CN112806477A - Method for producing feed by multi-strain solid-state fermentation of pumpkin seed meal - Google Patents
Method for producing feed by multi-strain solid-state fermentation of pumpkin seed meal Download PDFInfo
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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
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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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
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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
- A23K10/30—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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- 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
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- 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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- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a method for producing feed by multi-strain solid state fermentation of pumpkin seed meal, which comprises the step of respectively carrying out activated amplification culture on paecilomyces fibuligerus, geotrichum candidum, saccharomycetes and bacillus subtilis to obtain four liquid strains. Drying and crushing pumpkin seeds, adding a certain amount of water and a nitrogen source, mixing and sterilizing to obtain the solid fermentation medium. Inoculating the four liquid strains into a solid fermentation culture medium according to a certain proportion, fully and uniformly mixing, fermenting for 48-72 h at the natural pH value under the condition that the temperature is 25-32 ℃, and drying to obtain the pumpkin seed meal protein feed. The invention improves the immunity of animals, can improve the palatability of finished products, improves the digestion and absorption capacity of animals, has simple fermentation, reduces the fermentation cost, has little environmental pollution, does not contain any chemical additive because various nutrient components in the finished products are all generated by pure natural or microbial metabolism, does not generate substance accumulation after the animals eat the feed, and does not influence the safety of animal products.
Description
Technical Field
The invention relates to a method for producing single-cell protein feed by using pumpkin seed meal as a raw material and performing multi-strain solid-state mixed fermentation on the pumpkin seed meal, belongs to the technical field of bioengineering, and particularly relates to a method for producing feed by using the pumpkin seed meal through multi-strain solid-state fermentation.
Background
The pumpkin seeds are seeds of an annual vine plant pumpkin of Cucurbitaceae, the surface of the pumpkin seeds is light yellow and white to light yellow, the two surfaces of the pumpkin seeds are flat and slightly bulged, the two sides of the pumpkin seeds are rough edges, the hands of the pumpkin seeds are rough and uneven, the edges do not have a slight edge, one end of the pumpkin seeds is slightly pointed, the tip end of the pumpkin seeds is provided with a pearl hole, and the hilum of the pumpkin seeds is slightly protruded or not obvious.
The pumpkin seeds contain rich protein, fat, vitamins, mineral substances and the like, the oil content is as high as 41 percent, the oil is rich in palmitic acid, oleic acid, linoleic acid, linolenic acid and the like, the edible oil can be prepared by refining, and the oil quality can be comparable to that of soybean oil. The pumpkin seed meal is a byproduct after pumpkin seed oil extraction, contains a large amount of protein, vitamins, minerals and the like, is rich in nutritive value, is light yellow and granular, and has the protein content of up to 40 percent, but most of the pumpkin seed meal is used as feed or directly discarded at present, the feed fermented by the pumpkin seed meal has poor taste, and chemical components such as antibiotics are added to achieve certain functions, so that the use safety is increased, and the fermentation efficiency is poor at present, so that improvement is needed.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a method for producing feed by multi-strain solid state fermentation of pumpkin seed meal, which improves the biological effect of microecological bactericide.
In order to achieve the purpose, the invention designs a method for producing feed by multi-strain solid state fermentation of pumpkin seed meal, which takes pumpkin seeds, corn seeds and cucumber seeds as raw materials to carry out solid state fermentation, and adopts liquid strains to continue fermentation after activated amplification culture is carried out on the ascochyta, geotrichum candidum, saccharomycetes and bacillus subtilis, and specifically adopts the following steps:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 28-30 ℃, then inoculating into a triangular flask filled with 400mL of wort culture medium by using an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating into a large test tube filled with 4 mM MRS liquid culture medium, culturing at 28-30 deg.C for 24h, inoculating into a triangular flask filled with 400 mM MRS liquid culture medium at an inoculum size of 5%, and culturing at 28 deg.C for 36h by using a constant temperature shaking table with a rotation speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 7-10% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 121-124 ℃ for 20-30 min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2 percent of the liquid strain propagation liquid of the echinospora button-cyst, 1.0 percent of the liquid strain propagation liquid of the geotrichum candidum, 1.3 percent of the liquid strain propagation liquid of the saccharomycete and 0.1 percent of the liquid strain propagation liquid of the bacillus subtilis into the solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 48-72 hours at the natural pH value under the condition of the temperature of 25-32 ℃, and drying to obtain the multi-strain solid fermentation feed for the pumpkin seed meal.
