CN113736710B - Oxygen-resistant lactic acid bacteria and application thereof - Google Patents

Oxygen-resistant lactic acid bacteria and application thereof Download PDF

Info

Publication number
CN113736710B
CN113736710B CN202111134189.XA CN202111134189A CN113736710B CN 113736710 B CN113736710 B CN 113736710B CN 202111134189 A CN202111134189 A CN 202111134189A CN 113736710 B CN113736710 B CN 113736710B
Authority
CN
China
Prior art keywords
lactobacillus
oxygen
resistant
silage
lactic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111134189.XA
Other languages
Chinese (zh)
Other versions
CN113736710A (en
Inventor
张建国
田静
尹祥
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN202111134189.XA priority Critical patent/CN113736710B/en
Publication of CN113736710A publication Critical patent/CN113736710A/en
Application granted granted Critical
Publication of CN113736710B publication Critical patent/CN113736710B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • 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
    • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
    • 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
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention discloses an oxygen-resistant lactic acid bacterium and application thereof. The name of the oxygen-resistant lactobacillus is lactobacillus (Lactobacillus plantarum) SXC48, and the lactobacillus is preserved in the microorganism strain collection center of Guangdong province, the preservation place is the 5 th floor of Guangdong province microbiological institute of building No. 59 of Mitsui No. 100 of Guangzhou, the preservation number is GDMCC No. 61621, and the preservation date is 2021, 4 and 25 days. The lactic acid bacteria are gram-positive bacteria, are glucose homotype fermentation, are acid-resistant, have high growth speed and wide growth temperature range, are particularly oxygen-resistant, and can remarkably improve the fermentation quality of silage which is not easy to compact or is easy to air survival.

