CN115553380A - Fermented feed and fermentation process thereof - Google Patents
Fermented feed and fermentation process thereof Download PDFInfo
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- CN115553380A CN115553380A CN202211255232.2A CN202211255232A CN115553380A CN 115553380 A CN115553380 A CN 115553380A CN 202211255232 A CN202211255232 A CN 202211255232A CN 115553380 A CN115553380 A CN 115553380A
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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
- A23K10/30—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
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- 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
- A23V2400/169—Plantarum
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- 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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Abstract
The invention discloses a fermented feed and a fermentation process thereof, which are mainly prepared by fermenting mixed organic residue through mixed strains, wherein the mixed strains comprise 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws in parts by weight, and the mixed strains comprise bacillus subtilis and lactobacillus plantarum; the fermentation process mainly comprises the following steps: firstly, adding bacillus subtilis seed liquid into a fermentation substrate prepared by mixing organic matter residues, and performing first-stage fermentation to obtain a first-stage fermentation product; then adding lactobacillus plantarum seed liquid into the first-stage fermentation product, and performing second-stage fermentation to obtain a second-stage fermentation product; finally, freeze-drying and crushing the second-stage fermentation product to obtain a fermented feed; according to the fermented feed and the fermentation process thereof, the animal feed with high protease is obtained through a special compound formula, and the soluble protein and small peptide with high content are obtained through the special fermentation process, so that the animal feed is beneficial to the edible absorption of animals.
Description
Technical Field
The invention belongs to the field of animal feed, and particularly relates to fermented feed and a fermentation process thereof.
Background
The fermented feed is prepared by using the fermentation of microbe in aerobic or anaerobic condition and adding proper supplementary material into low cost industrial and agricultural by-products, and through decomposition and modification, the fermented feed has high nutritive value and nutrients essential for animal and fowl growth. The feed raw materials are rich in organic acid, vitamins, free amino acids, active small peptides and the like after being subjected to microbial fermentation, are easy to digest and absorb, and have good palatability, and beneficial bacteria used for feed fermentation are beneficial to adjusting intestinal flora, so that the intestinal health of livestock and poultry is promoted, the incidence of diseases is reduced, and the breeding cost of livestock and poultry breeding is reduced.
Disclosure of Invention
The invention aims to provide a fermented feed and a fermentation process thereof, wherein the animal feed with high protease is obtained through a special compound formula, and the high-content soluble protein and small peptide are obtained through the special fermentation process, so that the animal feed is beneficial to being eaten and absorbed by animals.
The fermented feed is mainly prepared by fermenting mixed organic matter residues through mixed strains, wherein the mixed organic residues mainly comprise 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws in parts by weight, and the mixed strains comprise bacillus subtilis and lactobacillus plantarum.
Preferably, the mixed organic residue comprises 90% of soybean meal, 4% of wheat bran, 3% of ginkgo leaf residues and 3% of garlic straws.
The technical scheme of the invention is a fermentation process of fermented feed, and the preparation of the fermented feed comprises the following steps: firstly, adding bacillus subtilis seed liquid into a fermentation substrate prepared by mixing organic residues, and performing first-stage fermentation to obtain a first-stage fermentation product; then adding lactobacillus plantarum seed liquid into the first-stage fermentation product, and performing second-stage fermentation to obtain a second-stage fermentation product; and finally, freeze-drying and crushing the second-stage fermentation product to obtain the fermented feed.
Preferably, the first stage fermentation time is at least when the colony number of the bacillus subtilis in the fermentation substrate reaches the maximum value, and the second stage fermentation time is at least when the colony number of the lactobacillus plantarum in the fermentation substrate reaches the maximum value.
Preferably, the first-stage fermentation time is 24-26 h, and the second-stage fermentation time is 48-56 h.
