobacillus plantarum (Lactobacillus plantarum) LBR-001.
The beneficial effects of the invention are mainly as follows:
1. lactobacillus plantarum LBR-001 has excellent acid resistance, shows good tolerance in the gastrointestinal tract, can resist the low-acid environment in the gastrointestinal tract, and can be rapidly planted in the intestinal tract, thereby being beneficial to regulating intestinal flora. In addition, the composition can play a remarkable antibacterial role in the gastrointestinal tract, and has the potential of developing a probiotic preparation for preventing and treating diarrhea and enteritis.
2. Lactobacillus plantarum LBR-001 shows remarkable acid production capability, produces a large amount of organic acid, and has excellent potential for conversion of fermented traditional Chinese medicines. The strain can enhance the content of active ingredients in fermented traditional Chinese medicine, and convert macromolecular substances into small molecules. This ability also improves the quality of the feed and improves the nutritional value of the feed. In addition, the lactobacillus plantarum LBR-001 also has a natural antiseptic effect, and is helpful for preventing the spoilage of fermented traditional Chinese medicines and feeds. Therefore, the strain has wide application potential in the field of traditional Chinese medicine fermentation and feed fermentation.
3. The lactobacillus plantarum LBR-001 has remarkable inhibition effect on staphylococcus aureus, escherichia coli, salmonella and the like, has the potential of being developed into a microecological traditional Chinese medicine preparation, and can be used for inhibiting pathogenic bacteria and reducing the pollution of the bacteria in feed.
4. The fermentation product of the lactobacillus plantarum LBR-001 is used as probiotics and feed additives, the problems of drug resistance and residue of antibiotic feed additives are avoided, and the application is safe.
Drawings
FIG. 1 shows the effect of different initial pH values on the growth of Lactobacillus plantarum LBR-001.
FIG. 2 shows the effect of different fermentation temperatures on the growth of Lactobacillus plantarum LBR-001.
FIG. 3 shows the effect of different fermentation times on the growth of Lactobacillus plantarum LBR-001.
FIG. 4 is a gram-stained microscopic morphology of Lactobacillus plantarum LBR-001.
FIG. 5 shows the growth of strains in different pH media and the pH change of the media after cultivation.
FIG. 6 shows the change in pH of the culture medium of LBP-012 strain at different culture times.
FIG. 7 shows the effect of LBR-001 of Lactobacillus plantarum on the effective components of the fermented traditional Chinese medicine part.
FIG. 8 shows the effect of Lactobacillus plantarum LBR-001 on the quality of fermented feed.
FIG. 9 shows the antibacterial effect of Lactobacillus plantarum LBR-001 and its fermented traditional Chinese medicine.
FIG. 10 shows feedback of the effect of lactobacillus plantarum LBR-001 fermented traditional Chinese medicine on a farm in the family of five figures.
FIG. 11 is a feedback chart showing the effect of the lactobacillus plantarum LBR-001 fermented traditional Chinese medicine on a certain farm in Xingan street in Anchu city of Weifang.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The embodiment of the invention adopts an oxford cup method to detect the antibacterial activity of the lactobacillus plantarum LBR-001 and the fermented traditional Chinese medicine thereof.
Fermentation traditional Chinese medicine culture medium: the traditional Chinese medicine composition comprises liquorice, dandelion, cordate houttuynia, bufo siccus, radix isatidis, radix scutellariae, epimedium, subprostrate sophora and honeysuckle in mass ratio: 2:2:2:1:1:1:1:1:1, crushing and sieving with a 40-mesh sieve, adding water to submerge the Chinese medicine, soaking for 1 hour, decocting for two times, concentrating, adding supplementary culture medium components according to the volume proportion, and sterilizing with high-pressure steam at 121 ℃ for 20min for later use, wherein the final concentration of the Chinese medicine extract is 0.5 g/ml.
Supplemented medium composition (g/L):
10.0 parts of peptone, 5.0 parts of beef extract powder, 4.0 parts of yeast extract powder, 20.0 parts of glucose, 2.0 parts of dipotassium hydrogen phosphate, 2.0 parts of tri-ammonium citrate, 5.0 parts of sodium acetate, 0.2 parts of magnesium sulfate and 0.05 parts of manganese sulfate.
