CN112877238A - Low-temperature-resistant pediococcus pentosaceus OL77 and application thereof - Google Patents
Low-temperature-resistant pediococcus pentosaceus OL77 and application thereof Download PDFInfo
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- CN112877238A CN112877238A CN202110128172.7A CN202110128172A CN112877238A CN 112877238 A CN112877238 A CN 112877238A CN 202110128172 A CN202110128172 A CN 202110128172A CN 112877238 A CN112877238 A CN 112877238A
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- silage
- pediococcus pentosaceus
- low
- low temperature
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
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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
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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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
- A23V2400/427—Pentosaceus
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Abstract
The invention discloses a low temperature resistant pediococcus pentosaceus OL77 and application thereof, wherein the preservation number of the low temperature resistant pediococcus pentosaceus OL77 in China center for type culture Collection is CCTCCNO: m2020290; the pediococcus pentosaceus OL77 has the characteristics of acid resistance, low temperature resistance and salt resistance, has high growth speed and high acid production rate at low temperature, can be rapidly propagated in the low-temperature ensiling process of the oat, rapidly produces acid and reduces the pH value, effectively inhibits the growth and propagation of harmful mixed bacteria, accelerates the ensiling fermentation process, improves the ensiling feed fermentation quality and retains more forage grass nutrient substances; the silage additive is prepared by mixing pediococcus pentosaceus OL77 with trehalose, so that the low-temperature adaptability of pediococcus pentosaceus OL77 can be further enhanced, the acid production rate is accelerated, the pH value is rapidly reduced, and the fermentation quality of oat silage is improved in the low-temperature silage process of oat.
Description
Technical Field
The invention relates to the technical field of microbial application and silage modulation and processing, in particular to low temperature resistant pediococcus pentosaceus OL77 and application thereof.
Background
The Qinghai-Tibet plateau is one of the main pasturing areas in China, the climate is severe, the withering period is as long as 7 months, and the insufficient forage grass supply and the unbalanced season caused by the climate are key problems influencing the long-term development of the animal husbandry of the Qinghai-Tibet plateau; one of the main ways to solve this problem is to increase the size of artificial grass planting and forage grass storage. However, under the influence of the overlapping of autumn forage grass harvesting season and rainy season, the hay is difficult to prepare, and the hay is easy to mildew and rot in the storage process. Ensiling is a simple, convenient and easy processing mode of pasture with low cost and little influence by climate environment, and is very suitable for popularization and application in Qinghai-Tibet plateau and areas with higher altitude and rainy harvest season around the Qinghai-Tibet plateau.
The oat is the forage grass with the largest planting area in the Qinghai-Tibet plateau area, taking Qinghai province as an example, the planting area is 1.63 multiplied by 105hm2Accounting for 83.3 percent of the planting area of the forage grass in the whole province. New silage pool of 1.42 multiplied by 10 in 20185m3Annual silage of forage up to 1X 106t, greatly improving the forage grass reserving capacity. Under the current situation of rapid development of grass husbandry, the planting area and the processing amount of oat forage grass still have a tendency of increasing continuously. However, the temperature in autumn and winter in the Qinghai-Tibet region is very low, the time required for completing ensiling fermentation is obviously increased, and the fermentation process can not be completed within 40-45 days generally considered. Oat is wrapped up in and ensiled in height above sea level 2517m, Gansu province, summer river county, king green pond town of Wang Ge can only finish fermentation for about 80 days, lactobacillus can not become dominant fermentation flora in a short time, pH value is maintained at a higher level all the time, harmful microbial activity can not be inhibited rapidly and effectively, and dry matter loss is large.
In order to solve the problems encountered in the actual production, it is necessary to develop a technology for promoting fermentation of silage in a severe cold area or under a low temperature condition. The use of the commercial lactic acid bacteria additive can obviously improve the quality of ensiling fermentation, but the improvement effect is not obvious under the low-temperature condition.
Disclosure of Invention
Aiming at the problems, the low-temperature resistant pediococcus pentosaceus OL77 and the application thereof have strong adaptability, can survive and propagate in an extreme environment, and can rapidly propagate and produce acid in the low-temperature ensiling process after being compounded and added with compatible solute trehalose, thereby effectively inhibiting the growth and propagation of harmful mixed bacteria and retaining more forage grass nutrient substances. The method can effectively solve the problems of slow fermentation, high pH value, large dry matter loss and the like caused by low temperature in the grass ensiling process of the current Qinghai-Tibet plateau area, and has the characteristics of rapid fermentation at low temperature, good storage effect, low cost, safety, reliability, simple operation and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a low temperature resistant Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 is Pediococcus pentosaceus OL77 which is Pediococcus pentosaceus OL77 and is separated from oat of autonomous state of Jade Tree Tibetan of China, Qinghai province, and is preserved in China center for type culture Collection CCTCC (CCTCC) in Wuhan university, Wuhan city, Hubei province, with the preservation number of CCTCC NO: m2020290.
