CN110835607B - Low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae and application thereof - Google Patents
Low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae and application thereof Download PDFInfo
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Abstract
The invention provides a low-temperature efficient degradation straw composite bacterium containing saccharomyces cerevisiae, which is prepared from 6 strains in an outdoor environment with the temperature of 1-6 ℃ to improve the decomposition days of sorghum straw by 2-3 days, and is particularly suitable for northern areas. The addition of a large amount of saccharomyces cerevisiae can reduce the odor in the degradation and decomposition process, the composite microbial inoculum is added into sorghum straw for fermentation to prepare feed, the fermented straw is soft in texture and has wine fragrance, the palatability is obviously improved, compared with untreated straw, the general feeding speed can be improved by 47%, the feeding amount can be increased by 21%, the digestion rate of cattle can be averagely improved by 60%, the daily weight gain is averagely improved by 32%, and as the cellulose and lignin after fermentation are partially degraded, rumen microorganisms can be fully contacted with straw fibers, and the conversion rate of the feed is increased.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a low-temperature efficient degradation straw composite bacterium containing saccharomyces cerevisiae and application thereof.
Background
The main utilization parts of the sorghum are grains, rice bran, stalks and the like. Wherein the main nutrient content in the seed particles is as follows: 3% of crude fat, 8-11% of crude protein, 2-3% of crude fiber and 65-70% of starch. The sorghum protein is slightly higher than corn, the quality is poor, the content of the protein in grains is generally 9-11%, lysine and tryptophan are absent, the protein digestibility is low, and the reason is that the intermolecular crosslinking of the sorghum protein is more, and strong bonding bonds exist between the protein and starch, so that the enzyme is difficult to enter into decomposition. The fat content is 3%, which is slightly lower than that of corn, and the saturated fatty acid in the fatty acid is slightly higher, so that the melting point of the fat is slightly higher. The nitrogen-free extract comprises starch and sugar, is a main component in the sorghum for feeding and is also a main energy source for livestock and poultry, and the content of the nitrogen-free extract in the sorghum for feeding is changed between 17.4 and 71.2 percent. The coarse fibers in the sorghum stalks and the sorghum shells are more, and the content of the coarse fibers is about 23.8 percent and 26.4 percent respectively. Starch content is comparable to corn, but sorghum starch particles are highly covered by protein, so that the digestibility of starch is lower than that of corn, and the effective energy value is equivalent to 90-95% of that of corn. The nutritional value of the sorghum stalks and the sorghum hulls is lower than that of the concentrate, but the sources are more, the price is low, and the raising cost can be reduced.
Because the air temperature in the north is very low for a long time, the sorghum straw is not easy to decompose in a short time, so that the sorghum straw resource is not reasonably developed for a long time, most of the sorghum straw is burnt as a living fuel except a small amount of the sorghum straw which is used for washers, livestock feeding and composting, and a small amount of the sorghum straw is also provided with a small amount of lignin, and the property is very stable at normal temperature, so the degradation is very good
Slow. The existing straw decomposing inoculants in China are many, but most of the straw decomposing inoculants are poor in straw degradation effect due to the limitation of the ambient temperature or the growth speed of thalli, and the decomposing utilization of microbial degradation straw is limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the low-temperature efficient degradation straw composite bacteria containing saccharomyces cerevisiae, which are prepared by mixing 6 strains domesticated at low temperature according to a certain mass ratio, and are particularly suitable for northern low-temperature weather and excellent in straw decomposition effect.
The invention aims at realizing the following technical scheme:
a low-temperature efficient degradation straw composite bacterium containing saccharomyces cerevisiae is characterized in that: the composite bacterial agent contains 6 strains, and is mixed according to a certain mass ratio, and contains 20 percent of cellulose-producing paenibacillus, 20 percent of xylanase-producing alternaria, 20 percent of laccase-producing verrucaria verrucosa, 10 percent of lignin peroxidase-producing bacillus mucilaginosus, 10 percent of manganese peroxide enzyme-producing actinomyces and 20 percent of saccharomyces cerevisiae,
the bacillus sp (Bacillus cellulosilyticus) for producing the cellulase is CGMCC 1.15312;
the xylanase-producing alternaria alternata (Alternaria humicola) CGMCC 3.2917;
the lignin peroxidase producing bacillus mucilaginosus (Bacillus mucilaginosus Krassilnikov) GIM 1.16;
said manganese peroxidase producing actinomycetes (Laceyella tengchongensis)
CCTCC AA 208050Laceyella；
The saccharomyces cerevisiae (Saccharomyces cerevisiae) is CGMCC 2.1366;
the strain can be purchased from China general microbiological culture collection center (CGMCC), china Center for Type Culture Collection (CCTCC) and Guangdong province microbiological collection center (GIM).