Description

Oxygen-resistant lactic acid bacteria and application thereof
Technical Field
The invention belongs to the technical field of microorganism application and feed modulation processing, and particularly relates to an oxygen-resistant lactic acid bacterium and application thereof.
Background
Silage is a commonly used forage processing and storing method at home and abroad, and is also an effective means for improving the production capacity and benefit of the modern grassland animal husbandry. Silage not only can improve the quality of meat and milk, but also can fully utilize plant materials which can not be directly fed, and reduce the waste of agricultural product processing byproducts. Silage has therefore been valued by countries around the world, particularly in developed countries of the animal industry. Silage relies on lactobacillus attached to the surface of silage raw material to convert water-soluble carbohydrate in raw material into organic acid mainly containing lactic acid under anaerobic condition, so that pH is reduced, thereby inhibiting growth and reproduction of harmful bacteria, and the fodder can be preserved for a long time. The existing lactobacillus additives are screened out under anaerobic conditions, namely the anaerobic environment shows better performance, and in the actual production of silage, the complete removal of air among raw material particles and in empty stems is impossible, particularly, the environment with more air exists in the first days after the cellar is filled to be sealed, the growth and the cell activity of the lactobacillus are often stressed by oxygen of active oxygen free radicals, and the reproduction and the acid production capacity of the lactobacillus are affected; meanwhile, oxygen stress can certainly increase the manufacturing cost in the process of producing the lactobacillus preparation. Once lactic acid is insufficient and the pH value is slowly reduced in the silage process, the activities of harmful microorganisms such as clostridium are promoted, so that excessive loss of nutrient substances is caused, and the quality and the utilization benefit of silage are reduced.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide an oxygen-resistant lactobacillus.
Another object of the invention is to provide the use of the above-mentioned oxygen-tolerant lactic acid bacteria in silage modulation.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
the strain of the oxygen-resistant lactobacillus is named as lactobacillus (Lactobacillus plantarum) SXC48, and is preserved in the microorganism strain collection center of Guangdong province, the preservation place is the institute of microorganisms of Guangdong province, building 5, of 100 th university of Mitsui, guangzhou, and the preservation date is 2021, 4, 25 days.
The biological characteristics of the lactobacillus (Lactobacillus plantarum) SXC48 are gram-positive bacillus, glucose homotype fermentation is performed, the acid resistance is strong (the strain can normally grow at the pH of 3.5), the growth speed is high (the pH of an MRS culture medium can be reduced to below 4.0 after being cultured for 24 hours), the growth temperature range is wide (the strain can normally grow at the temperature of 15-45 ℃), and the strain can normally grow under aerobic culture conditions.
The method for obtaining the lactobacillus Lactobacillus plantarum SXC comprises the following steps: enrichment culture of the strain, dilution plate method screening to obtain: sterilizing a conical flask containing 500mL distilled water, a test tube containing 9mL distilled water, MRS solid medium and a flat plate, sterilizing, and then subjecting MRS medium was poured into plates. Under aseptic condition, taking 10g sample in plastic bag, adding 90mL sterilized distilled water to make its final concentration 100g/L, standing at 200rpm shaking table for 30min, taking 1mL diluted sample solution, adding 9mL sterilized distilled water, and shaking uniformly in test tube to dilute 10 -1 The concentration is that 0.02mL of the liquid is coated on a flat plate, anaerobic culture is carried out for 24-48 hours at 37 ℃, then single colony is picked up by an inoculating needle, inoculated on an MRS liquid culture medium, anaerobic culture is carried out for 24-48 hours at 37 ℃, flat plate streaking separation is repeatedly carried out until single colony is obtained, and the single colony is inserted into the center of a test tube containing MRS solid culture medium by the inoculating needle and stored in a refrigerator at 4 ℃. Wherein the MRS culture medium is: 10.0g of peptone (protein peptone), 10.0g of Beef extract (Beef extract), 5.0g of Yeast extract (Yeast extract), 20.0g of glucose (Dextrose), 1mL of Tween (Polysorbate 80), 2.0g of Ammonium citrate (Ammonium citrate), 5.0g of sodium acetate (NaAc), 5.0g of magnesium sulfate (MgSO) 4 ·7H 2 O) 0.1g, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 2.0g of solid medium is added with 15g/L of Agar (Agar) and distilled water (H) 2 O) constant volume to 1000mL,121 ℃, and sterilizing for 20min.