Preferably, the strain of Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 is a gram-positive, catalase-negative, homofermentative coccus; the growth is good in the environment with pH of 3.0, 3.5, 4.0, 4.5 and 5.0, which shows that the acid-resistant performance is strong; the growth is good in the environment with the temperature of 20, 15, 10 and 5 ℃, which shows that the growth has better low temperature resistance; the growth is good under the NaCl concentration of 3.0 percent and 6.0 percent, which shows that the salt tolerance is stronger; the strain has strong growth adaptability and can survive and propagate in extreme environment, and the 16S rDNA sequence of the strain is shown as seq ID No. 1.
A microbial inoculum (lactic acid bacteria) contains low temperature resistant Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 strain as active ingredient, and optionally adjuvants such as MRS liquid culture medium (composed of peptone 10.0g, beef powder 5.0g, glucose 20.0g, yeast powder 4.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, ammonium citrate tribasic 2.0g, manganese sulfate 0.05g, Tween 801mL, distilled water 1L, pH 6.2 + -0.2).
A silage additive, the active ingredient of which is the low temperature resistant Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 strain of claim 1.
A compound additive for silage is prepared by adding 0.01mol L of the above bacterial agent (lactobacillus)-1Compatible solute trehalose.
A method for preparing a silage compound additive specifically comprises the following steps: fully mixing the Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 with compatible solute trehalose to obtain the composite silage additive, wherein the addition amount of the compatible solute trehalose is 0.01mol L-1And (5) bacterial liquid.
A silage, which contains the silage additive or the silage compound additive and silage raw materials;
preferably, the silage contains Pediococcus pentosaceus OL77 and trehalose at a ratio of 1013～1014CFU:0.01mol。
A method for preparing silage comprises mixing silage raw materials with silage additive or silage composite additive, performing solid anaerobic fermentation, and collecting all fermentation products to obtain silage;
wherein: the ensiling raw material is oat whole plant after mechanical kneading, and the water content is 668.3g kg-1FM；
In the method, the ratio of oat raw material to Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 is 1kg:108CFU；
The fermentation is low-temperature fermentation; under the condition of adding silage additives, the fermentation temperature is 10 ℃; under the condition of adding the silage compound additive, the fermentation temperature is 5 ℃;
the fermentation time was 45 days.
The invention has the beneficial effects that: the invention discloses a low temperature resistant pediococcus pentosaceus and application thereof, compared with the prior art, the improvement of the invention is as follows:
(1) the invention provides a low temperature resistant Pediococcus pentosaceus, which has stronger growth adaptability of OL77, can survive and reproduce in extreme environment, and can rapidly reproduce and produce acid after being compounded and added with compatible solute trehalose in the process of low temperature ensiling (5 ℃), effectively inhibits the growth and reproduction of harmful mixed bacteria, and reserves more forage grass nutrient substances;
(2) the method utilizes the microorganisms and the compatible solute trehalose to accelerate the silage fermentation process and improve the silage fermentation quality, and has the advantages of low cost, safety, reliability and simple operation;
(3) the Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 used in the invention has better effect than the existing commercial lactobacillus additive in a low-temperature environment, and has the advantages of low cost and good oat ensiling effect.
Drawings
FIG. 1 is a colony morphology of the pediococcus pentosaceus OL77 strain of the present invention.
FIG. 2 is a phylogenetic tree of Pediococcus pentosaceus OL77 according to the present invention.
FIG. 3 is an electrophoretogram of total DNA of pediococcus pentosaceus OL77 of the present invention.
FIG. 4 shows the rDNA electrophoretogram of pediococcus pentosaceus OL7716S of the present invention.
Fig. 5 is a graph of oat silage grass after treatment with the lactic acid bacteria additive of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.
Referring to the accompanying drawings 1-5, the low temperature resistant Pediococcus pentosaceus OL77 and the application thereof utilize that Pediococcus pentosaceus OL77 has strong growth adaptability, can survive and reproduce in extreme environments, and quickly reproduce and produce acid after being compounded and added with compatible solute trehalose in the low temperature ensiling (5 ℃) process, thereby effectively inhibiting the growth and reproduction of harmful mixed bacteria, retaining the characteristics of more forage grass nutrient substances and the like, and having better effect than the existing commercial lactobacillus additive in low temperature environments.