Example 1 screening, isolation and identification of lactobacillus plantarum LBR-001:
1. screening, separation and purification of lactobacillus plantarum LBR-001
Scraping 1g of sample from Sichuan acid cabbage, adding into 9ml of sterile distilled water, shaking for 2min by vortex to prepare suspension, diluting the suspension, coating the suspension on a lactobacillus selective culture medium MRS (MRS) culture medium, performing anaerobic culture at 37 ℃ for 36h, picking single colony on a plate, scribing again on the MRS culture medium plate, performing anaerobic culture at 37 ℃, and carrying out repeated scribing purification culture on the obtained colony to obtain a pure colony. The single colonies were designated LBP-001, LBT-002, LBT-003, … …, LBT-078 in this order. The single colony obtained by separation and purification is activated and cultured for 24 hours, 1 percent of the volume is taken as inoculum size to be respectively inoculated into MRS liquid culture mediums with pH values of 6.5, 5.0, 3.5 and 2.0, anaerobic culture is carried out for 24 hours at 37 ℃, meanwhile, sterile culture mediums with corresponding pH values are taken as blank control groups, and the absorbance value at 600nm and the pH value of each group of culture mediums are respectively measured.
Experimental results show that only three strains LBP-012, LBP-054 and LBP-068 selected by the applicant can normally grow in MRS liquid culture medium with pH value of 3.5-4.0, and the strain LBP-012 has stronger acid resistance, wherein the acid resistance of the strain LBP-012 is strongest, and the survival rate of viable bacteria is still up to 80% at pH value of 3.0, and the acid production capacity is strongest. The specific results are shown in FIG. 5.
2. Identification of lactobacillus plantarum LBP-012:
(1) Morphological characteristics and gram staining
LBP-012 strain is streaked and cultured for many times, and the colony is observed to be round, white, occasionally pale yellow, with the diameter of 1-2 mu m, smooth and moist surface, slightly convex in the middle, opaque, orderly and saw-toothed at the edge and no halo. Gram staining was performed and microscopic examination showed a straight bar shape at the round end. The strain LBP-012 was visualized in FIG. 4.
(2) And (3) physiological and biochemical characteristic identification:
the strain LBP-012 was subjected to physiological and biochemical characterization by glucose gas production test, catalase activity measurement, nitrate reduction test, hydrogen sulfide production test, gelatin liquefaction test, indole test and lactobacillus biochemical characterization strip (HBIG 11).
The identification result shows that: the strain LBP-012 is fermented to produce glucose and acid without producing gas, the catalase is negative, nitrate, hydrogen sulfide, gelatin and indole are negative, esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose are positive, and the strain LBP-012 is primarily determined to be lactobacillus plantarum (Lactobacillus plantarum).
(3) Molecular biology identification:
the DNA of the strain LBP-012 is used as a template, a 16S rRNA universal primer is used for amplification, the amplified fragment is subjected to sequence determination, the obtained 16S rDNA sequencing result of the strain LBR-001 is compared with the sequence in GenBank, and the result shows that the strain LBP-012 is compared with the sequence in GenBankLactobacillus plantarumThe homology was highest, and therefore, the strain LBR-001 was determined to be Lactobacillus plantarum.
The applicant has selected and selected strain LBP-012 designated as Lactobacillus plantarum (Lactobacillus plantarum) LBR-001 for strain preservation, preservation unit: china general microbiological culture Collection center (CGMCC); address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; preservation date: 2023, 03, 09; lactobacillus plantarum LBR-001 accession number: CGMCC No.26774.
Example 2 measurement of growth Properties of Lactobacillus plantarum LBR-001:
lactobacillus plantarum LBR-001 was inoculated at 1% inoculum size in MRS liquid medium at different initial pH values at OD 600 Drawing a growth curve for the measurement index, wherein the growth curve is shown in figure 1; inoculating Lactobacillus plantarum LBR-001 in 3% inoculum size into MRS liquid culture medium, fermenting at different temperatures for different times, and OD 600 The growth curves are respectively drawn for the measurement indexes and are shown in fig. 2 and 3. As can be seen from FIG. 1, the lactobacillus plantarum LBR-001 can grow at a pH value of 3.5-7.5, and the optimal initial pH value for growth is 6.5; as can be seen from FIG. 2, the lactobacillus plantarum LBR-001 can grow at 28-40 ℃, and the optimal growth temperature is 37 ℃; as is clear from FIG. 3, the growth lag phase of Lactobacillus plantarum LBR-001 is about the initial period of cultivation to about 6 hours, and then enters the logarithmic phase of cultivation, during which the bacterial cells increase exponentially and OD is obtained when cultivation is performed for 60 hours 600 Reaching the highest value, 24-60 h is the stable period of the growth of the lactobacillus plantarum LBR-001, and the bacterial growth is slow, and gradually enters into the decay period after 60h.
