CN111165653B - Potato residue and corn stalk mixture fermented feed, preparation method thereof and starter - Google Patents
Potato residue and corn stalk mixture fermented feed, preparation method thereof and starter Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/35—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from potatoes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Mycology (AREA)
- Sustainable Development (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
The invention discloses a potato residue and corn stalk mixture fermented feed, a preparation method thereof and a starter, wherein the starter comprises lactobacillus casei (Lactobacillus casei), bacillus subtilis (Bacillus subtilis) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and the starter can degrade crude fibers in potato residue and corn stalk at the temperature of 8-15 ℃, and also discloses a method for preparing the potato residue and corn stalk mixture fermented feed by using the starter and the obtained fermented feed. The invention can effectively utilize the abandoned potato residue and corn straw, reduce environmental pollution and improve the digestibility of the potato residue corn straw substrate feed.
Description
Technical Field
The invention relates to the technical field of microbial fermented feed, in particular to potato residue and corn stalk mixture fermented feed, a preparation method thereof and a lactic acid bacteria compound group starter used in the same.
Background
Corn stalks are one of the most abundant crop resources, and about 7 hundred million tons of corn stalks are harvested each year in China. Potatoes are the fourth largest grain crop in the world, with yields inferior to wheat, rice and maize. According to statistics of grain and agriculture organizations of united countries, the potato planting area of China exceeds 530 ten thousand hm 2 Annual yield has broken through 8000 ten thousand tons, and potato planting area and total yield all jump to the world's front. The annual straw amount of the annual resource utilization of China is less than 60 percent of the annual yield. The in-situ incineration treatment of the straw causes serious environmental problems and huge biomass resource waste.Meanwhile, 9 tons of starch residues are generated when 1 ton of potatoes are processed, the potato starch residues generated by potato starch production per year in China are about 72000 tons, basically no harmless treatment is carried out, and the potato starch residues are directly discarded, so that serious pollution is caused. Therefore, a technology capable of recycling straw and potato residues is developed, and the method has great significance in treating environmental pollution and promoting the development of related industries. The animal feed is produced by mixing and fermenting potato residues and corn stalks, and is one of the feasible technical approaches.
The traditional production process of the fermented feed is mature, but the fermentation is often required to be carried out in seasons with higher temperature due to the limitation of the growth conditions of the fermentation strain, and especially the rapid growth and propagation of most lactic acid bacteria are required to be carried out at about 20 ℃. However, in northern areas of China, corn and potato are harvested and processed in 9-10 months, the temperature difference between day and night is large, and the average temperature is reduced to below 15 ℃, so that the harvested corn stalks and potato residues are difficult to store and transport, difficult to fully utilize and difficult to convert into feed in a fermentation mode under a low-temperature condition. The method becomes a main limiting factor for producing the feed by mixing and fermenting the corn stalks and the potato residues in the north of China.
The corn stalk and potato residue contain a large amount of crude fibers, so that the corn stalk and potato residue are not beneficial to digestion and absorption of animals, and the characteristic of the corn stalk and potato residue is changed in a fermentation mode by means of strains degrading the crude fibers.
Disclosure of Invention
In view of the above, the present invention aims to provide a lactic acid bacteria complex group starter for degrading crude fibers in corn stalks and potato residues at low temperature, a method for preparing fermented feed from a mixture of potato residues and corn stalks by using the starter mainly using waste potato residues and corn stalks as base materials, and the fermented feed obtained thereby, so as to solve the technical problems that the corn stalks and potato residues are difficult to utilize and convert into fermented feed at low temperature in northern China.
The object of the present invention and the solution of the technical problems thereof can be achieved by the following technical solutions.
In a first aspect, the invention provides a lactic acid bacteria complex starter comprising lactobacillus casei (Lactobacillus casei), bacillus subtilis (Bacillus subtilis) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens), wherein the complex starter can degrade crude fibers in potato residues and corn stalks at a low temperature of 7-15 ℃.
In a second aspect, the invention provides a method for preparing a potato residue corn stalk mixture fermented feed by using the lactobacillus complex group starter, which comprises the following steps:
1) Activating and expanding the lactobacillus compound group ferment to obtain an expanding culture liquid,
2) Adding adjuvants into base material containing potato residue and crushed corn stalk to form a mixture, regulating water content of the mixture to 50% -60%, making into solid fermentation culture medium, and
3) Inoculating 1% -4% of the lactobacillus compound group starter culture expansion liquid into the solid state fermentation culture medium based on the weight of the solid state fermentation culture medium, and performing sealed fermentation.