Example 1: isolation and characterization of Pediococcus pentosaceus (Pediococcus pentosaceus) OL77, comprising the steps of:
s1: the method comprises the following steps of selecting and extracting Pediococcus pentosaceus (Pediococcus pentosaceus) OL 77:
collecting fresh samples of No. 5 Gansui oat varieties in 8-10 months at S101.2017 in Xining (2295m), Tianzhu (2797m), Shandan (2860m), Heiguan (2957m), Haiyan (3052m) Maqu (3474m), Gualoo (3765m) and Duo (4217m) at 8 different altitude test areas, packaging in a sample bag sterilized in advance, storing at low temperature by using an ice bag and a heat preservation box, sealing in a 1L sterile polyethylene bottle in a sterile environment after being taken back to a laboratory, ensiling at room temperature, and fermenting for collecting, separating, screening and identifying lactic acid bacteria after 3d and 60d ensiling;
s102, weighing 10g of oat silage which is ensiled for 3d and 60d in an aseptic environment, putting the oat silage into a conical flask, adding 90mL of aseptic normal saline, and shaking the oat silage on a shaking table for 2h (rotating speed of 120rpm min)-1) Gradient dilution is carried out by sterile physiological saline, 3 suitable gradient coating plates are taken, and each gradient is repeated for 3 times;
s103, carrying out isolated culture on lactobacillus on a GYP culture medium (10 g of glucose, 5g of yeast extract, 5g of peptone, 2g of sodium acetate, 805mL of Tween, 5g of calcium carbonate, 5g of sodium chloride, 5mL of salt solution, 15g of agar, 0.04g of bromocresol purple, 1000mL of distilled water and pH6.8), and carrying out colony selection and separation after culturing in an anaerobic incubator at 37 ℃ for 48 h;
wherein: the concentration of the Tween 80 is 50mg mL-1(ii) a The salt solution comprises: 0.2g of manganese sulfate 4 hydrate, 0.2g of ferrous sulfate 7 hydrate and 4g of magnesium sulfate 7 hydrate are dissolved in 100mL of distilled water;
s104, collecting single bacterial colonies according to the characteristics of the size, the color, the luster, the shape, whether a transparent ring exists or not;
s105, carrying out streak culture (37 ℃ and 1d) on an MRS solid culture medium (10.0 g of peptone, 5.0g of beef powder, 20.0g of glucose, 4.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 0.2g of magnesium sulfate, 2.0g of triammonium citrate, 0.05g of manganese sulfate, 801mL of Tween, 15g of agar, 1L of distilled water and 6.2 +/-0.2 of pH), and repeating the streak separation culture for 3 times to obtain a purified single strain;
s106, performing gram staining, microscopic observation and catalase test on the strains, and preliminarily determining gram staining positive strains and catalase negative strains as lactic acid bacteria; separating 232 lactic acid bacteria from oat attached lactic acid bacteria, wherein one strain is numbered as OL77, subpackaging sterilized MRS liquid culture medium, each test tube is 5mL, inversely placing into a Du's small tube, inoculating activated strains, the inoculum size is 3%, culturing for 3d at 37 ℃, and observing whether gas is produced; the results of the characterization test of strain OL77 are shown in table 1:
table 1: characterization of Strain OL77
| Bacterial strains | Shape of | Type of fermentation | Gram stain | Hydrogen peroxide enzymatic process | Glucose gas production |
| OL77 | Coccus bacterium | Same type of product | + | - | - |
As can be seen from table 1, strain OL77 is a gram-positive, catalase-negative, homofermentative coccus;
s107, enriching by using MRS liquid culture medium (37 ℃, 1d), adding glycerol (40%) with the same volume,subpackaging and storing at-20 deg.C for use; in subsequent experiments, the strain preserved at low temperature is thawed in flowing water quickly, placed in MRS liquid culture medium for activation culture (37 ℃ and 1d), subjected to subculture for 2 times, centrifuged to collect thalli, and added with physiological saline by turbidimetry to adjust the concentration of bacterial liquid to 108 cfu mL-1；