The laccase-producing verrucaria verrucosa is (Myrothecium verrucaria) mutant strain T2901, which is obtained by separating, screening and mutating a soil sample collected from Changbai mountain in the laboratory, and is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation date is 7 months and 5 days in 2017, the preservation unit address is university of Lopa nationality at Wu Shiwu Chang area of Hubei province in China, and the preservation number is CCTCC NO: M2017413.
A preparation method of a low-temperature efficient degradation straw composite bacterium containing saccharomyces cerevisiae comprises the following steps:
activating the above bacteria in conventional manner, and culturing until the number of viable bacteria in the bacterial liquid reaches 2.0X10 8 Individual/mL. The activated medium is PDA medium: 200g/L of potato, 20g/L of glucose and 15g/L of agar; mixing bacterial liquid according to the following mass ratio: 20% of cellulose-producing paenibacillus, 20% of xylanase-producing alternaria, 20% of laccase wart verrucaria, 10% of lignin peroxidase-producing bacillus mucilaginosus, 10% of manganese peroxidase-producing actinomyces and 20% of saccharomyces cerevisiae are fully mixed to obtain the composite microbial inoculum.
The application of the composite bacteria in degrading straw.
The beneficial effects obtained by the invention are as follows:
the composite bacteria for efficiently degrading the straw at low temperature, which is provided by the invention, is composed of 6 strains, improves the decomposing days of the sorghum straw by 2-3 days in the outdoor temperature of 1-6 ℃, and is particularly suitable for northern areas. Meanwhile, the composite microbial inoculum can also be applied to the addition of sorghum straw feed for pre-fermentation treatment, the addition of a large amount of saccharomyces cerevisiae can reduce odor in the degradation and decomposition process, the composite microbial inoculum is added into the sorghum straw for fermentation to prepare the feed, the fermented straw is soft in texture and has wine fragrance, the palatability is obviously improved, compared with untreated straw, the general feeding speed can be improved by 47%, the feeding amount can be increased by 21%, the digestion rate of cattle can be averagely improved by 60%, the daily weight increase is averagely improved by 32%, and as the cellulose and lignin after fermentation are partially degraded, rumen microorganisms can be fully contacted with straw fibers, and the conversion rate of the feed is increased.
EXAMPLE 1 mutagenesis screening of Verticillium verrucosum
(1) Morphological identification and screening
Collecting soil in a forest region of a natural protection region of a Changbai mountain, culturing the soil on a PDA culture medium by a conventional method, wherein bacterial strain hypha is initially white flocculent, grows outwards Zhou Fasan, has a bacterial colony which is approximately irregularly round, and has conidiophores after growing for 5 days on a flat plate, wherein the conidiophores are initially dark green, and after culturing for 8 days, the color of the conidiophores is continuously deepened, and a collagen cluster appears; after 10d of culture, the colony is concentric ring, the conidial gum is black, and the back of the colony is light brown and radial fold.
And (3) a bacterial block with the diameter of 1cm is picked up by a puncher, inoculated onto an aniline blue selection medium, cultured for 10d at the temperature of 30 ℃ in an aerobic incubator, observed to fade the aniline blue, and selected as a candidate bacterial strain with high fading rate and strong capability.
Meanwhile, in order to directionally screen strains degrading lignin, a liquid fermentation medium with lignin as a unique carbon source is designed as a culture medium, the lignin removal rate is determined through the limitation of the single carbon source, strains with good rise in the lignin culture medium and a certain lignin removal capacity are selected, and a subsequent pretreatment test of sorghum straw is carried out on the strains.
(2) ITS sequence amplification, sequence determination and molecular classification of strains
After the stable characters are confirmed, the strain is sent to a biological engineering (Shanghai) stock company for sequencing, and the strain is identified as the verrucaria verrucosa T2901.
(3) Mutagenesis and screening of strains
The mutant strain is mainly prepared by adopting an atmospheric pressure room temperature plasma mutagenesis (ARTP) method, and spore suspension is prepared by using the verrucaria verrucosa T2901. Then diluting to adjust the spore concentration to 10 7 And (3) per mL, 10 mu L of diluted bacterial suspension is dripped on an ARTP slide, mutagenesis is carried out, and the ARTP mutagenesis breeding system is used for irradiating with a 4mm range, wherein the optimal mutagenesis time is 75s.