The invention screens out lactobacillus Lactobacillus plantarum SXC with high oxygen resistance, acid resistance and growth speed by aerobic culture conditions. Extracting the full-length gene of lactobacillus Lactobacillus plantarum, amplifying 16S rDNA gene by using PCR amplification primers 25F (SEQ ID NO: 1) and 1492R (SEQ ID NO: 2), and comparing related sequences on NCBI to determine similar strains. Finally, the screened bacterial strain and the control bacteria (the most commercially available bacteria used in China) are respectively added into different forage grass, the silage fermentation quality is analyzed after unsealing and is compared with the control bacteria, and it is confirmed that Lactobacillus plantarum SXC can obviously improve the silage fermentation quality of the feed which is not easy to compact or easy to air remain.
The application of the oxygen-resistant lactobacillus in silage preparation preferably comprises the following steps:
(1) Cutting the feed to be fermented;
(2) Uniformly mixing the feed to be fermented after the chopping treatment with the oxygen-resistant lactobacillus;
(3) And (3) degassing and sealing the feed finally obtained in the step (2) and storing the feed.
As a preferred embodiment, the cut-down specification in the step (1) is preferably cut down to 3cm or less; more preferably to a length of 2.5cm or less; most preferably cut to 1-2 cm.
The addition amount of the oxygen-resistant lactobacillus in the step (2) is preferably 1.0x10 per kilogram of feed to be fermented 8 Calculation of the above-described oxygen-tolerant lactic acid bacteria.
Step (3) is preferably: the feed was degassed by a vacuum sealer and stored.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides the lactobacillus Lactobacillus plantarum SXC which can be used for silage in an aerobic environment and has high lactic acid yield, so that the microbial lactobacillus Lactobacillus plantarum SXC can be used for improving the silage fermentation quality of feed which is not easy to compact, and the invention has the advantages of low cost, safety, reliability and easy utilization.
(2) The lactobacillus Lactobacillus plantarum SXC used by the invention has better silage effect on feed which is not easy to compact than the effect of the lactobacillus additive sold in the market, and effectively improves silage fermentation quality of the feed which is not easy to compact.
Drawings
FIG. 1 shows the OD of different lactic acid bacteria cultured under aerobic conditions for 24h 600 A measurement result diagram; the different letters in the figures represent significant differences, P<0.05; SXC48, NF137, JF170, TN179, LFQ196, LN11, and NF134 are lactobacillus plantarum (l plant); LN4 is Pediococcus pentosaceus (Pediococcus pentosaceus); LN7 is Lactobacillus plantarum (L.paraloplantarium); LN10 is Pediococcus acidilactici (P.acidophilus); YZB1 is Lactobacillus acidophilus (L.acidophilus); GD1 is lactobacillus salivarius (l.salivarius); XC124 is enterococcus faecalis (Enterococcus faecalis).
FIG. 2 shows the OD of different lactic acid bacteria cultured at pH3.5 for 24h 600 A measurement result diagram; the different letters in the figures indicate that the differences are significant (P<0.05)。
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, but the examples are not intended to limit the present invention in any way.
Example 1: isolation, screening and physiological and biochemical determination of lactic acid bacteria
266 strains of lactic acid bacteria are separated from grasses such as grasses, italian ryegrass, alfalfa, coltsfoot, broadleaf paspalum and other pastures, silage such as grasses, corns and other materials, soil, cow dung and other 26 materials, gram staining and cell shape observation are carried out, and lactic acid bacteria SXC48 with high oxygen resistance, acid resistance and growth speed are screened according to aerobic culture, growth pH (3.5, 4.0, 4.5, 7.5, 8.0, 8.5) and other experiments. The method comprises the following specific steps: sterilizing a plurality of conical flasks filled with 500mL of distilled water, test tubes filled with 9mL of distilled water, MRS solid culture medium and a flat plate, and pouring the MRS solid culture medium into the flat plate after sterilization; under aseptic conditions, taking 10g of sample in a plastic bag, adding 90mL of sterilized distilled water, shaking uniformly to ensure that the final concentration is 100g/L, then taking 1mL of diluted sample, adding a test tube containing 9mL of sterilized distilled water, shaking uniformly to ensure that the final concentration is 10g/L, taking 0.02mL of the liquid at the moment, repeatedly carrying out plate scribing separation until single bacterial colonies are obtained, obtaining 266 bacterial colonies in total, inserting all the single bacterial colonies into the center of the test tube containing MRS solid culture medium by using an inoculating needle, and storing in a refrigerator at 4 ℃; wherein the MRS culture medium is: 10.0g of peptone (protein peptone), 10.0g of Beef extract (Beef extract), 5.0g of Yeast extract (Yeast extract), 20.0g of glucose (Dextrose), 1mL of Tween (Polysorbate 80), 2.0g of Ammonium citrate (Ammonium citrate), 5.0g of sodium acetate (NaAc), 5.0g of magnesium sulfate (MgSO) 4 ·7H 2 O) 0.1g, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 2.0g of solid medium is added with 15g/L of Agar (Agar) and distilled water (H) 2 O) constant volume to 1000mL,121 ℃, and sterilizing for 20min.