S2: the growth characteristics of OL77 under different salt concentrations, pH and temperature were studied, comprising the following steps:
s201, preparing an MRS liquid culture medium containing 3.0% and 6.0% NaCl, inoculating an activated strain, wherein the inoculation amount is 3%, culturing for 3 days at 37 ℃, measuring the light absorption value (OD600) at 600nm by using a spectrophotometer, and analyzing the salt tolerance of a strain OL77 to obtain the results shown in Table 2;
s202, using 4mol L-1Adjusting the pH value of the MRS liquid culture medium to 3.0, 3.5, 4.0, 4.5 and 5.0 respectively by using HCl and NaOH solutions, inoculating an activated strain with the inoculation amount of 3 percent, culturing for 3 days at 37 ℃, measuring the light absorption value (OD600) at 600nm by using a spectrophotometer, and analyzing the acid resistance of a strain OL77 to obtain the results shown in Table 2;
s203, inoculating an activated strain into an MRS liquid culture medium, wherein the inoculation amount is 3%, culturing at constant temperature of 20 ℃, 15 ℃, 10 and 5 ℃, respectively, culturing at 5 ℃ for 10 days, culturing at 10 ℃ and 15 ℃ for 7 days, culturing at 20 ℃ for 5 days, measuring the light absorption value (OD600) at 600nm by using a spectrophotometer, and analyzing the adaptability of the strain OL77 to the environmental temperature to obtain the results shown in Table 2;
table 2: growth characteristics of Strain OL77 under different environments
Note: "+" indicates good growth (OD600 greater than 0.3 is considered good growth)
As can be seen from table 2: (1) the strain OL77 grows well under the NaCl concentration of 3.0% and 6.0%, which shows that the strain OL77 has stronger salt tolerance; (2) the strain OL77 grows well in the environment with pH of 3.0, 3.5, 4.0, 4.5 and 5.0, which shows that the strain OL77 has stronger acid resistance; (3) the strain OL77 grows well in the environment with the temperature of 20, 15, 10 and 5 ℃, which shows that the strain OL77 has better low temperature resistance; (4) from the above results, it was found that the strain OL77 has a strong growth adaptability and can survive and propagate in extreme environments.
S3, screening low-temperature-resistant lactic acid bacteria, and specifically comprising the following steps:
through growth tests under different salt concentrations, pH values and temperatures, 18 strains of salt-resistant, acid-resistant and low-temperature-resistant lactic acid bacteria resources are obtained through screening, can grow under the stress of 3.0% and 6.0% NaCl, at a pH value of 3.0-5.0 and at a low temperature of 5-20 ℃, and have good environment adaptability;
s301, inoculating an activated strain in an MRS liquid culture medium, wherein the inoculation amount is 3%, culturing at 37 ℃ for 30h, sampling every 5h, measuring the pH value of a culture solution, and analyzing the acid production rate of the lactic acid bacteria strain, wherein the result is shown in Table 3;
s302, simultaneously, measuring the absorbance value (OD value) of the sample at the wavelength of 600nm by taking the culture solution as a reference, and analyzing the growth rate of the lactic acid bacteria, wherein the results are shown in Table 3;
table 3: acid production and growth rates of Strain OL77
| Incubation time (h) | 5 | 10 | 15 | 20 | 25 | 30 |
| Acid rate (pH) | 4.51 | 3.74 | 3.72 | 3.64 | 3.65 | 3.62 |
| Growth Rate (OD600) | 1.79 | 2.21 | 2.29 | 2.49 | 2.52 | 2.53 |
As can be seen from table 3: (1) the strain OL77 has high acid production and growth rate, and is suitable for serving as an alternative strain for silage fermentation.