In a third aspect, the invention provides a fermented feed of a potato residue and corn stalk mixture prepared by the method, which comprises a starter of the lactic acid bacteria complex group, a base material comprising potato residue and corn stalk and auxiliary materials.
Compared with the prior art, the invention has obvious beneficial technical effects. According to the technical scheme, the beneficial technical effects of the invention at least comprise:
in autumn, a great deal of abandoned corn stalks and potato residues containing sufficient nutrients exist in the north of China, so the raw materials of the invention have low cost and are easy to obtain.
The harvested corn stalks and potato starch residues are fed, so that the problem of insufficient feed can be solved, the biomass is fully utilized to create economic benefits, the income of farmers is increased, and the development of circulating economy and animal husbandry is realized.
The strain in the lactobacillus composite group starter is low in cost, easy to obtain and low in requirements on survival conditions.
The corn straw and potato starch residue mixture is fermented by the lactobacillus composite group starter, so that adverse reactions caused by pathogenic bacteria, mould and the like in the mixture after livestock ingestion are avoided, and the problems that the corn straw and potato starch residue are difficult to transport, store, ferment, recycle and the like due to the temperature conditions in the northern cold region are also avoided.
Compared with other treatment methods of corn stalks and potato starch residues, the method can directly use the potato residues after starch is extracted, so that pretreatment steps such as airing the potato residues are omitted, the pretreatment related cost is greatly reduced, and the time required by feed production is saved.
The method is more suitable for fermenting the corn stalks and the potato residues in northern cold areas, and the content of crude fibers in the corn stalks and the potato residues is reduced by the composite addition of the crude fiber degrading bacteria, so that the problem of lower digestibility of animals after eating the potato residue corn stalk substrate feed is solved. The lactic acid produced after fermentation of the lactic acid bacteria can inhibit the growth of harmful bacteria, and can increase the flavor of the feed and improve the palatability and the feed intake of animals.
Detailed Description
The technical solution of the present invention will be clearly and completely described in conjunction with the specific embodiments, but it should be understood by those skilled in the art that the embodiments described below are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on the embodiments of the present invention, are within the scope of the present invention.
Corn stover and potato residues contain a large amount of crude fiber. Crude fiber is the main component of plant cell wall, including cellulose, hemicellulose, lignin, cutin, etc. The food containing the crude fiber can promote gastrointestinal movement and help digestion to a certain extent, but the crude fiber can also prevent digestive enzymes in the digestive tract from contacting chyme so as to reduce the digestibility of nutrients, and can prevent the intestinal tract from absorbing some small molecular nutrient substances.
Potato residue is the residue of potato after squeezing starch, and has a water content of 84.78%, and has a soluble sugar content of 15.53g/kg, and contains abundant nutrients such as cellulose, hemicellulose, pectin, and free amino acids. The pH of the corn stalk is 6.1, and the dry matter content is 90%. Both contain large amounts of crude fiber which is detrimental to digestion and absorption by animals, and therefore it is necessary to modify this characteristic of corn stover and potato residues by fermentation with the aid of crude fiber degrading species.
Accordingly, in a first aspect the present invention provides a lactic acid bacteria complex starter comprising lactobacillus casei (Lactobacillus casei), bacillus subtilis (Bacillus subtilis) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the starter being capable of degrading potato residues and crude fibres in corn stover at low temperatures of 7-15 ℃.
The inventor surprisingly found that lactobacillus casei, bacillus subtilis and bacillus amyloliquefaciens composite group can rapidly consume oxygen in potato residue and corn straw base materials at low temperature to generate a low-oxygen environment, and then degrade crude fibers in feed under the action of cellulose degrading enzyme system synthesized by self metabolism and antibacterial substances such as subtilisin, polymyxin, nystatin, gramicidin and the like to reduce the content of harmful bacteria in the feed, and can also utilize reducing sugar generated after the crude fibers are decomposed to improve the acid production rate of lactobacillus casei and rapidly reduce the pH value in the fermentation process.
In a specific embodiment of the first aspect of the invention, the ratio of the number of viable bacteria of Lactobacillus casei, bacillus subtilis and Bacillus amyloliquefaciens is (3-5): 1-2, preferably 2:1:1.