Preferably, the preparation method of the fermentation substrate comprises the following steps: preparing an organic mixture dry material according to the mass fraction of the dry material, namely 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws, and adding water which is equal to the total amount of the organic dry material into the organic mixture dry material to obtain a fermentation substrate; the fermentation substrate is placed in a sterilization box for high-temperature sterilization at 120-125 ℃ for 15-20 min before inoculation of the bacillus subtilis, and then the bacillus subtilis is inoculated after the fermentation substrate is placed in a sterile environment and cooled to room temperature after sterilization.
Preferably, the addition ratio of the bacillus subtilis seed solution to the lactobacillus plantarum seed solution is 3:1, and the total addition amount of the bacillus subtilis seed solution and the lactobacillus plantarum seed solution is 4% of the weight of the fermentation substrate.
Preferably, the preparation method of the bacillus subtilis seed solution comprises the following steps: firstly activating the bacillus subtilis, wherein the activation method comprises the following steps: taking 100mLLB liquid culture medium, placing the medium in a 250mL conical flask, sealing, placing the conical flask in an autoclave for sterilization, taking out the conical flask after sterilization, aseptically cooling the conical flask to room temperature, inoculating the bacillus subtilis preserved by glycerol under the aseptic condition, sealing, placing the conical flask in a water bath shaker at 37 ℃ and 120r/min for culturing for 24-25 h, and obtaining activated bacillus subtilis strain after the culture is finished;
then preparing a bacillus subtilis seed solution by the activated bacillus subtilis strain, wherein the method comprises the following steps: taking 1ml of activated bacillus subtilis strain and 100ml of sterile LB liquid culture medium, placing the activated bacillus subtilis strain and the sterile LB liquid culture medium into a conical flask, then placing the conical flask on an alcohol lamp for shake culture, keeping the temperature at 27 ℃ for 120r/min, and culturing for 24-25 h to obtain bacillus subtilis seed liquid, and keeping the bacillus subtilis seed liquid at the body temperature under the condition of 3-4 ℃ for later use;
the LB liquid culture medium comprises 10 parts of tryptone, 5 parts of yeast extract powder, 5 parts of sodium chloride and 1000 parts of distilled water by weight, and the pH value is adjusted to 6.9-7.1.
Preferably, the preparation method of the lactobacillus plantarum seed solution comprises the following steps: firstly, activating lactobacillus plantarum, wherein the activation method comprises the following steps: placing 100mLMRS liquid culture medium in a 250mL conical flask, sealing, placing in an autoclave for sterilization, taking out after sterilization, aseptically cooling to room temperature, inoculating lactobacillus plantarum preserved in glycerol under aseptic condition, sealing, standing in a constant temperature incubator at 37 ℃, culturing for 24-25 h, and obtaining activated lactobacillus plantarum strain after culture;
then preparing lactobacillus plantarum seed liquid by the activated lactobacillus plantarum strain, wherein the method comprises the following steps: taking 1ml of activated lactobacillus plantarum strain and 100ml of sterile MRS liquid culture medium, placing the activated lactobacillus plantarum strain and the activated MRS liquid culture medium in a conical flask, then performing static culture at 37 ℃ for 12-13 h to obtain lactobacillus plantarum seed liquid, and keeping the lactobacillus plantarum seed liquid at the body temperature of 3-4 ℃ for later use;
the MRS liquid culture medium comprises, by weight, 10 parts of peptone 1.0g,10 parts of beef extract, 5 parts of yeast extract, 2 parts of diammonium hydrogen citrate, 20 parts of glucose, 1 part of Tween 80, 2 parts of dipotassium hydrogen phosphate, 1.16 parts of magnesium sulfate, 0.5 part of ammonium sulfate and 1000 parts of distilled water, and the pH value is adjusted to be 6.2-6.
The fermented feed has the beneficial effects that: the soybean meal and the wheat bran are compounded by the ginkgo leaf residues and the garlic straws, so that on one hand, the deep utilization of the garlic straws is realized, and on the other hand, the protease activity in the fermented feed can be effectively improved by adding the garlic straws for fermentation.