Then, 266 purified strains are respectively inoculated into MRS liquid culture medium, aerobically cultured (5 mL culture medium is placed in a 50mL centrifuge tube and placed in a shaking table at 200 rpm) for 24 hours, and OD is carried out 600 Measuring; meanwhile, 266 strains are respectively inoculated to MRS liquid culture medium with pH of 3.5,anaerobic culture was carried out for 24 hours, and OD was carried out 600 13 strains were selected for measurement, and the measurement results obtained are shown in FIGS. 1 and 2: the OD value of the SXC48 is obviously higher than that of other strains, and the result shows that the SXC48 strain can grow under aerobic culture and pH3.5 and has stronger oxygen resistance and acid resistance.
The separation, physiological and biochemical test and culture medium of lactobacillus SXC48 and the preparation method thereof are as follows:
1) Gram staining, fermentation type and shape observations are referred to the handbook of the identification of common bacterial systems, by the main code of Dongxiu beads.
2) The pH of the lactic acid bacteria growth was adjusted using 4mol/L NaOH and 4mol/L HCl.
3) Sugar fermentation was analyzed using API 50 CHL (bioMerieux, l' EtOile, france).
4) The culture medium was as follows:
MRS medium (for culture of lactic acid bacteria):
10.0g of peptone (protein peptone), 10.0g of Beef extract powder (Beef extract), 4.0g of Yeast extract powder (Yeast extract), 20.0g of glucose (Dextrose), 1mL of Tween (Polysorbate 80), 2.0g of tri-Ammonium citrate (Ammonium citrate), 5.0g of sodium acetate (NaAc), 5.0g of magnesium sulfate (MgSO) 4 ·7H 2 O) 0.1g, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 2.0g. Dissolving the components by using distilled water, and then fixing the volume to 1000mL by using distilled water to obtain an MRS liquid culture medium; the MRS solid culture medium is prepared by adding 15g/L Agar (Agar) and then distilled water to 1000mL, and sterilizing at 121deg.C for 20min.
API 50 CHL medium:
10.0g of peptone (protein peptone), 5.0g of Yeast extract powder (Yeast extract), 1mL of Tween (Polysorbate 80), 5.0g of sodium acetate (NaAc), 2.0g of tri-Ammonium citrate (Ammonium citrate), and magnesium sulfate (MgSO) 4 ·7H 2 O) 0.2g of dipotassium hydrogen phosphate (K) 2 HPO 4 ) 2.0g of manganese sulfate (MnSO 4 ·4H 2 O) 0.05g, bromocresol purple (Bromcresol purple) 0.17g. Dissolving the above components with distilled water, then metering with distilled water to 1000mL, and packaging into 15mL test tubes with volume of 10m eachL, sterilizing at 121deg.C for 20min.
The test results are shown in Table 1:
TABLE 1 physiological and biochemical characteristics of strain SXC48
Index (I) SXC48 Index (I) SXC48 Index (I) SXC48
Shape and shape Rod-shaped Fermented sugar Fermented sugar
Gram staining + Mannitol (mannitol) + D-cellobiose +
Type of fermentation Homotypic type L-arabinose + D-melibiose +
Growth temperature D-ribose + Inulin ±
10℃ + D-xylose ± D-melezitose +
15℃ +++ L-rhamnose ± D-raffinose +
45℃ +++ Mannitol (mannitol) + Starch ±
50℃ ± Sorbitol + D-gentiobiose +
Growth pH value Methyl-alpha D mannopyranoside ± D-Tulun pond +
3.50 + Methyl-alpha D glucopyranoside ± D-arabitol ±
4.00 +++ N-acetylglucosides + Potassium gluconate +
4.50 +++ Amygdalin +
7.50 +
8.00 +
8.50 -
+++: the growth is vigorous; ++: the growth is good; +: growing; and (3) the following steps: weak growth; -: does not grow.
Example 2: identification of lactic acid bacteria
The strain obtained in example 1 was cultured overnight at 37℃in 5mL of MRS medium, the bacterial solution was transferred into a 1.5mL centrifuge tube, centrifuged at 10000rpm for 3min to 5min to collect the bacteria, washed twice with TE (10 mmol/L Tris-HCl, 0.1mmol/L EDTA, pH 8.0) solution, the full length gene of lactobacillus SXC48 was extracted, then the 16S rDNA gene was amplified using PCR amplification primers 25F (5'-AACTGAAGAGTTTGATCCTGGCTC-3') and 1492R (5'-TACGGCTACCTTGTTACGACT-3') (specifically as follows), and transferred to Hua major gene (China) for sequencing, and the relevant sequences were compared on NCBI to determine that SXC48 was lactobacillus plantarum (Lactobacillus plantarum).
GCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTTGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCA。
Example 3: effect of addition to silage
Silage addition experiments were performed using southwest grasses and stylosanthes (the nutritional characteristics before silage are shown in table 2) as materials. Cutting the materials to 2.5cm below, mixing, adding control, SXC48 and commercial bacteria (Lactobacillus plantarum bacteria), and adding strain 1.0X10 per kg of fresh materials 8 cfu, control added an equal amount of sterile water. Polyethylene silage bags of 30cm x 20cm were filled, each treatment was repeated 3 times, each bag was about 200g, and deaerated with a vacuum sealer (SINBO Vacuum Sealer, hong Tai Home Electrical Appliance co.ltd.) to a degree of heavy deaeration (substantially no residual air), medium deaeration (residual 300 to 400mL air), light deaeration (residual 600 to 800mL air), and stored in a dark place for 60d after sealing, and the silage fermentation quality was analyzed and subjected to comparative analysis after unsealing.