In a specific embodiment of the first aspect of the invention, the lactobacillus casei is lactobacillus casei cctccc AB 2013130 strain, the bacillus subtilis is bacillus subtilis cctccc AB 130002 strain, and the bacillus amyloliquefaciens is bacillus amyloliquefaciens cctccc AB 205140 strain, all purchased from the chinese collection of typical cultures (cctccc).
The second aspect of the invention provides a method for preparing potato residue and corn stalk mixture fermented feed by using the lactobacillus complex group starter, which comprises the following steps:
1) Activating and expanding the lactobacillus compound group ferment to obtain an expanding culture liquid,
2) Adding adjuvants into base material containing potato residue and crushed corn stalk to form a mixture, regulating water content of the mixture to 50% -60%, making into solid fermentation culture medium, and
3) Inoculating 1% -4% of the lactobacillus compound group starter culture expansion liquid into the solid state fermentation culture medium based on the weight of the solid state fermentation culture medium, and performing sealed fermentation.
In an embodiment of the second aspect of the invention, the strain-activated propagation described in step 1) is well known to the person skilled in the art. For lactobacillus casei, bacillus subtilis and bacillus amyloliquefaciens of the invention, the activation and propagation of the strain can be performed in the following manner: activating lactobacillus casei with MRS culture medium, and performing expansion culture with the culture medium to obtain an expansion culture solution; activating bacillus subtilis and bacillus amyloliquefaciens respectively by using LB culture medium, and performing expansion culture respectively by using the culture medium to obtain the expansion culture solution.
In one embodiment, in step 1), the activating and expanding culture specifically comprises: activating low Wen Ganlao lactobacillus with MRS culture medium, and performing expansion culture by using the MRS culture medium, wherein the culture conditions are as follows: standing and culturing for 7d at 8-12 ℃ to obtain a culture expanding liquid; activating bacillus subtilis and bacillus amyloliquefaciens respectively by using LB culture media, and performing expansion culture by using the LB culture media respectively, wherein the culture conditions are as follows: shake culturing at 12deg.C for 7d at 150r/min to obtain the culture expanding liquid. In one embodiment, the bacterial concentration of each of the culture broths can be controlled to be 10 in order to facilitate the operation of the culture at a predetermined ratio during inoculation 10 cfu/g。
In an embodiment of the second aspect of the invention, in the substrate of step 2), the ratio of potato pulp to corn stover is between 1:2 and 1:4. In particular embodiments, the ratio of potato residue to corn stover can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, as well as any direct ratio, such as 1:2.4, 1:2.6, etc., preferably 1:3.
The length of the corn straw has a significant effect on fermentation. The shorter the length of the corn straw is, the larger the relative contact area between the fermentation strain and the straw is, the crude fiber degrading enzyme system can act on the cell wall components of the matrix more fully, so that the crude fiber content is reduced better. Thus, in an embodiment of the second aspect of the invention, the crushed corn stover has a length of less than or equal to 6cm. In particular embodiments, the length of the crushed corn stover can be less than or equal to 5.5cm, 5.0cm, 4.5cm, 4.0cm, 3.5cm, 3.0cm, 2.5cm, 2.0cm, 1.5cm, 1.0cm, 0.5cm, or even lower. The grain size of the potato after starch is squeezed to form residues is not uniform, and is usually far smaller than the length size of the crushed corn straw.
In a specific embodiment of the second aspect of the invention, the auxiliary materials are wheat bran, corn flour and urea, and the content of the auxiliary materials is 0.5-3% of the weight of the mixture respectively. In particular embodiments, the wheat bran, corn meal, urea may each be present in an amount of 0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, preferably 1% by weight of the mixture.