The fermentation process of the fermented feed has the beneficial effects that: the bacillus subtilis is added firstly and then is added, the bacillus subtilis is firstly selected to perform quick and well-maintained fermentation, after oxygen in a fermentation substrate is consumed, the lactobacillus plantarum is added, the lactobacillus plantarum performs anaerobic fermentation, the high-efficiency fermentation time is prolonged, the protease activity in the fermented feed is increased, and the obtained fermented feed has high-content soluble protein and small peptide and is beneficial to animal absorption.
Drawings
FIG. 1 is a schematic diagram showing the protease activity of fermentation substrate with the same ratio and fermentation products of Bacillus subtilis and Lactobacillus plantarum with different ratios, and the ratio of Bacillus subtilis and Lactobacillus plantarum is determined according to the results shown in FIG. 1.
FIG. 2 is a schematic diagram showing the conditions of protease activity in the fermented product obtained by preparing a fermentation substrate according to the mixture ratio of examples 1 to 8 and comparative groups 1 to 3, adding Bacillus subtilis and Lactobacillus plantarum according to the mixture ratio of 3:1, and fermenting, and determining the optimal mixture ratio of dry materials of the fermentation substrate according to the actual optimal embodiment in FIG. 2.
FIG. 3 is a schematic diagram showing the results of preparing a fermentation substrate according to the mixture ratio of example 6, adding Bacillus subtilis and Lactobacillus plantarum according to the ratio of the second group, fermenting, and measuring the viable count of Bacillus subtilis and Lactobacillus plantarum in the fermentation product during the fermentation process, and the adding time of Bacillus subtilis and Lactobacillus plantarum is determined by the graph of FIG. 3.
FIG. 4 is a schematic diagram showing results of fermentation of a fermentation substrate prepared according to the mixture ratio of example 6, fermentation of a second group of Bacillus subtilis and Lactobacillus plantarum added according to the ratio of the second group, and detection of the degree of proteolysis during the fermentation process.
FIG. 5 is a diagram showing the results of fermentation of a fermentation substrate prepared according to the blending ratio of example 6, fermentation with Bacillus subtilis and Lactobacillus plantarum added according to the second group of ratios, and determination of free amino acid content during the fermentation process.
FIG. 6 is a schematic diagram of the results of fermentation of a fermentation substrate prepared according to the mixture ratio of example 6, fermentation of a second group of Bacillus subtilis and Lactobacillus plantarum added according to the ratio of the second group, and measurement of the content of small peptides in the fermentation process.
Detailed Description
In order to facilitate the understanding of the technical solutions of the present invention for those skilled in the art, the technical solutions of the present invention will be further described with reference to the specific embodiments and the drawings.
The technical scheme of the invention is that the fermented feed is mainly prepared by fermenting mixed organic matter residues through mixed strains. The mixed organic residue mainly comprises 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residue and 0.5-5% of garlic straw by weight. The mixed strain comprises bacillus subtilis and lactobacillus plantarum.
Preferably, the mixed organic residue comprises 90% of soybean meal, 4% of wheat bran, 3% of ginkgo leaf residues and 3% of garlic straws.
The technical scheme of the invention is a fermentation process of fermented feed, and the preparation of the fermented feed mainly comprises the following steps: firstly, adding bacillus subtilis seed liquid into a fermentation substrate prepared by mixing organic residue, and carrying out first-stage fermentation to obtain a first-stage fermentation product. And then adding lactobacillus plantarum seed liquid into the first-stage fermentation product for second-stage fermentation to obtain a second-stage fermentation product. And finally, freeze-drying and crushing the second-stage fermentation product to obtain the fermented feed.
The first stage fermentation time is at least when the colony number of the bacillus subtilis in the fermentation substrate reaches the maximum value, and the second stage fermentation time is at least when the colony number of the lactobacillus plantarum in the fermentation substrate reaches the maximum value. Generally, the fermentation time of the first stage is 24h-26h, preferably 24h, and the fermentation time of the second stage is 48h-56h, preferably 48h.