Example 3 evaluation of the acid generating ability of Lactobacillus plantarum LBR-001:
to further verify the acid-producing ability of lactobacillus plantarum LBR-001, the applicant inoculated lactobacillus plantarum LBR-001 and lactobacillus plantarum ATCC 14917 strain (control strain) respectively in equal amounts in MRS liquid medium at pH6.5, cultured at 37 ℃ and the pH of the culture was measured every 2 hours, and the specific results are shown in fig. 6.
As can be seen from the data of FIG. 6, after 12 hours of cultivation, the pH of the medium inoculated with Lactobacillus plantarum ATCC 14917, the control strain, was reduced from 6.50 to 4.67, while the pH of the medium inoculated with Lactobacillus plantarum LBR-001 was reduced from 6.50 to 3.68; after 24h of incubation, the pH of the medium inoculated with the control strain Lactobacillus plantarum ATCC 14917 was reduced to 4.28, while the pH of the medium inoculated with Lactobacillus plantarum LBR-001 was reduced to 3.16; therefore, compared with a control strain, the lactobacillus plantarum LBR-001 provided by the invention has stronger acid production capacity and faster acid production speed.
Example 4 evaluation of the fermentation conversion ability of Lactobacillus plantarum LBR-001 to a drug:
(1) Preparing a fermentation traditional Chinese medicine culture medium:
the traditional Chinese medicine is prepared by the following steps: glycyrrhrizae radix, herba Taraxaci, herba Houttuyniae, herba Momordicae Charantiae, herba Potentillae chinensis, radix Isatidis, scutellariae radix, herba Epimedii, radix Sophorae Tonkinensis, and flos Lonicerae by the following proportion: 2:2:2:1:1:1:1:1:1, crushing and sieving with a 40-mesh sieve, adding water to submerge the Chinese medicine, soaking for 1 hour, decocting for two times, concentrating, adding supplementary culture medium components according to the volume proportion, and sterilizing with high-pressure steam at 121 ℃ for 20min for later use, wherein the final concentration of the Chinese medicine extract is 0.5 g/ml.
Supplemented medium composition (g/L):
10.0 parts of peptone, 5.0 parts of beef extract powder, 4.0 parts of yeast extract powder, 20.0 parts of glucose, 2.0 parts of dipotassium hydrogen phosphate, 2.0 parts of tri-ammonium citrate, 5.0 parts of sodium acetate, 0.2 parts of magnesium sulfate and 0.05 parts of manganese sulfate.
(2) Lactobacillus plantarum LBR-001 is inoculated into a fermentation traditional Chinese medicine culture medium according to 5% of the volume, the pH value is regulated to 6.5 by 30% sodium hydroxide solution, the fermentation temperature is 37 ℃, and the shaking fermentation is carried out for 60 hours at 100 r/min.
The fermented Chinese medicinal culture medium contains flavonoid active ingredients, herba Epimedii and Scutellariae radix contains glycoside active ingredients, the fermented Chinese medicinal culture medium without inoculating any strain is used as control group, the fermented Chinese medicinal culture medium with lactobacillus plantarum LBR-001 is added as treatment group, pH value of control group is 5.19, pH value of treatment group is 3.76, and content determination of effective ingredients in the fermented Chinese medicinal culture medium is shown in figure 7.
As can be seen from FIG. 7, compared with the control group, the effective components of the traditional Chinese medicine fermented by the lactobacillus plantarum LBR-001 are improved by 1-2 times compared with the unfermented traditional Chinese medicine, and the improvement range of the glycoside active components is the largest, so that the lactobacillus plantarum LBR-001 provided by the invention has extremely strong fermentation conversion capability on the traditional Chinese medicine, and releases the effective components which are difficult to dissolve in the traditional Chinese medicine.