In the embodiment of the second aspect of the invention, the auxiliary materials are added first and then the water content is regulated, so that the water content is ensured to be about 50% -60%, the growth of the fermentation strain and the degradation effect of the crude fiber can be influenced if the water content is higher or lower than the water content, and the growth of the composite strain can be inhibited when the water content is higher than 60% in batch fermentation, so that other microorganisms become dominant strains. Thus, in particular embodiments of the second aspect of the invention, the moisture content may be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, and values between any two of the foregoing, for example 50.5%, 51.5%, 51.8%, etc. The too high or too low inoculum size of the culture-expanding liquid can influence the fermentation rate of the feed and the pathogenic bacteria content in the feed, and the proper inoculum size of the culture-expanding liquid can shorten the delay period of strain growth, so that bacillus subtilis and bacillus amyloliquefaciens can quickly synthesize cellulose degrading enzyme systems, and lactobacillus casei can quickly produce acid to reduce the pH of the culture environment. However, too high inoculation amount can cause too fast consumption of nutrient substances in the solid state fermentation medium, influence various normal life activities in the later period of the strain, and meanwhile, metabolites produced by the strain can be increased in a short period, so that the feed fermentation rate is reduced. Therefore, the inoculation amount of the strain expansion culture solution is preferably controlled to be 1-4% of the weight of the solid fermentation culture medium, and the ratio of the viable count of lactobacillus casei, bacillus subtilis and bacillus amyloliquefaciens is (3-5): 1-2. In specific embodiments, the inoculum size of the seed expansion medium may be 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0% by weight of the solid state fermentation medium, and any value therebetween, such as 1.4%, 1.6%, 1.8%, 2.2%, 2.8%, 3.7%, 4.8%, etc., and the ratio of the viable count of lactobacillus casei, bacillus subtilis, bacillus amyloliquefaciens is 3:2:1, 2:1:1, 3:2:2, 4:2:1, 5:2:1, 5:2:2, etc., preferably the inoculum size of the seed expansion medium is 3% by weight of the solid state fermentation medium, and the ratio of the viable count of lactobacillus casei, bacillus subtilis, bacillus amyloliquefaciens is 2:1:1.
In an embodiment of the second aspect of the invention, the sealed fermentation is carried out at a temperature of from 7 ℃ to 15 ℃ for at least 10 days, for example at least about 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, typically at least about 30 days. Therefore, the method is more suitable for fermenting potato residues and corn stalks in autumn and night in northern cold regions when the temperature difference between autumn and night is large.
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The strains used in the following examples are all purchased from China Center for Type Culture Collection (CCTCC), specifically, the lactobacillus casei is lactobacillus casei CCTCC AB 2013130 strain, the bacillus subtilis is bacillus subtilis CCTCC AB 130002 strain, and the bacillus amyloliquefaciens is bacillus amyloliquefaciens CCTCC AB 205140 strain.
Example 1
Strain activation and culture
Activating low Wen Ganlao Lactobacillus with MRS culture medium, performing amplification culture with the culture medium at 10deg.C for 7d under standing condition, and controlling bacterial concentration of bacterial liquid at 10 10 cfu/g; activating bacillus subtilis and bacillus amyloliquefaciens respectively with LB culture medium, performing amplification culture with the culture medium respectively, shake culturing at 12 deg.C and 150r/min for 7d, and controlling bacterial concentration of bacterial liquid at 10 10 cfu/g。
Solid state fermentation medium
The air-dried corn stalks are crushed by a crusher and then pass through a 1cm sieve, 45g of crushed corn stalks are mixed with 22.5g of potato residues, 0.85g of bran, 0.85g of corn flour and 0.85g of urea are added, the water content is adjusted to be 50%, the mixture is filled into a 1L screw bottle for compaction, and the mixture is sterilized at 121 ℃ for 15min.
Inoculating and fermenting technological parameters
Based on the weight of the solid fermentation medium, 3 percent of mixed bacteria-expanding liquid (the ratio of lactobacillus casei, bacillus subtilis and bacillus amyloliquefaciens is 3:1:1) is inoculated, and the mixture is fermented for 15 days at the temperature of 10 ℃.
Fermentation results
The pH value of the corn stalk and potato residue after fermentation of the composite microbial inoculum is reduced from 7.1 to 5.3, the degradation rate of crude fiber reaches 13.1% as measured by a normal form washing fiber measuring method, and the crude protein content is increased from 4.1% to 6.5% before fermentation as measured by a Kjeldahl nitrogen method.
Example 2
Activating and culturing strains: same as in example 1
Solid state fermentation medium: about 60 jin of harvested corn stalks are crushed by a silk kneading machine, the length of the crushed corn stalks is generally less than 6cm, the crushed corn stalks are uniformly mixed with 15 jin of potato residues, and about 1 jin of bran, 1 jin of corn flour and 1 jin of urea are added to adjust the moisture to 60 percent.
Inoculating and fermenting technological parameters
Inoculating 2% of mixed culture-expanding bacteria liquid (ratio of lactobacillus casei, bacillus subtilis and bacillus amyloliquefaciens is 5:2:2) based on the weight of the solid fermentation culture medium, packaging with a packaging machine, wherein the number of packaging layers is 5, the packaging weight is 50 jin, and fermenting for 30d at 10 ℃.