S4, identifying the excellent low-temperature-resistant lactic acid bacteria, which comprises the following specific steps:
s401, carrying out enrichment culture on the activated strain OL77 in an MRS culture medium for 24h, centrifuging and collecting thalli, and extracting DNA according to the instruction of a Bacteria DNA Kit (Shanghai Biotechnology Co., Ltd., Shanghai, China) Kit;
s402, amplifying the 16S rDNA of the activated strain OL 77;
wherein: the primer sequence is as follows: 27f (5'-AGA GTT TGA TCC TGG CTC AG-3'), 1492r (5'-TAC CTT GTT ACG ACT-3'); the PCR amplification reaction system is as follows: 2 XPCR Master Mix, upstream and downstream primers 1. mu.l each, DNA template 1. mu.l, 9.5. mu.l sterile ddH2O, total volume 25. mu.l; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 51 ℃ for 45s, extension at 72 ℃ for 1min, and 30 cycles; finally, extending for 10min at 72 ℃, and storing at 4 ℃;
s403, taking 5 mu l of PCR product to perform electrophoresis on 1% agarose gel to detect a target band, as shown in figure 4;
s404, sending the amplification product to the forward and reverse sequencing of Shanghai Biotechnology limited, wherein the sequence of the amplification product is shown as seq ID No. 1;
s405, searching the gene sequence of the lactobacillus strain 16S rDNA in GenBank by using BLAST (http:// blast.ncbi.nlm.nih.gov/blast.cgi), comparing the gene sequence with the strain with the known classification position with the highest similarity of the strain to be tested, and preliminarily determining the genus of the strain to be tested, wherein the test result is shown in Table 4;
table 4: 16S rDNA Gene sequence analysis of Strain OL77
As can be seen from table 4: the access number of 16S rDNA of the strain OL77 in NCBI database is MT359243, and the similarity with its kindred species of Pediococcus pentosaceus (Pediococcus pentosaceus) DSM 20336 is up to 99.72%;
s406, determining the difference of the strain OL77 in utilization of different carbon sources by using an API 50 CHL fermentation kit (BioMerieux, France) according to kit instructions, and identifying the strains, wherein the test results are shown in Table 5:
table 5: sugar fermentation characteristics of Strain OL77
Note: "+" indicates available; "-" indicates unavailable; "W" indicates weak utilization;
as can be seen from the results of table 5: strain OL77 can utilize L-arabinose, ribose, D-xylose, galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, D-raffinose, beta-gentiobiose, D-tagatose and gluconate; erythritol, D-arabinose, L-xylose, adonitol, beta-methyl-xyloside, L-sorbose, galactitol, inositol, alpha-methyl-D-glucoside, starch, glycogen, xylitol, D-turanose, D-lyxose, D-trehalose, L-fucose, D-arabitol, L-arabitol, 2-keto-gluconate, and 5-keto-gluconate cannot be utilized;
according to the analysis results of the morphology, the physiological and biochemical characteristics, the 16s rDNA sequence homology and the sugar fermentation characteristics, the strain OL77 is identified as Pediococcus pentosaceus (Pediococcus pentosaceus) and is preserved in China center for type culture Collection (CCTCC for short, address: eight-channel 299 Wuhan university school in Wuchang district, Wuhan university Collection, Zip code 430072, Hubei province, Wuhan province, 7.22.2020), and the preservation number is CCTCC NO: m2020290, which is classified and named Pediococcus pentosaceus OL 77.
Example 2: the influence of the compatible solute on the low-temperature adaptability of the lactic acid bacteria is researched
S1, researching the influence of a compatible solute on the low-temperature growth of lactic acid bacteria, and the method specifically comprises the following steps:
s101, respectively adding betaine, proline, trehalose and sorbitol into an MRS liquid culture medium, wherein the addition amount of each of the betaine, the proline, the trehalose and the sorbitol is 0.01mol L-1；
S102, inoculating activated and modulated Pediococcus pentosaceus OL77 into a culture medium added with solutes with different compatibility and no additive (CK), culturing in a constant-temperature incubator at 5 ℃, culturing for 30h at 37 ℃, sampling once every 5h, measuring the pH value of a culture solution, and analyzing the acid production rate of the lactic acid bacteria strain, wherein the results are shown in Table 6;
s103, simultaneously, measuring the absorbance value (OD value) of the sample at the wavelength of 600nm by taking the culture solution as a reference, and analyzing the growth rate of the lactic acid bacteria, wherein the results are shown in Table 6;
table 6: effect of compatible solute on Low temperature acid production and growth Rate of Strain OL77
Note: different lower case letters indicate that the strains showed significant differences in acid production or growth rate between treatments (P < 0.05).
As can be seen from the results of table 6: compatible solutes have obvious influence on the acid production and growth rate of Pediococcus pentosaceus OL77, and the difference between different compatible solutes is obvious; the effects of betaine and trehalose on improving the acid production and growth rate of the strain OL77 are more obvious, particularly, the trehalose treatment group is optimal, the pH value is reduced to 4.04 after 30 hours, the OD values are all increased to more than 1.0, and the increase is 1 time compared with the control.
Example 3: low-temperature-resistant pediococcus pentosaceus and preparation and application thereof
S1, planting oats in 2019 in 23 days 4 months and with the seeding rate of 150 kg.hm-2Applying urea (containing 46% of N) before sowing, and applying 75 kg. hm of fertilizer-2Weeding for 2 times in the growth period without irrigation; oats are harvested at the milk stage, mechanically shredded for ensiling conditioning, and the chemical and microbial composition of the oat material is shown in table 7:
table 7: chemical components and microorganism composition of oat silage raw material
| Item | |
| DM(g kg-1FM) | 331.7 |
| CP(g kg-1DM) | 97.9 |
| WSC(gkg-1 DM) | 198.5 |
| NDF(g kg-1DM) | 575.2 |
| ADF(gkg-1DM) | 334.1 |
| pH value | 6.15 |
| LAB(log10 cfu g-1FM) | 4.13 |
| Aerobic bacteria (log)10 cfu g-1FM) | 7.56 |
| Mold (log)10 cfu g-1FM) | 3.94 |
| Yeast (log)10 cfu g-1FM) | 4.63 |
Note: FM: fresh weight; DM: and (4) dry matter.