The results were as follows: silage under moderate and mild deaeration conditions of southernwood and coltsfoot, silage with SXC48 added, pH reduced below 4.8 and 5.3, respectively, lower than control and commercial microbial agent treatments (table 3), and silage fermentation quality also better than control and commercial microbial agent treatments (table 3). Under the seriously degassed silage condition, the fermentation quality of the treatment by adding SXC48 is better, the pH and ammonia nitrogen content are lower than those of a control without addition and a commercial microbial inoculum, the lactic acid content is higher, and the fermentation quality of the stylosanthes guianensis is obviously improved. So the strain SXC48 can improve silage fermentation quality of feed which is not easy to compact or air-entrapping.
TABLE 2 chemical Properties of Inula albopicta and Inula linearis and microbial counts
Note that: FM, silage fresh material; DM, silage dry matter.
TABLE 3 influence of the addition of different lactic acid bacteria on the quality of silage from different degassed grasses and cylindrical flowers
Note that: DM, dry matter of silage; TN represents total nitrogen; the same pasture is obviously different from the lower case letters in the same column.
The method for measuring the nutritional ingredients and silage fermentation indexes comprises the following steps:
1) Determination of forage grass nutrient components and microbial analysis: the Dry Matter (DM) content was determined by oven drying at 70deg.C in the feed analysis of Zhang Liying, the crude protein content was determined by Kjeldahl nitrogen determination method (azotometer KN680, ALVA instruments Co., ltd.), the neutral washing fiber and the acidic washing fiber content was determined by filter bag method, the crude fat content was determined by diethyl ether extraction method (SLF-06, hangzhou Topu instruments Co., ltd.), and the crude ash content was determined by burning method. The Water Soluble Carbohydrates (WSC) content was determined by anthrone-sulfuric acid method, the ammoniacal nitrogen content was determined by Kjeldahl method, the buffering energy was determined by hydrochloric acid, sodium hydroxide titration, and the amounts of lactobacillus, bacteria, yeast and mold were counted by MRS (de-Man Rogosa Sharpe) agar medium, nutrient agar medium (Nutrient agar, guangdong Cyclo Kawau Co., ltd.), potato dextrose agar medium (Potato-dextrose agar, guangdong Cyclo Kawau Co., ltd.). Culturing lactobacillus in an anaerobic box at 37 ℃ for 1-2 d; bacteria, saccharomycetes and mould are cultured for 2-4 d at 30 ℃ in a biochemical incubator.
2) Fermentation quality analysis: after the silage bag is unsealed, 20g of the silage material which is uniformly mixed is taken to be placed into a self-sealing bag, 80mL of distilled water is added, the silage bag is placed into a refrigerator with the temperature of 4 ℃ to be soaked for 18 hours and then filtered, and the pH value of the leaching solution is measured by a pH meter (Mettler Toledo FE pH meter). The amounts of lactobacillus, bacteria, saccharomycetes and mildew are determined as above, and the content of the organic acid is determined by using an Shimadzu LC-20AT type high performance liquid chromatograph: chromatographic conditions: chromatographic column (Eleven Organic Acids on Transgenomic COREGel 87H 3), detector: RID-10A, phosphoric acid solution with mobile phase of 0.1mmol/L, flow rate of 1mL/min, column temperature of 40 ℃, detection wavelength of 210nm and sample injection amount of 20 mu L.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> an oxygen-tolerant lactic acid bacterium and use thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer 25F
<400> 1
aactgaagag tttgatcctg gctc 24
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer 1492R
<400> 2
tacggctacc ttgttacgac t 21
<210> 3
<211> 761
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<220>
<223> 16S rDNA Gene
<400> 3
gcgtcagtta cagaccagac agccgccttc gccactggtg ttcttccata tatctacgca 60
tttcaccgct acacatggag ttccactgtc ctcttctgca ctcaagtttc ccagtttccg 120
atgcacttct tcggttgagc cgaaggcttt cacatcagac ttaaaaaacc gcctgcgctc 180
gctttacgcc caataaatcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 240
acgtagttag ccgtggcttt ctggttaaat accgtcaata cctgaacagt tactctcaga 300
tatgttcttc tttaacaaca gagttttacg agccgaaacc cttcttcact cacgcggcgt 360
tgctccatca gactttcgtc cattgtggaa gattccctac tgctgcctcc cgtaggagtt 420
tgggccgtgt ctcagtccca atgtggccga ttaccctctc aggtcggcta cgtatcattg 480
ccatggtgag ccgttacccc accatctagc taatacgccg cgggaccatc caaaagtgat 540
agccgaagcc atctttcaaa cttggaccat gcggtccaag ttgttatgcg gtattagcat 600
ctgtttccag gtgttatccc ccgcttctgg gcaggtttcc cacgtgttac tcaccagttc 660
gccactcact caaatgtaaa tcatgatgca agcaccaatc aataccagag ttcgttcgac 720
ttgcatgtat taggcacgcc gccagcgttc gtcctgagcc a 761