Preferably, the nitrogen source in step (2) is urea or ammonium sulfate.
Preferably, the ratio of inoculated strains of the paecilomyces deductius liquid strain, the geotrichum candidum liquid strain, the saccharomycete liquid strain and the bacillus subtilis liquid strain in the step (3) is 2: 1-2: 1-2: 1.
preferably, the solid-state fermentation medium further comprises sunflower seed meal, and the weight of the sunflower seed meal is 1/2 pumpkin seeds.
The amylase type of the Neurospora capsulata is mainly alpha-amylase and glucoamylase, alpha-1, 4 and alpha-1, 6 glucosidic bonds of starch are hydrolyzed, and the final product is glucose with high purity.
Geotrichum candidum is a kind of fungi of Deuteromycotina, class Hyphomycetes, order Hyphomycetales, family Moniliaceae, genus Geotrichum. The growth temperature range of the strain is 5-38 ℃, the optimum temperature is 25 ℃, the growth pH range is 3-11, and the optimum pH is 5-7. The geotrichum candidum has high nutritive value of mycoprotein, and can be used for food, feed and nucleic acid extraction. The geotrichum candidum can also synthesize fat, and can utilize organic wastewater of sugar factories, wineries and other food factories to produce feed protein.
The yeast is a unicellular fungus, can survive in aerobic and anaerobic environments, and belongs to facultative anaerobes. The optimum growth temperature is generally 20-30 ℃. It is rich in protein (about 30-40%), B vitamins and amino acids, and can be widely used as protein supplement for animal feed. It can promote growth and development of animals, shorten breeding period, increase meat and egg amount, improve meat quality and lean meat percentage, improve glossiness of fur, and enhance disease resistance of young livestock.
Bacillus subtilis is a kind of Bacillus. Active substances such as subtilin, polymyxin, nystatin, gramicidin and the like generated in the growth process of bacillus subtilis have obvious inhibiting effect on pathogenic bacteria or conditional pathogenic bacteria of endogenous infection. The bacillus subtilis can also rapidly consume free oxygen in the environment to cause hypoxia in intestinal tracts, promote the growth of beneficial anaerobic bacteria, generate organic acids such as lactic acid and the like, reduce the pH value of the intestinal tracts and indirectly inhibit the growth of other pathogenic bacteria. It also can stimulate growth and development of animal immune organs, activate T, B lymphocytes, increase immunoglobulin and antibody levels, enhance cellular immunity and humoral immunity, and improve immunity. Self-synthesized enzymes such as alpha-amylase, protease, lipase, cellulase and the like, and play a role together with digestive enzymes in animal bodies in the digestive tract. Can synthesize various B vitamins such as vitamin B1, B2, B6, nicotinic acid, etc., and improve the activity of interferon and macrophage in animals.
The method for producing the feed by the multi-strain solid state fermentation of the pumpkin seed meal has the beneficial effects that: by utilizing the characteristics of the paecilomyces capsulatus, the geotrichum candidum, the microzyme, the bacillus subtilis and the like, the growth of intestinal pathogenic bacteria can be directly or indirectly inhibited, the immunity of animals can be improved, the mouthfeel of finished products can be improved, and the digestion and absorption capacity of the animals can be improved.