S2, researching the influence of temperature and lactobacillus additives on the nutrient content and ammonia nitrogen content of oat silage, and specifically comprising the following steps:
s201, setting a Control (CK) for experiments, commercial silage additive Synlac I (SLI, Su core Biotechnology Co., Ltd.), an artificial screening strain OL77, trehalose additive (T) and trehalose + OL77(T + OL77), and 5 additive treatment groups in total;
s202, the addition amount of the lactobacillus additive is based on the fresh weight of the silage, and the addition amount of the lactobacillus is 108CFU·kg- 1FM, adding equal amount of clear water as control, adding trehalose into OL77 bacterial solution in advance to obtain compound addition group with addition amount of 0.01mol L-1Fully and uniformly mixing the bacterial liquid; mixing all additives and silage raw materials, and loading into 5L polyethylene silage barrels with a loading density of 210kg DM/m and 3.2kg of each barrel3(ii) a Each treatment was divided into 3 groups, each group was repeated 3 times, and stored in a constant temperature incubator at 15 ℃, 10 ℃ and 5 ℃, respectively, and after 45 days, the nutritional ingredients, fermentation quality and microbial composition of the silage were measured by sampling, and the measurement results are shown in table 8;
wherein the Dry Matter (DM), Crude Protein (CP), soluble sugar (WSC), ammoniacal Nitrogen (NH)3-N) the determination method is referred to feed analysis and feed quality detection techniques;
table 8: effect of temperature and lactic acid bacteria additives on oat silage nutrient content and ammoniacal nitrogen content
Note: different lower case letters indicate significant differences between different treatments in the same column (P <0.05), FM: fresh weight; DM: dry matter; TN: total nitrogen.
As can be seen from table 8: (1) under the condition of low-temperature ensiling, the DM content of the OL77+ T and OL77 added treatment groups is always higher, particularly the OL77+ T treatment group is the highest; (2) when ensiled at 10 ℃, the crude protein contents of OL77 and OL77+ T are high, 97.1 and 96.6g kg respectively-1DM, all are significant (P)<0.05) higher than CK; (3) the CP content of the OL77+ T treatment group is highest when the strain is ensiled at 5 ℃; (4) the WSC content of the OL77 treated group is the highest at 10 ℃ and 5 ℃ for silage, and is respectively more remarkable than that of the CK treated group (P)<0.05) higher by 25.56% and 22.80%, OL77+ T times, no significant difference from the OL77 treatment group (P)>0.05); (5) NH in the form of OL77+ T at treatment temperatures of 15 ℃, 10 ℃ and 5 DEG C3Minimum N content (18.2, 19.8 and 17.6g kg)-1TN), significant (P)<0.05) lower than CK treated group; the method shows that the Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 treatment can retain more forage nutrients when the strain is ensiled at the low temperature of 10 ℃ and 5 ℃, and the compound addition effect of the Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 and trehalose is better.
S3, researching the influence of temperature and lactic acid bacteria additives on the fermentation quality of oat silage, and specifically comprising the following steps:
s301, adding 180mL of deionized water into 20g of silage samples, leaching for 24 hours in a refrigerator at 4 ℃, filtering by using 4 layers of gauze, and then finely filtering by using qualitative filter paper to obtain silage leaching liquor;
s302, taking one part of the mixture, and measuring the pH value by using an acidimeter; the other part was filtered through a 0.22 μm filter and then subjected to determination of Lactic Acid (LA), Acetic Acid (AA), Propionic Acid (PA) and Butyric Acid (BA) by agilent 1260 hplc, the results of which are shown in table 9;
wherein the chromatographic conditions are: SB-AQ C18Chromatography columns (4.6 mm. times.250 mm); mobile phase a (methanol): mobile phase B [0.01 mol. L-1(NH4)2HPO4,pH＝2.70]97 at a flow rate of 1 mL/min-1The sample introduction amount is 20 mu L, the detection wavelength is 210nm, and the column temperature is 25 ℃;
table 9: influence of temperature and lactic acid bacteria additive on oat silage fermentation quality
Note: different lower case letters indicate significant differences between different treatments in the same column (P <0.05), ND: it was not detected.