Claims (1)

1. The application of the strain of the oxygen-resistant lactobacillus in silage modulation is characterized in that: the name of the oxygen-resistant lactobacillus is lactobacillus plantarumLactobacillus plantarum) SXC48 is preserved in the microorganism strain collection center of Guangdong province, the preservation place is the 5 th floor of Guangdong province institute of microorganisms, the preservation number is GDMCC No. 61621, and the preservation date is 2021, 4 months and 25 days;
the application of the oxygen-resistant lactobacillus in silage modulation comprises the following steps:
(1) Cutting the feed to be fermented;
(2) Uniformly mixing the feed to be fermented after the chopping treatment with the oxygen-resistant lactobacillus;
(3) Degassing and sealing the feed obtained in the step (2), and storing;
the cut-down specification in the step (1) is cut-down to below 3 cm;
the addition amount of the oxygen-resistant lactobacillus in the step (2) is 1.0x10 per kilogram of feed to be fermented 8 Calculating the oxygen-resistant lactobacillus;
and (3) degassing the feed by a vacuum sealing machine and storing.
CN202111134189.XA 2021-09-27 2021-09-27 Oxygen-resistant lactic acid bacteria and application thereof Active CN113736710B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111134189.XA CN113736710B (en) 2021-09-27 2021-09-27 Oxygen-resistant lactic acid bacteria and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111134189.XA CN113736710B (en) 2021-09-27 2021-09-27 Oxygen-resistant lactic acid bacteria and application thereof