In the technical scheme, the preparation method of the fermentation substrate comprises the following steps: preparing an organic mixture dry material according to the mass fraction of the dry material, namely 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws, and adding water which is equal to the total amount of the organic dry material into the organic mixture dry material to obtain a fermentation substrate. The fermentation substrate is placed in a sterilization box for high-temperature sterilization at 120-125 ℃ for 15-20 min before inoculation of the bacillus subtilis, and then the bacillus subtilis is inoculated after the sterilization is finished and the fermentation substrate is placed in a sterile environment to be cooled to room temperature.
In the technical scheme, the addition ratio of the bacillus subtilis seed liquid to the lactobacillus plantarum seed liquid is 3:1, and the total addition amount of the bacillus subtilis seed liquid and the lactobacillus plantarum seed liquid is 4% of the weight of the fermentation substrate.
In the technical scheme, the preparation method of the bacillus subtilis seed liquid comprises the following steps: firstly activating the bacillus subtilis, wherein the activation method comprises the following steps: placing 100mLLB liquid culture medium in a 250mL conical flask, sealing, and sterilizing in an autoclave. And taking out after the sterilization is finished, and cooling to room temperature in a sterile manner. Inoculating the Bacillus subtilis preserved by glycerol under aseptic condition, sealing, culturing in water bath shaker at 37 deg.C and 120r/min for 24-25 h, and obtaining activated Bacillus subtilis strain after culturing.
Then preparing a bacillus subtilis seed solution by the activated bacillus subtilis strain, wherein the method comprises the following steps: 1ml of activated bacillus subtilis strain and 100ml of sterile LB liquid culture medium are placed in a conical flask. And then placing the conical flask on an alcohol lamp for shake culture, keeping the temperature at 27 ℃ at 120r/min, and culturing for 24-25 h to obtain the bacillus subtilis seed solution. Keeping the temperature of the bacillus subtilis seed liquid at 3-4 ℃ for later use.
In the technical scheme, the LB liquid culture medium comprises 10 parts of tryptone, 5 parts of yeast extract powder, 5 parts of sodium chloride and 1000 parts of distilled water by weight, and the pH value is adjusted to 6.9-7.1.
In the technical scheme, the preparation method of the lactobacillus plantarum seed liquid comprises the following steps: firstly, activating lactobacillus plantarum, wherein the activation method comprises the following steps: placing 100mL of liquid culture medium of LMRS into a 250mL conical flask, sealing, and placing in an autoclave for sterilization. And taking out after the sterilization is finished, and cooling to room temperature in a sterile manner. Inoculating lactobacillus plantarum preserved in glycerol under aseptic condition, sealing, standing in a constant temperature incubator at 37 ℃, culturing for 24-25 h, and obtaining activated lactobacillus plantarum strain after the culture is finished.
Then preparing lactobacillus plantarum seed liquid by the activated lactobacillus plantarum strain, wherein the method comprises the following steps: 1ml of activated lactobacillus plantarum strain and 100ml of sterile MRS liquid culture medium are taken and placed in a conical flask. And then standing and culturing for 12-13 h at 37 ℃ to obtain lactobacillus plantarum seed liquid, and keeping the body temperature of the lactobacillus plantarum seed liquid to 3-4 ℃ for later use.
In the technical scheme, the MRS liquid culture medium comprises, by weight, 10 parts of peptone 1.0g,10 parts of beef extract, 5 parts of yeast extract, 2 parts of diammonium hydrogen citrate, 20 parts of glucose, 1 part of Tween 80, 2 parts of dipotassium hydrogen phosphate, 1.16 parts of magnesium sulfate, 0.5 part of ammonium sulfate and 1000 parts of distilled water, and the pH value is adjusted to be 6.2-6.