Meanwhile, the paecilomyces capsulatus, the geotrichum candidum, the saccharomycetes and the bacillus subtilis are applied to solid state fermentation, the fermentation is simple, the fermentation cost is reduced, the environmental pollution is less, various nutrient components in a finished product are all purely natural or generated by microbial metabolism, no chemical additive is contained, no matter accumulation is generated after animals eat the product, and the safety of animal products is not influenced.
Detailed Description
The present invention will be further described with reference to the following examples.
The microbial species used in the experiments can be purchased.
Example 1:
the pumpkin seed meal multi-strain solid-state fermentation feed provided by the embodiment is prepared by performing solid-state fermentation on pumpkin seeds, corn seeds and cucumber seeds as raw materials, performing activated amplification culture on fusarium oxysporum, geotrichum candidum, saccharomycetes and bacillus subtilis, and then continuing to ferment as liquid strains, and specifically adopts the following steps:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 28-30 ℃, then inoculating into a triangular flask filled with 400mL of wort culture medium by using an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating into a large test tube filled with 4 mM MRS liquid culture medium, culturing at 28-30 deg.C for 24h, inoculating into a triangular flask filled with 400 mM MRS liquid culture medium at an inoculum size of 5%, and culturing at 28 deg.C for 36h by using a constant temperature shaking table with a rotation speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 7-10% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 121-124 ℃ for 20-30 min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2 percent of the liquid strain propagation liquid of the echinospora button-cyst, 1.0 percent of the liquid strain propagation liquid of the geotrichum candidum, 1.3 percent of the liquid strain propagation liquid of the saccharomycete and 0.1 percent of the liquid strain propagation liquid of the bacillus subtilis into the solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 48-72 hours at the natural pH value under the condition of the temperature of 25-32 ℃, and drying to obtain the multi-strain solid fermentation feed for the pumpkin seed meal.
The amylase type of the Neurospora capsulata is mainly alpha-amylase and glucoamylase, alpha-1, 4 and alpha-1, 6 glucosidic bonds of starch are hydrolyzed, and the final product is glucose with high purity.
Geotrichum candidum is a kind of fungi of Deuteromycotina, class Hyphomycetes, order Hyphomycetales, family Moniliaceae, genus Geotrichum. The growth temperature range of the strain is 5-38 ℃, the optimum temperature is 25 ℃, the growth pH range is 3-11, and the optimum pH is 5-7. The geotrichum candidum has high nutritive value of mycoprotein, and can be used for food, feed and nucleic acid extraction. The geotrichum candidum can also synthesize fat, and can utilize organic wastewater of sugar factories, wineries and other food factories to produce feed protein.
The yeast is a unicellular fungus, can survive in aerobic and anaerobic environments, and belongs to facultative anaerobes. The optimum growth temperature is generally 20-30 ℃. It is rich in protein (about 30-40%), B vitamins and amino acids, and can be widely used as protein supplement for animal feed. It can promote growth and development of animals, shorten breeding period, increase meat and egg amount, improve meat quality and lean meat percentage, improve glossiness of fur, and enhance disease resistance of young livestock.
Bacillus subtilis is a kind of Bacillus. Active substances such as subtilin, polymyxin, nystatin, gramicidin and the like generated in the growth process of bacillus subtilis have obvious inhibiting effect on pathogenic bacteria or conditional pathogenic bacteria of endogenous infection. The bacillus subtilis can also rapidly consume free oxygen in the environment to cause hypoxia in intestinal tracts, promote the growth of beneficial anaerobic bacteria, generate organic acids such as lactic acid and the like, reduce the pH value of the intestinal tracts and indirectly inhibit the growth of other pathogenic bacteria. It also can stimulate growth and development of animal immune organs, activate T, B lymphocytes, increase immunoglobulin and antibody levels, enhance cellular immunity and humoral immunity, and improve immunity. Self-synthesized enzymes such as alpha-amylase, protease, lipase, cellulase and the like, and play a role together with digestive enzymes in animal bodies in the digestive tract. Can synthesize various B vitamins such as vitamin B1, B2, B6, nicotinic acid, etc., and improve the activity of interferon and macrophage in animals.