Two groups were simultaneously subjected to an acceleration test: the temperature is 40 ℃ +/-2 ℃ and the relative humidity is 75% +/-5% under the same condition for 6 months. The control group is a dark brown uniform liquid initially, has astringency of traditional Chinese medicine cooking, has floccules in 1 st month, has a large number of sediments, has obvious change of color in 2 nd month, has layering phenomenon, has putrefactive smell, has cyan mould-like substances slightly and completely putrefactive in 3 rd month and has a large number of cyan bacterial spots in 4 th month. The treatment group is light brown uniform liquid initially, has lactic acid taste and flavor of traditional Chinese medicines, and has no change in color and smell, no obvious change in sediment, color and smell in the 1 st month, and slight shaking and scattering of sediment in the 2 nd month. No significant change was observed in month 3 and month 6. From the accelerated test results, the lactobacillus plantarum LBR-001 has a preservative and quality guaranteeing effect on fermented traditional Chinese medicines, and can be used as a feed additive.
According to the results of a control test and an acceleration test, the lactobacillus plantarum (Lactobacillus plantarum) LBR-001 has strong acid production capacity, generates a large amount of organic acid, has extremely strong fermentation conversion capacity on fermented traditional Chinese medicines, can effectively improve the content of active ingredients of the fermented traditional Chinese medicines, and converts macromolecules into small molecules. Meanwhile, the natural preservative has the function of effectively preventing the fermented traditional Chinese medicine from spoilage, and can be widely applied to the field of traditional Chinese medicine fermentation.
Example 5 effect of lactobacillus plantarum LBR-001 on fermented feed quality:
the lactobacillus plantarum LBR-001 suspension is streaked in MRS solid culture medium, and is respectively subjected to static culture at 37 ℃ for 36 hours, and single colony is obtained through separation. Selecting single bacterial colony to inoculate in MRS liquid culture medium, culturing at 37deg.C for 2d at 100deg.C for 100 r.min < -1 >, to obtain seed liquid for fermentation; inoculating lactobacillus plantarum LBR-001 into MRS liquid culture medium with 1% of inoculum size, and standing at 37 ℃ for 24 hours; according to mass fraction, the bacterial liquid is inoculated into coarse fodder composed of wheat bran 30%, rice bran 15% and soybean meal 10% according to 5% inoculation amount, and sterile water is added to adjust the final water content to 40% -45%. The mixture was placed in a closed fermentation bag and incubated at 37 ℃. Taking the above coarse fodder without inoculating any strain as control group, fermenting for 60 hr, sampling, and respectively measuring pH value, viable count, total acid, crude protein and acid soluble protein content of fermented fodder, and specifically, the specific results are shown in figure 8.
As can be seen from the data in fig. 8, compared with the control group, the content of crude protein and acid soluble protein in the fermented group feed fermented by lactobacillus plantarum LBR-001 is respectively increased by 76.77% and 64.44%, the content of organic acid is obviously increased, and the pH is obviously reduced. Therefore, the lactobacillus plantarum LBR-001 provided by the invention can degrade part of macromolecular substances such as polyprotein, fat and the like to generate micromolecular substances such as organic acid, soluble polypeptide and the like, so that the biological feed with rich nutrition, good palatability and high viable bacteria content is formed, and the palatability and the nutritional value of the fermented feed are obviously improved.
Example 6 Lactobacillus plantarum LBR-001 and evaluation of antibacterial properties of fermented traditional Chinese medicine:
1. materials and test instruments:
(1) Materials:
LB liquid medium, agar powder, purchased from Qingdao sea Bo Biotechnology Co.
LB solid medium: adding 15.0g of agar powder into LB liquid medium, and sterilizing at 121deg.C for 20 min.
MRS liquid medium: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 5.0g of glucose, 5.0g of sodium acetate, 2.0g of citric acid diamine, 1.0g of tween-80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate heptahydrate, 20.0g of calcium carbonate, 20.0g of agar and distilled water with constant volume of 1000ml, and performing high-pressure sterilization at the temperature of 121 ℃ for 20 min.
Sterile straw: 1ml (with 0.01ml scale), 10ml (with 0.1ml scale), micropipettes and tips.
Sterile conical flask: capacity 250ml, 500ml.
Sterile culture dish: the diameter is 90mm.
Sterile test tube: 18cm.
Reagent: sterile physiological saline.
Instrument: an autoclave, a centrifuge, a mixer, a constant temperature incubator, a water bath, an ultra-clean operation table, an acidometer and the like.
2. The test method comprises the following steps:
(1) Preparation of pathogenic bacteria suspension:
the 3 pathogenic bacteria of Staphylococcus aureus ATCC 29213, escherichia coli ATCC 259922 and Salmonella ATCC 12002 were used as test strains, and cultured in LB medium at 37 ℃.