In order to further understand and verify the technical scheme of the invention, the compounding ratio of the fermented feed is provided below, and the following table 1 shows several raw material compounding ratios of the fermented feed.
TABLE 1
1. Any one of the above embodiments 1 to 8 is selected, and the fermentation substrate is prepared by compounding the selected embodiment 5, the prepared fermentation substrate is divided into 5 groups, and strains are added according to the proportion in the following table 2 respectively for fermentation.
TABLE 2
| First group | Second group | Third group | Fourth group | Fifth group | |
| Bacillus subtilis |
4％ | 3％ | 2％ | 1％ | 0％ |
| Lactobacillus |
0％ | 1％ | 2％ | 3％ | 4％ |
The base prepared in example 5 was fermented in 5 groups in accordance with the addition ratio of the strains in Table 2, and the protease activity of the fermented product was measured by the Folin phenol method after 72 hours of continuous fermentation, and the protease activity of the fermented product of 5 groups after fermentation is shown in FIG. 1.
As can be seen from FIG. 1, when the total amount of inoculated bacteria is 4%, the number of fermentation strains is 3% of Bacillus subtilis and 1% of Lactobacillus plantarum, the activity of the produced protease is the highest, and can reach 340U/g, which is obviously higher than that of the other 4 groups, i.e., in the technical scheme, the adding ratio of the Bacillus subtilis seed solution to the Lactobacillus plantarum seed solution is 3:1, and the total amount of the Bacillus subtilis seed solution and the Lactobacillus plantarum seed solution is 4% of the weight of the fermentation substrate.
2. Fermentation substrates were prepared according to examples 1 to 8 in table 1 and comparative groups 1 to 3, fermentation seed solutions were added according to the ratio of group 2 in table 2 (the addition ratio of bacillus subtilis seed solution to lactobacillus plantarum seed solution was 3:1), and the fermentation processes were performed according to the above technical solutions, respectively, and after 72 hours of fermentation, lyophilization was performed to measure protease activities of the examples, and the specific results are shown in fig. 2.
As can be seen from fig. 2, it can be seen from examples 1 and 5 and comparative groups 1 to 3 that the substrate to which the garlic straw and the ginkgo biloba leaves residue are added (example 1) is fermented under the same fermentation conditions, and the obtained fermented product has a higher protease activity than the substrate to which the garlic straw and the ginkgo biloba leaves residue are not added (comparative group 3). Compared with the fermentation substrate (comparative group 2) with separately added ginkgo biloba leaf residue, the fermentation substrate (comparative group 1) with separately added garlic straw has higher protease activity in the obtained fermentation product after fermentation. The fermentation substrate with garlic straw added alone (comparative group 1) has higher protease activity in the obtained fermentation product than the fermentation substrate with equal amount of garlic straw and ginkgo biloba leaf residue added (example 5).
As can be seen from fig. 2, from example 1 to example 8, as the addition amount of the garlic straw and the ginkgo leaf residue increases, the protease activity as a whole tends to increase first and then decrease, and when both the garlic skin and the ginkgo leaf residue are 3%, the protease activity is the highest, and the highest is 367.83U/g, which is obviously higher than the protease activity of the addition amount of other garlic skin and ginkgo leaf residue, and as shown in fig. 2, the protease activity after fermentation of the fermentation substrate configured according to example 6 is the highest, thus determining the proportion of each component of the optimal fermentation substrate in the dry basis as: 90% of soybean meal, 4% of wheat bran, 3% of garlic skin and 3% of ginkgo leaf residue; the water-material ratio is 1:1.
3. Preparing a substrate according to the proportion of example 6 in table 1, fermenting the prepared substrate according to the fermentation process of the invention, and adding strain seed liquid according to the proportion of the second group of strains in table 2. During the fermentation process, samples are taken every 12h, and the change condition of the number of viable bacteria in 72h of fermentation is dynamically detected. The change of viable count during fermentation is shown in FIG. 3.