The diluted bacterial suspension is evenly coated on a guaiacol selective culture medium (the concentration of the guaiacol is 0.4 percent and the guaiacol is added into a PDA culture medium), and the culture is carried out for 10 days in an aerobic incubator at the temperature of 30 ℃, the condition of a color-changing ring is observed, and a large strain of the color-changing ring is selected as a candidate strain. The laccase activity of the forward mutant strain T2901 is improved by about 50% compared with that of a wild strain, and the strain T2901 can be stably inherited after multiple subcultures.
Example 2 Low temperature domestication of strains
(1) Enrichment culture of strains
Inoculating the strain into a triangular flask containing 150mL PDA culture medium, adding filter paper sheets with length of 5cm and width of 2cm, shake culturing at 30deg.C for 1 hr, mixing, and collecting supernatant to obtain 10 -1 -10 -9 Diluting, inoculating 1mL of each diluted solution into PDA culture medium with filter paper as the only carbon source, and standing and enriching at 28 ℃ for 4 generations. Screening out the materials with short breaking time and high ulceration degree of the filter paper. PDA medium: potato 200g/L, glucose 20g/L, agar 15g/L, and enrichment culture for 4 generations.
(2) Primary screening and domestication of low-temperature degradation cellulose composite bacterial system
And (3) inoculating the enrichment culture into a filter paper strip culture medium to domesticate a low-temperature cellulose decomposition bacterial system by adopting a filter paper disintegration method, performing secondary culture for 4 generations at the initial culture temperature of 28 ℃, and selecting a culture with high filter paper ulcer degree, short breaking time and nearly neutral pH for continuous passage. And reducing the culture temperature by 2 ℃ every time of subculture until the culture temperature is reduced to 4 ℃, judging the cellulose decomposition capacity of the culture by observing the fracture condition of the filter paper, selecting the culture with better degradation capacity while carrying out passage, continuously culturing for 8 generations under the temperature condition of 4 ℃ in the domestication process, and stopping low-temperature domestication when the fracture time of the filter paper strip is consistent.
After preliminary low-temperature domestication, the result shows that the obtained 6 strains grow well under the condition of 4 ℃, which indicates that the strain can grow normally under the low-temperature condition.
The 6 strains are as follows: paenibacillus, alternaria, myrothecium, bacillus mucilaginosus, actinobacillus, saccharomyces cerevisiae,
the bacillus sp (Bacillus cellulosilyticus) for producing the cellulase is CGMCC 1.15312;
the xylanase-producing alternaria alternata (Alternaria humicola) CGMCC 3.2917;
the lignin peroxidase producing bacillus mucilaginosus (Bacillus mucilaginosus Krassilnikov) GIM 1.16;
the manganese peroxidase producing actinomycetes (Laceyella tengchongensis) CCTCC AA 208050Laceyella; the saccharomyces cerevisiae (Saccharomyces cerevisiae) is CGMCC 2.1366;
the strain can be purchased from China general microbiological culture collection center (CGMCC), china Center for Type Culture Collection (CCTCC) and Guangdong province microbiological collection center (GIM).
The laccase-producing verrucaria verrucosa is (Myrothecium verrucaria) mutant strain T2901, is obtained by separating, screening and mutating a soil sample collected from Changbai mountain in the laboratory, and is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation date is 2017, 7, 5 and the preservation number is CCTCC NO: M2017413.
Example 3 preparation of Complex microbial inoculant
Activating Paenibacillus, alternaria xylan, paecilomyces laccase and Bacillus mucilaginosus, producing lignin peroxidase, producing manganese peroxidase, culturing Saccharomyces cerevisiae in conventional manner until the number of viable bacteria in bacterial liquid reaches 2.0X10 8 And each mL.
Mixing bacterial liquid according to the following mass ratio: 20% of cellulose-producing paenibacillus, 20% of xylanase-producing alternaria, 20% of laccase wart verrucaria, 10% of lignin peroxidase-producing bacillus mucilaginosus, 10% of manganese peroxidase-producing actinomyces and 20% of saccharomyces cerevisiae, and fully stirring and mixing to obtain mixed bacterial liquid.