As can be seen from table 9: (1) the pH values of OL77+ T and OL77 treated groups were significant when ensiled at 10 ℃ (P)<0.05) less than CK, SLI and T; (2) the OL77+ T treated group pH dropped to 3.87 at 5 deg.C, significantly (P)<0.05) lower than that of each of the other treatment groups, while the pH of the other treatment groups is still kept at a higher level (4.38-5.29); (3) the LA content in the groups treated with OL77+ T and OL77 was high when they were ensiled at 10 ℃ (P)<0.05) higher than the other treatment groups; (4) the highest LA content was achieved with OL77+ T treatment at 5 deg.C (91.4g kg)-1DM), significant (P)<0.05) higher than all treatment groups; (5) AA content was minimized by treatment with OL77+ T at 5 ℃ (7.3g kg)-1DM); (6) the PA content and the BA content show the same change rule, and are not detected in all treatments at 5 ℃;
the method proves that the fermentation quality of oat silage can be effectively improved by treating Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 when the oat is ensiled at 10 and 5 ℃, the composite addition effect of the Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 and trehalose is better, more lactic acid can be generated at low temperature of 5 ℃, the pH value is reduced, and the acceleration of the fermentation process is facilitated.
S4, researching the influence of temperature and lactic acid bacteria additives on the number of oat ensiling microorganism groups, and specifically comprising the following steps:
s401, counting Lactic Acid Bacteria (LAB), aerobic bacteria (aerobic bacteria), mold (mold) and yeast (yeast) by using a gradient dilution and spreading method and respectively adopting an MRS culture medium, a common agar culture medium and a tiger red agar culture medium, wherein each sample is divided into 3 samples in parallel, and the results are shown in a table 10;
wherein: the MRS culture medium comprises: 10.0g of peptone, 5.0g of beef powder, 20.0g of glucose, 4.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 2.0g of triammonium citrate, 0.05g of manganese sulfate, 801mL of tween, 15g of agar, 1L of distilled water, pH: 6.2 plus or minus 0.2; common agar media include: peptone 10.0g, beef extract powder 3.0g, sodium chloride 5.0g, agar 15.0g, distilled water 1L, pH: 7.3 plus or minus 0.1; the tiger red agar medium comprises: peptone 5.0g, glucose 10.0g, potassium dihydrogen phosphate 1.0g, Bengal 0.033g, magnesium sulfate 0.5g, chloramphenicol 0.1g, agar 18.5g, pH: 6.3 plus or minus 0.1;
table 10: effect of temperature and lactic acid bacteria additives on the number of oat silage microbial populations (Log)10 cfu g-1FM)
Note: different lower case letters indicate significant differences between different treatments in the same column (P < 0.05).
As can be seen from table 10: (1) at 5 ℃ for ensiling, all placesThe number of the LAB in the physical group is obviously reduced compared with that at 15 ℃ and 10 ℃ (P)<0.05), highest OL77+ T (8.87 Log)10 cfu g-1FM), significant (P)<0.05) higher than the other treatments; CK. The difference between SLI and T treatments was not significant (P)>0.05); (2) the numbers of aerobic bacteria in the OL77 and OL77+ T addition treatment groups were always lower, both lower than 5.10Log10 cfu g-1FM; (3) the number of the mould and the yeast shows the same change rule; none of the OL77+ T treated groups appeared on colony culture plates with mold and yeast seen at 3 treatment temperatures.