(2) Preparation of lactobacillus plantarum LBR-001 bacterial suspension:
inoculating pure lactobacillus plantarum LBR-001 into 100ml MRS liquid culture medium under aseptic condition, standing at 37deg.C for 24 hr, and culturing to obtain bacterial solution with concentration of 10 8 CFU/ml。
(3) Preparation of lactobacillus plantarum LBR-001 fermentation traditional Chinese medicine culture medium:
the fermentation temperature is 37 ℃, the inoculation amount is 5%, the initial pH is 6.5, the fermentation time is 60 hours, and the final lactobacillus plantarum LBR-001-containing viable bacteria is 5.32X10 9 CFU/ml. Diluting lactobacillus plantarum LBR-001 fermentation Chinese medicinal culture medium for 50 times under aseptic operation, and diluting unfermented Chinese medicinal culture medium for 50 times after sterilization.
(4) Lactobacillus plantarum LBR-001 and antibacterial property measurement of fermented traditional Chinese medicine:
taking cultured pathogenic bacteria by oxford cup method, and diluting the bacteria solution with a concentration of about 10 5 CFU/ml, coating on solid LB culture medium and placing oxford cup in turn; the activated lactobacillus plantarum LBR-001 and a fermentation traditional Chinese medicine culture medium thereof are subjected to centrifugation for 10min at 4 ℃ and 10000 Xg, supernatant is filtered and sterilized by a microporous filter membrane of 0.22 mu m, 100 mu L of supernatant is added into an oxford cup, the mixture is placed at 4 ℃ for 3h, and then the mixture is transferred into a constant temperature incubator for culture at 37 ℃ for 24h, and the result is observed, and the diameter of a bacteriostasis ring is measured. The control group was added with 100. Mu.L of sterile LB liquid medium, 3 replicates per group, and the test was repeated 3 times, and the results are shown in FIG. 9.
As shown in FIG. 9, the lactobacillus plantarum LBR-001 has obvious inhibition effect on staphylococcus aureus ATCC 29213, escherichia coli ATCC 259922 and salmonella ATCC 12002, the lactobacillus plantarum LBR-001 fermentation traditional Chinese medicine culture medium is obviously higher than the unfermented traditional Chinese medicine culture medium, the lactobacillus plantarum LBR-001 and the fermented traditional Chinese medicine thereof have synergistic effect, and compared with antibiotics, the lactobacillus plantarum LBR-001 can replace partial antibiotics in terms of the diameter interpretation standard of the common drug-sensitive bacteriostasis circle for clinical microbiological examination, and can be applied to developing a microecological traditional Chinese medicine preparation for preventing diseases caused by bacterial pathogenic bacteria.
Example 7 application of Lactobacillus plantarum LBR-001 fermented traditional Chinese medicine in livestock and poultry cultivation.
1. The method comprises the steps of (1) diagnosing acute respiratory tract air sac inflammation caused by escherichia coli infection by veterinarian, dying 50 animals in the same day, fermenting traditional Chinese medicine with lactobacillus plantarum LBR-001 for 5 days, adding 1000L of water every L, concentrating for 3-4 hours in the whole day, and dying 48 animals in the 1 st day, wherein the farm is a high-rise farm of five pictures in Changle county, the age of 20 is 600, and the respiratory tract symptoms such as depression, draughts, dyspnea, cough and the like appear in 2021, and the respiratory tract symptoms are proved by 2021; 35 deaths occurred on day 2, and respiratory symptoms were significantly reduced; 20 deaths on day 3; 5 deaths on day 4; on day 5, 0 deaths occurred, respiratory symptoms were essentially absent, and only sporadic dyspnea and cough sounds. As is clear from the above and FIG. 10, the use of Lactobacillus plantarum LBR-001 for fermentation of traditional Chinese medicine can rapidly and effectively relieve respiratory symptoms of poultry.