In this embodiment, pumpkin seeds, corn seeds, and cucumber seeds are used as raw materials to perform solid state fermentation, and activated amplification culture is performed on endosporium fibuligerum, geotrichum candidum, yeast, and bacillus subtilis to be used as liquid strains to continue fermentation, specifically, the following are used:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 30 ℃, then inoculating into a triangular flask filled with 400mL of wort culture medium by using an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24 hours at 30 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 30 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium in an inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating into a large test tube filled with 4 mM MRS liquid culture medium, culturing at 30 ℃ for 24h, then inoculating into a triangular flask filled with 400 mM MRS liquid culture medium by 5% of inoculum size, and culturing for 36h at 28 ℃ by using a constant temperature shaking table with the rotating speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1 (800 g of pumpkin seeds, 200g of corn seeds and 200g of cucumber seeds), drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 7% of nitrogen source to the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 125 ℃ for 30min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2% of a liquid strain propagation solution of the echinospora capsulata, 1.0% of a liquid strain propagation solution of geotrichum candidum, 1.3% of a liquid strain propagation solution of saccharomycetes and 0.1% of a liquid strain propagation solution of bacillus subtilis into a solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 72 hours at a natural pH value under the condition of a temperature of 32 ℃, and drying to obtain the pumpkin seed meal multi-strain solid fermentation feed. In this example, the nitrogen source in step (2) is urea.
Example 2:
the method for producing the pumpkin seed meal multi-strain solid-state fermentation feed provided by the embodiment is characterized in that pumpkin seeds, corn seeds and cucumber seeds are used as raw materials to be subjected to solid-state fermentation, and activated amplification culture is performed on endophyta capsulata, geotrichum candidum, saccharomycetes and bacillus subtilis to be used as liquid strains to be continuously fermented, and the following steps are specifically adopted:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 28 ℃, then inoculating the Neurospora capsulata into a triangular flask filled with 400mL of wort culture medium by 5% of inoculum size, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain the Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24h at 28 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 28 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium in an inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating the activated yeast into a large test tube filled with 4 mM MRS liquid culture medium, culturing for 24h at 28 ℃, then inoculating the activated yeast into a triangular flask filled with 400 mM MRS liquid culture medium in an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 8% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 123 ℃ for 20min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2% of a liquid strain propagation culture solution of the echinospora capsulata, 1.0% of a liquid strain propagation culture solution of geotrichum candidum, 1.3% of a liquid strain propagation culture solution of saccharomycetes and 0.1% of a liquid strain propagation culture solution of bacillus subtilis into a solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 48h at the temperature of 32 ℃ and the natural pH value, and drying to obtain the multi-strain solid fermentation feed for the pumpkin seed meal. The nitrogen source described in step (2) in this example was ammonium sulfate.
Example 3:
in the production method of the pumpkin seed meal multi-strain solid-state fermentation feed provided by the embodiment, preferably, the ratio of inoculated strains of the paecilomyces capsulatus liquid strain, the geotrichum candidum liquid strain, the yeast liquid strain and the bacillus subtilis liquid strain in the step (3) is 2: 1-2: 1-2: 1, the inoculation ratios are therefore 2: 1: 1: 1. 2: 1: 2: 1. 2: 2: 1: 1. 2: 2: 2: four types, so that according to the requirements of the specific embodiment, pumpkin seeds, corn seeds and cucumber seeds are used as raw materials to perform solid state fermentation, and activated amplification culture is performed on the pseudoendospora capsulata, geotrichum candidum, saccharomycetes and bacillus subtilis to be used as liquid strains to continue fermentation, and the following specific steps are adopted:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 28 ℃, then inoculating the Neurospora capsulata into a triangular flask filled with 400mL of wort culture medium by 5% of inoculum size, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain the Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24h at 28 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 28 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium in an inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating the activated yeast into a large test tube filled with 4 mM MRS liquid culture medium, culturing for 24h at 28 ℃, then inoculating the activated yeast into a triangular flask filled with 400 mM MRS liquid culture medium in an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 8% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 123 ℃ for 20min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2 percent of the liquid strain propagation liquid of the echinospora button-cyst, 1.0 percent of the liquid strain propagation liquid of geotrichum candidum, 1.3 percent of the liquid strain propagation liquid of saccharomycete and 0.1 percent of the liquid strain propagation liquid of bacillus subtilis into the solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 48 hours at the natural pH value under the condition of the temperature of 25-32 ℃, and drying to obtain the multi-strain solid fermentation feed for the pumpkin seed meal. The nitrogen source described in step (2) in this example was ammonium sulfate.