Fermentation results
After the corn stalks and potato residues crushed by the silk kneading machine are fermented by the composite microbial inoculum for 30 days, the pH value of the corn stalks and potato residues is reduced to 4.8, the degradation rate of crude fibers reaches 17.39% as measured by a normal form washing fiber measurement method, and the crude protein content is increased to 8.7% as measured by a Kjeldahl nitrogen measurement method.
Example 3
(1) Activating and culturing strains: same as in example 1
(2) Solid state fermentation medium: the corn stalks are crushed by a silk kneading machine, 60 jin of the corn stalks are uniformly mixed with 20 jin of potato residues, and wheat bran, corn flour and urea are added to adjust the water content to about 55 jin of the corn stalks.
(3) Inoculating and fermenting technological parameters
Inoculating 4% of mixed culture-expanding bacteria liquid (the ratio of lactobacillus casei to bacillus subtilis to bacillus amyloliquefaciens is 2:1:1) according to the weight of the solid fermentation culture medium, packaging by a packaging machine, wherein the number of packaging layers is 5, the packaging weight is 50 jin, and fermenting for 30d under the outdoor environment temperature (10-20 ℃) of 10 months in Heilongjiang province.
Fermentation results
After the fermentation for 30d in the outdoor environment of 10 months in Heilongjiang, the pH of the fermented feed is reduced to 4.3, the degradation rate of crude fiber reaches 15.85% and the crude protein content is improved to 7.7% as measured by a paradigm-type washing fiber assay method.
Example 4
The experiment also researches the influence of the lactobacillus composite group starter on the microbial diversity of a fermentation system, and the specific experiment is as follows:
1. test design
1.1 materials and methods
Preparation of test group fermented feed: the method for preparing the fermented feed in example 3 was the same.
Preparation of control group feed: the corn stalks are crushed by a silk kneading machine, 60 jin of the corn stalks are uniformly mixed with 20 jin of potato residues, and the wheat bran, the corn flour and the urea are added to adjust the water content to be 55 percent after 1 jin of the corn stalks are respectively added. Inoculating 4% of MRS culture medium and LB culture medium (the ratio of MRS culture medium to LB culture medium is 1:1) according to the weight of the solid fermentation culture medium, packaging by a packer, wherein the number of layers of the packer is 5, the packing weight is 50 jin, and fermenting for 30d under the outdoor environment temperature (10-20 ℃) of 10 months in Heilongjiang province. After 30d the microbial diversity of both feeds was determined simultaneously.
1.2 sample collection of fermentation System
Samples were taken at the beginning and end of the fermentation, A1: control group fermentation initial sample, A2: test group fermentation initial sample, B1: control end-of-fermentation samples, B2: end-of-fermentation samples from the test group. After mixing the fermented samples, three bottles of samples were added 20ml Extraction Buffer each time and stored at-20 ℃ for later testing.
1.3 extraction of total DNA from fermentation samples
Taking 5g of A1, A2, B1 and B2 samples respectively, adding 50mL of sterile water, oscillating for 60min at 200r/min, taking supernatant to a 50mL centrifuge tube, centrifuging at 12000r/min for 1min, transferring the supernatant to another 50mL centrifuge tube, and centrifuging at 12000r/min for 10min to obtain thalli. The total DNA was extracted by benzyl chloride method.
1.4PCR amplification, product purification and quantification
The diluted genome DNA is used as a template, and a specific primer with Barcode and high-efficiency high-fidelity enzyme (TaKaRa, dalia) are used for PCR according to the selection of a sequencing region, so that the amplification efficiency and accuracy are ensured. Primer corresponding region: 16SV4 thin region primer 515F (5 '-GTGCCAGCMGCCGCGGTAA-3'), and
909R(5'-CCCCGYCAATTCMTTTRAGT-3')。
the PCR conditions were as follows:
PCR amplification System (25. Mu.L): 10 Xbuffer 2.5 muL；dNTP 0.5μL；MgCl 2 2μL；Primer 515F 0.25μL；
Primer 909R 0.25. Mu.L; DNA template 0.5. Mu.L; rTaq DNA polymerase 0.25. Mu.L; dd H 2 O 18.75μL；
PCR amplification conditions: denaturation at 94℃for 3min,30 cycles; annealing at 56 ℃ for 60s, and extending at 72 ℃ for 60s; finally, the extension is carried out for 1min at 72 ℃.
The PCR products were electrophoretically detected using 1% concentration agarose gel; samples with the same product concentration are mixed, after uniform mixing, agarose gel with 1% concentration is used for product detection, a target strip is recovered by using a gel recovery kit (Sangon Biotech, china, cat#SK 8132) provided by the manufacturing company, and the concentration and the quality are measured by using Nanodrop.