2. In a certain farm on the Xingan street in Anchu city in Fang, 9000 ducks are put on, 10 months and 3 days in 2021, respiratory symptoms such as cold and cough appear in 33 days, appetite is reduced, 45 bags are taken a day, the lactobacillus plantarum LBR-001 is used for fermenting the traditional Chinese medicine, 1000L of water is added every L, the traditional Chinese medicine is concentrated for 3 to 4 hours in the whole day, the traditional Chinese medicine is shown in fig. 11, the respiratory symptoms are relieved after two days, the materials are obviously added, 52 bags are taken a day, and the cough symptoms are obviously relieved after three days are used, and the traditional Chinese medicine has no secondary infection and no injury. As can be seen from the above and FIG. 11, the use of Lactobacillus plantarum LBR-001 for fermenting the traditional Chinese medicine can not only rapidly and effectively relieve the respiratory tract infection of poultry, but also improve the feed intake of meat ducks and promote the growth of the meat ducks.
3. 3 ten thousand ducks are planted in a certain farm in Linghe in Anchu city, 3 sheds, 10 months and 3 days in 2021 and 23 days old, and about 18000 of the two sheds have symptoms of mental depression, cold, cough, anorexia and white thin feces. The two sheds are divided into a western medicine group and a lactobacillus plantarum LBR-001 fermentation traditional Chinese medicine experimental group, the two groups of feeds and the management conditions are the same, the western medicine group uses antibiotic treatment medicines, the experimental group uses 1000L of water added every L of the invention, and the two groups of feeds and the management conditions are concentrated for 3-4 hours in the whole day to drink. On the 2 nd day, the experimental group is recovered to be normal compared with the western medicine group, the mental state is good, the excrement is obviously improved, the cough symptoms of the western medicine group and the experimental group are not developed, and the casualties of the sick ducks are reduced. On the 3 rd day, the number of cough symptoms of two large groups of western medicine groups and experimental groups is reduced, the western medicine groups have the condition of material reduction, and the experimental groups increase material. On day 4, the two groups of large groups of cough symptoms are few, no casualties exist, the western medicine group is subjected to material reduction, and the experimental group still continues to increase materials. Therefore, lactobacillus plantarum LBR-001 is used for fermenting traditional Chinese medicine to replace antibiotics for preventing and treating poultry respiratory tract infection, and meanwhile, the traditional Chinese medicine is refreshing and material increasing, and has no side effect.
The invention discloses lactobacillus plantarum and application thereof, and the above embodiments are only used for illustrating the technical scheme of the invention and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. The lactobacillus plantarum (Lactobacillus plantarum) LBR-001 is characterized in that the lactobacillus plantarum is separated from Sichuan acid Chinese cabbage, the preservation unit is China general microbiological culture collection center, and the preservation number is CGMCC No.26774.
2. Lactobacillus plantarum (Lactobacillus plantarum) LBR-001 according to claim 1, characterised in that the suitable growth temperature is 28-40 ℃; the pH value is 5.5-7.5; the fermentation time is 24-72 hours.
3. Lactobacillus plantarum (Lactobacillus plantarum) LBR-001 according to claim 2, characterised in that the suitable growth temperature is 37 ℃; the pH value is 6.5; fermentation time was 60h.
4. A composite microbial inoculum is characterized by being prepared by compounding lactobacillus plantarum (Lactobacillus plantarum) LBR-001 according to claim 1 with clostridium butyricum, enterococcus faecium, enterococcus faecalis, lactobacillus thermophilus, lactobacillus acidophilus and saccharomyces cerevisiae.
5. The use of the composite microbial agent of claim 4 in the preparation of a feed additive.
6. A fermented Chinese medicinal preparation comprising the Lactobacillus plantarum (Lactobacillus plantarum) LBR-001 or a fermented product thereof according to claim 1.
7. The fermented Chinese medicinal preparation according to claim 6, wherein the fermented Chinese medicinal preparation is a fermented Chinese medicinal preparation for inhibiting staphylococcus aureus, escherichia coli and salmonella.
8. The fermented traditional Chinese medicine preparation according to claim 6, wherein the mass ratio of liquorice, dandelion, houttuynia cordata, common cold herb, radix isatidis, baikal skullcap root, epimedium herb, subprostrate sophora and honeysuckle is 2:2:2:1:1:1:1:1:1 is prepared by fermenting lactobacillus plantarum (Lactobacillus plantarum) LBR-001.
9. Use of lactobacillus plantarum (Lactobacillus plantarum) LBR-001 according to claim 1 for the preparation of a feed additive.
10. Use of lactobacillus plantarum (Lactobacillus plantarum) LBR-001 and its fermentation products according to claim 1 for the preparation of fermented feed for livestock and poultry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311022692.5A CN116731939B (en) | 2023-08-15 | 2023-08-15 | Lactobacillus plantarum and application thereof |
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