Example 4:
in the production method of the pumpkin seed meal multi-strain solid-state fermentation feed provided by the embodiment, the solid-state fermentation medium further comprises sunflower seed meal, and the weight of the sunflower seed meal is 1/2 pumpkin seeds.
Example 5:
the pumpkin seed meal multi-strain solid-state fermentation feed provided by the embodiment is prepared by performing solid-state fermentation on pumpkin seeds, corn seeds and cucumber seeds as raw materials, performing activated amplification culture on fusarium oxysporum, geotrichum candidum, saccharomycetes and bacillus subtilis, and then continuing to ferment as liquid strains, and specifically adopts the following steps:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 30 ℃, then inoculating into a triangular flask filled with 400mL of wort culture medium by using an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24 hours at 30 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 30 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium in an inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating into a large test tube filled with 4 mM MRS liquid culture medium, culturing at 30 ℃ for 24h, then inoculating into a triangular flask filled with 400 mM MRS liquid culture medium by 5% of inoculum size, and culturing for 36h at 28 ℃ by using a constant temperature shaking table with the rotating speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 9% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 122 ℃ for 30min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2% of a liquid strain propagation culture solution of the echinospora capsulata, 1.0% of a liquid strain propagation culture solution of geotrichum candidum, 1.3% of a liquid strain propagation culture solution of saccharomycetes and 0.1% of a liquid strain propagation culture solution of bacillus subtilis into a solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 60 hours at a natural pH value under the condition of a temperature of 30 ℃, and drying to obtain the pumpkin seed meal multi-strain solid fermentation feed.
The pumpkin seed meal multi-strain solid-state fermentation feed prepared by the embodiment is proved to have better taste compared with other feeds, the poultry is more lovely to eat, the poultry rarely suffers from diseases in the using process, animals using the feed can increase the meat quantity and the egg quantity, improve the meat quality and the lean meat percentage, and the poultry can improve the glossiness of skin and hair after half a month and enhance the disease resistance of young poultry.