As can be seen from FIG. 3, after the Bacillus subtilis is inoculated, the Bacillus subtilis rapidly grows, and when the bacillus subtilis grows for about 24 hours, the viable count of the Bacillus subtilis reaches the highest value, the maximum value is 11.5log (CFU/g), at this time, most of the nutrient components in the fermentation substrate are utilized by the Bacillus subtilis, and the accumulation of various metabolites also makes the growth environment of the Bacillus subtilis thallus after the time period not good, so the colony count of the Bacillus subtilis generally shows a trend of decreasing in the subsequent fermentation process. At about 24h, the lactobacillus plantarum is inoculated, and because the lactobacillus plantarum is facultative anaerobic, most of oxygen in the matrix is utilized by the bacillus subtilis after fermentation for 24h, the lactobacillus plantarum rapidly grows within 24h after the lactobacillus plantarum is added, and the maximum value is 9.8log (CFU/g) at 48h of fermentation. After 48 hours, the number of Lactobacillus plantarum decreased after 48 hours of fermentation due to the lack of nutrients until the end of fermentation.
According to the fermented feed provided by the technical scheme of the invention, the garlic straws are added, so that the problems of resource utilization and treatment of the garlic straws in the prior art are solved, and the nutritional ingredients in the feed are enriched by adding the garlic straws.
According to the fermentation process of the fermented feed, the garlic straws are added into the fermentation substrate and are byproducts of harvested garlic, researches show that the nutrient substances and effective components in the garlic straws are very rich and almost comparable to those of garlic, and the garlic straws are enlarged, so that the garlic straws have an antibacterial effect in the fermentation process, inhibit the growth of mixed bacteria, ensure the growth and fermentation of bacillus subtilis and lactobacillus plantarum, ensure the fermentation time and component fermentation decomposition, and improve the protease content in a fermentation product.
According to the fermentation process of the fermented feed, the bacillus subtilis is firstly inoculated, and after the bacillus subtilis is inoculated for 24 hours, a proper amount of lactic acid bacteria is added for continuous operation. The two strains are taken as fermentation strains, and the bacillus subtilis is a common aerobic fermentation strain, oxygen in a tissue culture bottle is consumed after the bacillus subtilis is fermented for a period of time, and the lactobacillus plantarum is facultative anaerobic, so that the lactobacillus plantarum is inoculated after the bacillus subtilis is fermented for a period of time, better growth of the lactobacillus plantarum is facilitated, meanwhile, due to the existence of the lactobacillus plantarum, the acidic environment in the fermentation process is facilitated to be maintained, the palatability of the feed is improved, and the like.
According to the fermentation process of the fermented feed, bacillus subtilis is firstly inoculated, lactobacillus plantarum is inoculated after 24, the effective fermentation time is prolonged, the high-speed fermentation time is prolonged, and the proteolysis degree is improved, as can be seen from figure 4, the proteolysis degree generally tends to increase along with the increase of the fermentation time in the fermentation process, and the proteolysis degree reaches the highest and is 24% at the highest when the fermentation is carried out for 60 h. A slow decrease occurred after 60 h. In the early stage of fermentation, strains are added just before, the strains are in the growth and reproduction stage, the capability of hydrolyzing protein is limited due to the lack of various enzymes, the number of viable bacteria in a fermentation substrate is high after 12 hours of fermentation, and some enzymes are generated through early growth and metabolism, so that the capability of hydrolyzing protein is improved, the degree of hydrolysis of protein is further increased, and in the 24 th hour of fermentation, due to the addition of lactobacillus plantarum and the highest number of viable bacteria of bacillus subtilis, the capability of hydrolyzing protein is further enhanced, and the degree of hydrolysis of protein is highest in the 60 th hour. In the later stage of fermentation, thalli autolysis and change of growth environment are no longer beneficial to thalli growth and metabolism, so that the degree of hydrolysis is reduced.