Example 4 degradation of straw
The experimental place is selected in a Jilin agricultural large test field in Jilin province, the day and night temperature is 1-6 ℃, the sorghum straw is paved in the test field after being harvested, 1.5kg of the microbial inoculum prepared in the example 1 is applied to the test field per mu, the composite microbial inoculum is not used for straw fermentation in a control group, then the water is filled, straw decomposition is observed, and the change condition is changed:
TABLE 1 straw decomposition conditions for different treatments
The result shows that compared with the method without adding the composite microbial inoculum, the composite microbial inoculum can advance the degradation and decomposition time of the straws to 8-10 days, can effectively decompose the straws even in the low-temperature weather of 1-6 ℃, and meanwhile, the odor of the compost can be reduced by adding a large amount of saccharomycetes.
Example 5 composite bacteria fermented sorghum straw feed
Crushing sorghum straw, cutting the crushed sorghum straw to a length of 1-3 cm, mixing the crushed sorghum straw with the composite bacterial liquid of the embodiment 3 according to a mass ratio of 200:1, and stirring for 10min, wherein the composite bacterial is not added in a control group. And (3) packaging the straws mixed with the fermentation broth, sealing the straws, and fermenting the straws in a constant-temperature solid fermentation workshop. After 3-5 days, the fermented straw feed has bouquet smell, which indicates that the straw feed is successfully fermented, and can be used for feeding cattle and sheep. Production practice shows that the digestibility of cattle after the sorghum straw is fermented is improved by 60%, daily gain is improved by 32%, and the fermented straw is soft in texture and has wine fragrance, so that the palatability is obviously improved, and the appetite of livestock is increased. Compared with untreated straw, the general feeding speed can be increased by 47%, the feeding capacity can be increased by 21%, and as the fermented cellulose and lignin are partially degraded, rumen microorganisms can be fully contacted with straw fibers, so that the conversion rate of the feed is increased.
Claims (1)
1. A low-temperature efficient degradation straw composite bacterium containing saccharomyces cerevisiae is characterized in that: the composite microbial inoculum comprises 6 strains, wherein bacterial solutions are mixed according to the following mass ratio, and the bacterial solutions comprise 20% of cellulose-producing paenibacillus, 20% of xylanase-producing alternaria, 20% of laccase-producing verrucaria verrucosa, 10% of lignin peroxidase-producing bacillus mucilaginosus, 10% of manganese peroxidase-producing actinomycetes and 20% of saccharomyces cerevisiae;
the bacillus sp (Bacillus cellulosilyticus) for producing the cellulase is CGMCC 1.15312;
the xylanase-producing alternaria alternata (Alternaria humicola) CGMCC 3.2917;
the lignin peroxidase producing bacillus mucilaginosus (Bacillus mucilaginosus Krassilnikov) GIM 1.16;
the manganese peroxidase producing actinomycetes (Laceyella tengchongensis) CCTCC AA 208050Laceyella;
the saccharomyces cerevisiae (Saccharomyces cerevisiae) is CGMCC 2.1366;
the laccase-producing verrucaria verrucosa (Myrothecium verrucaria) mutant strain T2901 has a preservation number of CCTCC NO: M2017413.
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| US5747029A (en) * | 1995-08-07 | 1998-05-05 | Walker; Harrell L. | Control of weeds with a fungal pathogen derived from M. verrucaria |
| CN103710282A (en) * | 2013-12-03 | 2014-04-09 | 福州农博士生物技术有限公司 | Straw decomposing agent for quick start of decomposition at low temperature and its preparation method |
| CN107828667A (en) * | 2017-11-27 | 2018-03-23 | 吉林农业大学 | A kind of myrothecium verrucaria mutant strain T2901 and its application |
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Patent Citations (4)
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|---|---|---|---|---|
| US5747029A (en) * | 1995-08-07 | 1998-05-05 | Walker; Harrell L. | Control of weeds with a fungal pathogen derived from M. verrucaria |
| CN103710282A (en) * | 2013-12-03 | 2014-04-09 | 福州农博士生物技术有限公司 | Straw decomposing agent for quick start of decomposition at low temperature and its preparation method |
| CN106701621A (en) * | 2013-12-03 | 2017-05-24 | 福州农博士生物技术有限公司 | Straw decomposing additive for decomposition in low-temperature state and preparation method of straw decomposing additive |
| CN107828667A (en) * | 2017-11-27 | 2018-03-23 | 吉林农业大学 | A kind of myrothecium verrucaria mutant strain T2901 and its application |
Non-Patent Citations (1)
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