With reference to tables 8, 9, and 10, it is demonstrated that the compound addition treatment of Pediococcus pentosaceus (Pediococcus pentosaceus) OL77 and trehalose can effectively inhibit the growth of mold, yeast, and other miscellaneous bacteria, rapidly reduce the pH value, effectively retain more forage grass nutrients, and improve the forage grass silage fermentation quality in a low temperature environment;
wherein, in the preparation process of the silage additive, the ratio of the pediococcus pentosaceus OL77 to the trehalose is 1013～1014CFU 0.01mol, the optimal proportion is 5 multiplied by 1013CFU:0.01mol。
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> university of agriculture in Gansu province
<120> low temperature resistant pediococcus pentosaceus OL77 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
cgcgggctat catgcaagtc gaacgaactt ccgttaattg attatgacgt acttgtactg 60
attgagattt taacacgaag tgagtggcga acgggtgagt aacacgtggg taacctgccc 120
agaagtaggg gataacacct ggaaacagat gctaataccg tataacagag aaaaccgcat 180
ggttttcttt taaaagatgg ctctgctatc acttctggat ggacccgcgg cgtattagct 240
agttggtgag gtaaaggctc accaaggcag tgatacgtag ccgacctgag agggtaatcg 300
gccacattgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 360
cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt 420
aaagctctgt tgttaaagaa gaacgtgggt aagagtaact gtttacccag tgacggtatt 480
taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540
gttatccgga tttattgggc gtaaagcgag cgcaggcggt cttttaagtc taatgtgaaa 600
gccttcggct caaccgaaga agtgcattgg aaactgggag acttgagtgc agaagaggac 660
agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720
ggcggctgtc tggtctgcaa ctgacgctga ggctcgaaag catgggtagc gaacaggatt 780
agataccctg gtagtccatg ccgtaaacga tgattactaa gtgttggagg gtttccgccc 840
ttcagtgctg cagctaacgc attaagtaat ccgcctgggg agtacgaccg caaggttgaa 900
actcaaaaga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960
acgcgaagaa ccttaccagg tcttgacatc ttctgacagt ctaagagatt agaggttccc 1020
ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080
gttaagtccc gcaacgagcg caacccttat tactagttgc cagcattaag ttgggcactc 1140
tagtgagact gccggtgaca aaccggagga aggtggggac gacgtcaaat catcatgccc 1200
cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagtcgc gagaccgcga 1260
ggttaagcta atctcttaaa accattctca gttcggactg taggctgcaa ctcgcctaca 1320
cgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380
ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagccgg tggggtaacc 1440
ttttaagagc tagcctcaaa 1460
Claims (8)
1. A low temperature resistant Pediococcus pentosaceus OL77 is characterized in that: the low-temperature resistant Pediococcus pentosaceus OL77 is Pediococcus pentosaceus OL77, is separated from oat of autonomous State of Jade Tree Tibetan of China, Qinghai province, and is preserved in China center for type culture Collection CCTCC (China center for type culture Collection) in Wuhan university of Wuhan City, Hubei province, and the preservation number is CCTCC NO: m2020290.
2. The strain of pediococcus pentosaceus OL77 of claim 1, wherein: the 16S rDNA sequence of Pediococcus pentosaceus OL77 is shown as seq ID No. 1.
3. A microbial inoculum, which is characterized in that: the active ingredient of the microbial inoculum is the low temperature resistant pediococcus pentosaceus OL77 strain of claim 1.
4. An additive for silage, which is characterized in that: the active ingredient of the silage additive is the low temperature resistant pediococcus pentosaceus OL77 strain of claim 1.
5. A silage composite additive, which is characterized in that: the preparation method of the silage composite additive comprises the following steps: the microbial preparation according to claim 3, wherein 0.01mol L of the microbial preparation is added-1Compatible solute trehalose.
6. An ensilage characterized in that: the silage comprising the silage additive according to any one of claims 4 to 5.
7. A silage according to claim 6, characterised in that: in the silage, the ratio of pediococcus pentosaceus OL77 to trehalose is 1013～1014CFU:0.01mol。
8. A method of preparing the silage of claim 6, wherein: mixing silage raw materials with the silage additive of claim 4 or 5, performing solid anaerobic fermentation, and collecting all fermentation products to obtain silage;
wherein: the ensiling raw material is specifically oat whole plant after mechanical shredding;
in the method, the ratio of oat raw material to pediococcus pentosaceus OL77 is 1kg:108CFU；
The fermentation is low-temperature fermentation; the low temperature is 10 ℃ with the silage additive of claim 4; the low temperature is 5 ℃ with the silage additive of claim 5;
the fermentation time was 45 days.
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| CN115287232A (en) * | 2022-08-10 | 2022-11-04 | 四川轻化工大学 | Pediococcus pentosaceus and application thereof in fermentation of choerospondias axillaris lactobacillus beverage |
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| KR20160063025A (en) * | 2014-11-26 | 2016-06-03 | 대한민국(농촌진흥청장) | Pediococcus pentosaceus KCC-23 and composition comprising the same |
| CN108060103A (en) * | 2018-01-29 | 2018-05-22 | 四川农业大学 | A kind of Pediococcus pentosaceus and its application, screening calibration method |
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| CN114854642A (en) * | 2022-05-30 | 2022-08-05 | 中国农业大学 | Alfalfa endogenous pediococcus pentosaceus EL5 and application thereof |
| CN115287232A (en) * | 2022-08-10 | 2022-11-04 | 四川轻化工大学 | Pediococcus pentosaceus and application thereof in fermentation of choerospondias axillaris lactobacillus beverage |
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