Claims (5)
1. A method for producing feed by multi-strain solid state fermentation of pumpkin seed meal is characterized by comprising the following steps: the method comprises the following steps of carrying out solid state fermentation on pumpkin seeds, corn seeds and cucumber seeds as raw materials, carrying out activated amplification culture on the pseudoendospora capsulata, geotrichum candidum, saccharomycetes and bacillus subtilis, and then continuing fermentation by taking the cultured pseudoendospora capsulata, geotrichum candidum, saccharomycetes and bacillus subtilis as liquid strains, wherein the method specifically comprises the following steps:
(1) liquid strain culture
a. And (3) performing amplification culture on the ascochyta: inoculating the Neurospora capsulata into a large test tube filled with 4mL of wort culture medium, culturing for 24h at 28-30 ℃, then inoculating into a triangular flask filled with 400mL of wort culture medium by using an inoculation amount of 5%, and culturing for 36h at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a Neurospora capsulata liquid strain;
b. and (3) amplification culture of geotrichum candidum: activating geotrichum candidum, inoculating the activated geotrichum candidum into a large test tube filled with 4 mLCzapek's liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated geotrichum candidum into a triangular flask filled with 400 mLCzapek's liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a geotrichum candidum liquid strain;
c. amplification culture of bacillus subtilis: activating bacillus subtilis, inoculating the activated bacillus subtilis into a large test tube filled with a 4mLBPY liquid culture medium, culturing for 24 hours at 28-30 ℃, then inoculating the activated bacillus subtilis into a triangular flask filled with a 400mLBPY liquid culture medium according to the inoculation amount of 5%, and culturing for 36 hours at 28 ℃ by using a constant-temperature shaking table with the rotating speed of 180r/min to obtain a bacillus subtilis liquid strain;
d. and (3) amplification culture of the yeast: activating yeast, inoculating into a large test tube filled with 4 mM MRS liquid culture medium, culturing at 28-30 deg.C for 24h, inoculating into a triangular flask filled with 400 mM MRS liquid culture medium at an inoculum size of 5%, and culturing at 28 deg.C for 36h by using a constant temperature shaking table with a rotation speed of 180r/min to obtain yeast liquid strain;
(2) preparation of solid fermentation Medium
Mixing pumpkin seeds, corn seeds and cucumber seeds according to a ratio of 4: 1: 1, then drying and crushing, adding water which is 3 times of the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, fully and uniformly mixing, adding 7-10% of nitrogen source in the total weight of the pumpkin seeds, the corn seeds and the cucumber seeds, uniformly mixing, sterilizing at 121-124 ℃ for 20-30 min under high pressure, and cooling to obtain a solid fermentation culture medium;
(3) preparation of multi-strain solid-state fermentation feed by mixed solid-state fermentation
Then adding 3.2 percent of the liquid strain propagation liquid of the echinospora button-cyst, 1.0 percent of the liquid strain propagation liquid of the geotrichum candidum, 1.3 percent of the liquid strain propagation liquid of the saccharomycete and 0.1 percent of the liquid strain propagation liquid of the bacillus subtilis into the solid fermentation culture medium according to the solid fermentation culture medium, fully and uniformly mixing, fermenting for 48-72 hours at the natural pH value under the condition of the temperature of 25-32 ℃, and drying to obtain the multi-strain solid fermentation feed for the pumpkin seed meal.
2. The method for producing the feed by the multi-strain solid state fermentation of the pumpkin seed meal according to claim 1, wherein the nitrogen source in the step (2) is urea or ammonium sulfate.
3. The method for producing the feed by the multi-strain solid state fermentation of the pumpkin seed meal according to claim 1 or 2, wherein the ratio of inoculated strains of the paecilomyces capsulatus liquid strain, the geotrichum candidum liquid strain, the yeast liquid strain and the bacillus subtilis liquid strain in the step (3) is 2: 1-2: 1-2: 1.
4. the method for producing the feed by the multi-strain solid state fermentation of the pumpkin seed meal according to claim 1 or 2, wherein the solid state fermentation culture medium further comprises sunflower seed meal, and the weight of the sunflower seed meal is 1/2 pumpkin seeds.
5. The method for producing the feed by the multi-strain solid state fermentation of the pumpkin seed meal according to claim 3, wherein the solid state fermentation culture medium further comprises sunflower seed meal, and the weight of the sunflower seed meal is 1/2 pumpkin seeds.
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| CN102630813A (en) * | 2012-05-10 | 2012-08-15 | 麻名汉 | Preparation method of probiotics mixed formulation for milk cows |
| CN103478413A (en) * | 2013-09-18 | 2014-01-01 | 中国林业科学研究院林产化学工业研究所 | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues |
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| CN102630813A (en) * | 2012-05-10 | 2012-08-15 | 麻名汉 | Preparation method of probiotics mixed formulation for milk cows |
| CN103478413A (en) * | 2013-09-18 | 2014-01-01 | 中国林业科学研究院林产化学工业研究所 | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues |
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