During the fermentation process, the content of free amino acid is measured by adopting a Folin phenol method, as shown in figure 5, the content of free amino acid in the fermentation process generally increases along with the prolonging of the fermentation time, and tends to be stable after 48h, and the content of free amino acid reaches 1031.21 mug/g at the end of the fermentation. The strain is still in the growth and propagation stage and lacks of various metabolic enzyme systems 12 hours before fermentation, so that the capability of decomposing free amino acid is not strong; from 12h, because the number of the viable bacteria in the fermentation substrate reaches a certain number and various enzyme systems are provided through early growth and metabolism, the capability of decomposing protein to produce free amino acid is greatly improved compared with the early stage, and the concentration of the free amino acid is increased quickly; after 48 hours, because most of the nutrient components in the fermentation substrate are utilized and the growth and metabolism of the strains are limited, the capability of decomposing protein to produce free amino acid is weak, and the content of the free amino acid in the substrate tends to be stable.
In the fermentation process, the content of small peptides in the fermentation product is measured by using an OPA method. As can be seen from FIG. 6, the content of small peptides in the fermentation sample increases more slowly in the early stage of fermentation, rises more rapidly after 24h, and increases more slowly after 60h, reaching 230.30mg/g at the end of fermentation. The strain is not high in activity and lacks of various enzyme systems in the early stage of fermentation, so that the capability of decomposing protein to produce small peptides is weak, and from the 24 th hour of fermentation, the capability of hydrolyzing protein to produce small peptides is strong due to the high viable count in the fermentation substrate and the new addition of lactobacillus plantarum. In the later stage of fermentation, most of the nutrients in the fermentation substrate are consumed, and the growth metabolism of the fermentation strain is inhibited along with the change of various physicochemical properties, so that the content of the small peptide tends to be stable in the later stage.
Therefore, by inoculating the strains step by step, the effective fermentation time is prolonged, the content of hydrolyzed protein in the fermented feed is improved, the degree of proteolysis is improved, the content of small peptides is improved, the small peptides are beneficial to animal absorption, are not easy to saturate in absorption, and are better than both protein and amino acid.
Technical solution of the invention is described above by way of example with reference to the embodiments and the accompanying drawings, and it is obvious that the specific implementation of the invention is not limited by the above-mentioned manner, and it is within the scope of the invention as long as various insubstantial improvements are made to the concept and technical solution of the method of the invention, or the concept and technical solution of the invention are directly applied to other occasions without improvements.
Claims (9)
1. A fermented feed is characterized by being mainly prepared by fermenting mixed organic matter residues through mixed strains, wherein the mixed organic residues mainly comprise 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws in parts by weight, and the mixed strains comprise bacillus subtilis and lactobacillus plantarum.
2. The fermented feed according to claim 1, wherein the mixed organic residue comprises 90% of soybean meal, 4% of wheat bran, 3% of ginkgo biloba leaves residue and 3% of garlic straw.
3. A fermentation process of a fermented feed, characterized in that the fermented feed as claimed in any one of claims 1 or 2 is prepared by the steps of: firstly, adding bacillus subtilis seed liquid into a fermentation substrate prepared by mixing organic matter residues, and performing first-stage fermentation to obtain a first-stage fermentation product; then adding lactobacillus plantarum seed liquid into the first-stage fermentation product, and performing second-stage fermentation to obtain a second-stage fermentation product; and finally, freeze-drying and crushing the second-stage fermentation product to obtain the fermented feed.
4. The process of fermenting a fermented feed according to claim 3, wherein the first stage fermentation time is at least when the number of colonies of the Bacillus subtilis in the fermentation substrate reaches a maximum value, and the second stage fermentation time is at least when the number of colonies of the Lactobacillus plantarum in the fermentation substrate reaches a maximum value.
5. The fermentation process of fermented feed according to claim 4, wherein the first stage fermentation time is 24-26 h, and the second stage fermentation time is 48-56 h.
6. The fermentation process of fermented feed according to claim 3, wherein the fermentation substrate is prepared by the following steps: preparing an organic mixture dry material according to the mass fraction of the dry material, namely 86-95% of soybean meal, 4% of wheat bran, 0.5-5% of ginkgo leaf residues and 0.5-5% of garlic straws, and adding water which is equal to the total amount of the organic dry material into the organic mixture dry material to obtain a fermentation substrate; the fermentation substrate is placed in a sterilization box for high-temperature sterilization at 120-125 ℃ for 15-20 min before inoculation of the bacillus subtilis, and then the bacillus subtilis is inoculated after the fermentation substrate is placed in a sterile environment and cooled to room temperature after sterilization.
7. The fermentation process of fermented feed according to claim 3, wherein the ratio of the Bacillus subtilis seed solution to the Lactobacillus plantarum seed solution is 3:1, and the total amount of the Bacillus subtilis seed solution and the Lactobacillus plantarum seed solution added is 4% of the weight of the fermentation substrate.
8. The fermentation process of fermented feed according to claim 3, wherein the preparation method of the Bacillus subtilis seed solution comprises: firstly activating the bacillus subtilis, wherein the activation method comprises the following steps: taking 100mLLB liquid culture medium, placing the medium in a 250mL conical flask, sealing, placing the conical flask in an autoclave for sterilization, taking out the conical flask after sterilization, aseptically cooling the conical flask to room temperature, inoculating the bacillus subtilis preserved by glycerol under the aseptic condition, sealing, placing the conical flask in a water bath shaker at 37 ℃ and 120r/min for culturing for 24-25 h, and obtaining activated bacillus subtilis strain after the culture is finished;
then preparing a bacillus subtilis seed solution by the activated bacillus subtilis strain, wherein the method comprises the following steps: 1ml of activated bacillus subtilis strain and 100ml of sterile LB liquid culture medium are taken and placed in a conical flask, then the conical flask is placed on an alcohol lamp for shake culture, the temperature is kept at 27 ℃ and 120r/min, the culture is carried out for 24-25 h, bacillus subtilis seed liquid is obtained, and the temperature of the bacillus subtilis seed liquid is kept at 3-4 ℃ for standby application;
the LB liquid culture medium comprises 10 parts of tryptone, 5 parts of yeast extract powder, 5 parts of sodium chloride and 1000 parts of distilled water by weight, and the pH value is adjusted to 6.9-7.1.
9. The fermentation process of fermented feed according to claim 3, wherein the preparation method of the Lactobacillus plantarum seed solution comprises the following steps: firstly, activating lactobacillus plantarum, wherein the activation method comprises the following steps: placing 100mLMRS liquid culture medium in a 250mL conical flask, sealing, placing in an autoclave for sterilization, taking out after sterilization, aseptically cooling to room temperature, inoculating lactobacillus plantarum preserved in glycerol under aseptic condition, sealing, standing in a constant temperature incubator at 37 ℃, culturing for 24-25 h, and obtaining activated lactobacillus plantarum strain after culture;
then preparing lactobacillus plantarum seed liquid by the activated lactobacillus plantarum strain, wherein the method comprises the following steps: taking 1ml of activated lactobacillus plantarum strain and 100ml of sterile MRS liquid culture medium, placing the activated lactobacillus plantarum strain and the activated MRS liquid culture medium in a conical flask, then performing static culture at 37 ℃ for 12-13 h to obtain lactobacillus plantarum seed liquid, and keeping the lactobacillus plantarum seed liquid at the body temperature of 3-4 ℃ for later use;
the MRS liquid culture medium comprises, by weight, 10 parts of peptone 1.0g,10 parts of beef extract, 5 parts of yeast extract, 2 parts of diammonium hydrogen citrate, 20 parts of glucose, 1 part of Tween 80, 2 parts of dipotassium hydrogen phosphate, 1.16 parts of magnesium sulfate, 0.5 part of ammonium sulfate and 1000 parts of distilled water, and the pH value is adjusted to be 6.2-6.
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