Table 1: bacterial changes in fermentation systems at different times
A1: fermentation initiation of the control group; a2: fermentation initiation of test group; b1: end of fermentation in control group; b2: end of fermentation in test group
It can be seen from Table 1 that the dominant bacterial groups in the control group were Yersinia pestis (Yersinia pestis), leuconostoc and enterococcus, all belonging to the harmful pathogenic bacteria, at the initial stage of fermentation. Yersinia pestis parasitic in mammals causes intestinal diseases in humans and animals. Leuconostoc is associated with serious infections and can cause bacteremia, endocarditis, and the like. Enterococcus can cause urinary tract, abdominal cavity and other infections of human and animal. In the test group, the dominant bacterial group is lactobacillus belonging to lactobacillus casei due to the inoculation of exogenous bacterial; the bacillus genus to which the bacillus subtilis and the bacillus amyloliquefaciens belong has less harmful bacteria.
At the end of fermentation, the dominant bacterial groups in the control group and the test group are lactobacillus, but a large amount of Yersinia pestis and enterococcus still exist in the control group, and the content of harmful bacteria in the test group is lower than that of the control group except for the lactobacillus, and the bacillus content of bacillus subtilis and bacillus amyloliquefaciens for degrading crude fibers is relatively higher.
Therefore, the fermented feed can effectively reduce the content of harmful bacteria in the feed, and the lactobacillus becomes dominant bacteria in a fermentation system, so that the feeding value and the nutritional value of the corn stalk and the potato residue serving as the feed are improved.
In view of the above, the fermented feed developed by the inventor breaks through the defect that the traditional fermentation process is easily affected by low-temperature conditions, realizes the feed conversion of corn stalks and potato residues which are difficult to recycle by the combined fermentation of lactobacillus casei for producing lactic acid, bacillus subtilis for degrading crude fibers and bacillus amyloliquefaciens, changes the defects of low nutritive value and difficulty in digestion of raw materials by the degradation of lactic acid produced by fermentation strains, simultaneously reduces the reproduction of harmful bacteria in the fermented feed to the greatest extent, and provides guarantee for feed safety.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. The method for preparing the potato residue and corn stalk mixture fermented feed by using the lactobacillus composite group starter comprises the following steps:
1) Activating and expanding the lactobacillus compound group ferment to obtain an expanding culture liquid,
2) Adding adjuvants into base material containing potato residue and crushed corn stalk to form a mixture, regulating water content of the mixture to 50% -60%, making into solid fermentation culture medium, and
3) Inoculating 1% -4% of lactobacillus compound group starter culture expansion liquid into the solid state fermentation culture medium based on the weight of the solid state fermentation culture medium, performing sealed fermentation,
wherein the lactobacillus composite group starter consists of lactobacillus caseiLactobacillus casei) Bacillus subtilisBacillus subtilis) Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) The composition of the microbial agent, the starter can degrade crude fibers in potato residues and corn stalks at the temperature of 7-15 ℃, the lactobacillus casei is a lactobacillus casei CCTCC AB 2013130 strain, the bacillus subtilis is a bacillus subtilis CCTCC AB 130002 strain, the bacillus amyloliquefaciens is a bacillus amyloliquefaciens CCTCC AB 205140 strain, the viable count ratio of the lactobacillus casei, the bacillus subtilis and the bacillus amyloliquefaciens is 2:1:1,
the ratio of the potato residues to the corn stalks is 1:3,
the auxiliary materials comprise wheat bran, corn flour and urea, the content of which is 0.5-3% of the weight of the mixture respectively, and
the sealed fermentation is carried out at 7-15 ℃ for at least 10 days.
2. The method according to claim 1, wherein in step 1), the activating and expanding culture specifically comprises: activating lactobacillus casei with MRS culture medium, and performing expansion culture by using the MRS culture medium, wherein the culture conditions are as follows: standing and culturing for 7d at 8-12 ℃ to obtain a culture expanding liquid; activating bacillus subtilis and bacillus amyloliquefaciens respectively by using LB culture media, and performing expansion culture by using the LB culture media respectively, wherein the culture conditions are as follows: shake culturing at 12deg.C and 150r/min for 7d to obtain culture medium.
3. The method of any of claims 1-2, wherein the length of the crushed corn stover is less than or equal to 6cm.
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