Publications (2)

Publication Number Publication Date
CN113736710A CN113736710A (en) 2021-12-03
CN113736710B true CN113736710B (en) 2024-02-09

Family

ID=78741342

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111134189.XA Active CN113736710B (en) 2021-09-27 2021-09-27 Oxygen-resistant lactic acid bacteria and application thereof

Country Status (1)

Country Link
CN (1) CN113736710B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004041064A (en) * 2002-07-11 2004-02-12 National Agriculture & Bio-Oriented Research Organization New lactobacillus strain, microbial additive containing the same and method for preparing rice plant-fermented crude feed using the additive
CN104073454A (en) * 2014-06-18 2014-10-01 兰州大学 Low-temperature-resistant lactobacillus planetarium and application thereof
CN105524867A (en) * 2016-01-08 2016-04-27 中国农业大学 Lactobacillus plantarum and application thereof in native grass silage
CN106148249A (en) * 2016-09-12 2016-11-23 江苏省农业科学院 A kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof
CN108570421A (en) * 2017-03-09 2018-09-25 北京大北农科技集团股份有限公司 One lactobacillus plantarum strain and its ensilage fermentation agent

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004041064A (en) * 2002-07-11 2004-02-12 National Agriculture & Bio-Oriented Research Organization New lactobacillus strain, microbial additive containing the same and method for preparing rice plant-fermented crude feed using the additive
CN104073454A (en) * 2014-06-18 2014-10-01 兰州大学 Low-temperature-resistant lactobacillus planetarium and application thereof
CN105524867A (en) * 2016-01-08 2016-04-27 中国农业大学 Lactobacillus plantarum and application thereof in native grass silage
CN106148249A (en) * 2016-09-12 2016-11-23 江苏省农业科学院 A kind of lactic acid bacteria microbial inoculum being applicable to grass silage and application thereof
CN108570421A (en) * 2017-03-09 2018-09-25 北京大北农科技集团股份有限公司 One lactobacillus plantarum strain and its ensilage fermentation agent

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Effect of re-ensiling on the quality of sorghum silage;G. V. S. dos Anjos et al.;Journal of dairy science;第101卷(第7期);摘要，第1页右栏第2段至第7页左栏第3段 *
乳酸菌的分离鉴定及其在玉米秸秆青贮中的应用;张大伟;中国优秀硕士学位论文全文数据库 农业科技辑(第4期);摘要，第1页第1段至第40页第3段，表2-2至表2-3，图2-1至图2-4 *
袁嘉丽 等.微生物学与免疫学.中国中医药出版社,2017,194. *

Also Published As

Publication number Publication date
CN113736710A (en) 2021-12-03

Similar Documents

Publication Publication Date Title
CN102851233B (en) Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof
CN102640885B (en) Applications of Lactobacillus plantarum in complete-strain Triticumspp silage
CN102864095A (en) Lactobacillus plantarum used for silage alfalfa and use method thereof
CN102851232B (en) Lactobacillus strain lactobacillus parafarraginis ZH1 and application thereof
CN107034157A (en) A kind of Lactobacillus plantarum and its application
CN110791460B (en) Strain for inhibiting growth of aspergillus flavus and generation of toxin
CN113969243A (en) Lactobacillus plantarum capable of degrading tannin and application thereof
CN113073064B (en) Bacterial strains capable of efficiently degrading cellulose, silage bacterial agent and application of silage bacterial agent
CN111575213A (en) Microbial compound bacterium agent and application thereof in preparation of fermented feed
CN114921375B (en) Bacillus capable of producing cellulase at high yield and application thereof
CN113151032A (en) Bacillus subtilis with efficient gossypol degradation capability and application thereof
CN111676170B (en) Lactobacillus fermentum and application thereof in preparation of conjugated fatty acid
CN113736710B (en) Oxygen-resistant lactic acid bacteria and application thereof
CN112877238A (en) Low-temperature-resistant pediococcus pentosaceus OL77 and application thereof
CN115418337B (en) Lignin degrading bacterium and application thereof in rice straw micro-storage
CN108060102A (en) A kind of cibarium Wei Si Salmonellas and its application, screening calibration method
CN102640886B (en) Applications of Lactobacillus plantarum in Brassica oleracea outer wrapper leaf silage
CN113755383B (en) Low-nutrition-resistant lactic acid bacteria and application thereof
CN113308418B (en) Lactobacillus chaff for fermentation and fermentation preparation process thereof
CN111557377B (en) Method for preparing fruit and vegetable waste fermented feed
CN111040969B (en) Compound lactobacillus agent and application thereof in buffalo silage
CN111925972A (en) Lactobacillus hilgardii and application thereof
CN111557378A (en) Method for preparing fermented feed by using bifidobacterium longum
CN117210365B (en) Bacillus bailii and application thereof in improving digestion and antioxidation capability
CN113943667B (en) Lactobacillus plantarum isolated from camel rumen and application of lactobacillus plantarum in silage

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant