CN113892556A

CN113892556A - Biological protein feed prepared from waste vinasse of Jimo old wine and preparation method thereof

Info

Publication number: CN113892556A
Application number: CN202111153193.0A
Authority: CN
Inventors: 邓小晨; 曹月刚; 于红美
Original assignee: Qingdao Research Institute Of Sichuan University
Current assignee: Qingdao Research Institute Of Sichuan University
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-01-07

Abstract

The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a biological protein feed prepared from waste vinasse of Jimo old wine and a preparation method thereof. The vinasse of the old wine and the peanut skin are screened under the fermentation condition, so that the palatability, the acid-soluble protein content and the protein digestion utilization rate are improved, and the anti-nutritional factors are decomposed to a certain degree. The two raw materials are subjected to compound fermentation, the amino acid composition is complementary and improved, the contents of crude protein, lysine, methionine and crude fiber can be complementary, and the nutritional value and the feeding value are improved. The product of the invention can reduce the breeding cost and increase the income; after enzyme treatment and fermentation, the feed contains a large amount of probiotics, is rich in growth factors, peptides and the like, can improve the health level of animal organisms and intestinal tracts, enhances the disease resistance, can be used without antibiotics conventionally, and is beneficial to the promotion of antibiotic-free healthy culture.

Description

Biological protein feed prepared from waste vinasse of Jimo old wine and preparation method thereof

Technical Field

The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a biological protein feed prepared from waste vinasse of Jimo old wine and a preparation method thereof.

Background

At present, the breeding feed in China seriously depends on corn-soybean meal type daily ration, particularly, the protein raw material is mainly soybean meal, most of the protein raw material is imported from North America and is limited by the international market, the breeding cost is greatly fluctuated, and the risk is increased. In addition, the imported soybean meal contains transgenic components, which do not meet the requirements of organic agricultural products.

A large number of local high-quality biomass resources in China are not fully utilized because the unconventional raw materials often contain more anti-nutritional factors, have poor palatability and even contain certain toxic substances, so that the application of the unconventional raw materials in animal daily rations is limited, and the nutritional value of the unconventional raw materials can be exerted after the unconventional raw materials are treated.

The treatment of unconventional raw materials is carried out by physical, chemical and biological methods. Physical and chemical methods generally result in material denaturation and a reduction in nutritional value. The biological method includes an enzymatic method and a microbial fermentation method. The enzyme method has mild conditions and can not cause the material to deteriorate. Due to the limited enzyme source, only part of the anti-nutritional factors can be decomposed to a certain degree. The microbial fermentation method adopts proper strain combination, can continuously grow and produce enzyme in the material, can resist the degradation of nutrient factors, improve the palatability, can degrade toxic substances to a certain extent, and can also improve the content of nutrient substances and increase active factors.

The Jimo wine belongs to yellow wine, is a local brand name wine in Jimo area, and is different from other wine in raw material, starter propagation, production process and wine taking mode, so that the vinasse of the Jimo wine is also obviously different from other vinasse. The vinasse after the fermentation of the Jimo old wine has the advantages of high protein content and high residual precipitation; the disadvantage is that the production process brings obvious scorched flavor and affects the palatability; the residual precipitation is high but partial coking is difficult to utilize; in addition, the amino acid composition is very unbalanced and lysine is very low. If the product is directly used, the nutrition value is not high. The vinasse is used for feeding animals, and the situations of vinasse alcoholism and vinasse acidification mildewing need to be avoided.

Peanuts are an important oil crop in our country. The peanut skin refers to the purplish red seed coat on the outer surface of the peanut seed, and is also called peanut skin and peanut skin. The peanut skin is a byproduct of peanut processing, is rich in flavonoids, polyphenols, pigments and the like, has high protein content and has certain edible value. But the bitter and astringent taste is heavy, the palatability of the feed is poor, cattle are not sensitive to the bitter and astringent taste compared with pigs, and the feed is often directly added into cattle feed in rural areas to feed cattle, so that the cost is reduced. Another disadvantage of peanut skin is the amino acid imbalance and the very low methionine.

Under the application background, the inventor of the invention aims to improve the nutritional composition and taste of the waste lees of the Jimo old wine and the peanut skin, comprehensively utilizes the wastes in the food processing link, improves the economic value and is environment-friendly. The method can complement the components in the two wastes, improves the feeding value through microbial fermentation, and provides a better way for effectively utilizing the local waste biomass.

Disclosure of Invention

Aiming at the defects of the old wine lees and the peanut skin, the invention provides a biological protein feed and a preparation method thereof. In the biological protein feed prepared by the invention, the main raw material is the waste vinasse of the Jimo old wine, only the waste vinasse of the Jimo old wine can be adopted, and partial peanut skin can be added as the raw material. The two raw materials can complement each other in the content of crude protein, lysine, methionine and crude fiber, and the nutritive value is improved. The two raw materials are both low-price and high-protein, and can obviously reduce the breeding cost and increase the income after the negative factors are reduced through reasonable treatment.

The first technical problem to be solved by the invention is to provide a bioprotein feed prepared by taking fermented spent grains of the Jimo wine as raw materials through microbial fermentation and enzymatic treatment, wherein the fermented spent grains of the Jimo wine are hereinafter referred to as the biological proteins of the Jimo wine.

The fermented waste vinasse of the Jimo old wine, also called as Jimo old vinasse biological protein, is prepared by fermenting the following raw materials in parts by weight: 100 parts of waste lees of the Jimo old wine, 0.2-0.4 part of solid strains of mixed yeast and 0.02-0.04 part of mixed enzyme preparation.

Preferably, the fermented waste wine lees of the Jimo old wine, also called the biological protein of the Jimo old wine lees, is prepared by fermenting the following raw materials in parts by weight: 100 portions of waste lees of the black aged wine, 0.3 portion of solid strains of the mixed bacteria and 0.03 portion of mixed enzyme preparation.

The water content of the waste vinasse of the Jimo old wine is about 55-65%, and the water content is 55% after the waste vinasse of the Jimo old wine is placed for a little dryness, so that the waste vinasse of the Jimo old wine cannot be placed for a long time and can be mildewed; preferably, the spent distiller's grains of the Jimo old wine with the water content of 60 percent are adopted.

Further, the strains used in the fermentation of the waste lees of the Jimo old wine comprise: candida utilis (Candida utilis) and Kluyveromyces marxianus (Kluyveromyces marxianus).

Specifically, the preparation method of the mixed yeast solid strain comprises the following steps: respectively selecting 1 ring (inoculating ring) from slant preserved strains of candida utilis and kluyveromyces marxianus, respectively inoculating into 100mLPDA (potato sucrose culture medium), performing shake flask culture (150-200 rpm) at 28-32 ℃ for 2-3 d, and respectively obtaining a candida utilis culture solution and a kluyveromyces marxianus culture solution, wherein the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/ml; the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml preferably, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml preferably; and mixing the two culture solutions according to a ratio of 2.5-3.5: 1 in sequence, and transferring into a bran corn culture medium.

In the preparation method, the weight ratio of the candida utilis to the kluyveromyces marxianus is 3-3.5: 1.

Preferably, the weight ratio of the candida utilis to the kluyveromyces marxianus is 2.5-3: 1.

Optimally, the weight ratio of the candida utilis to the kluyveromyces marxianus is 3: 1.

The preparation method of the bran corn culture medium comprises the following steps: mixing bran, corn and water at a weight ratio of 7-8: 2-3: 8, sterilizing at 118-126 ℃ for 25-40 min, and cooling.

The bran corn culture medium comprises the following components in percentage by weight: and (3) mixing the mixed yeast culture solution at a ratio of 10: 0.1-0.2 uniformly, and culturing at 28-32 ℃ for 2-3 d to obtain the mixed yeast solid strain.

Specifically, the preparation method of the mixed enzyme preparation comprises the following steps: amylase, glucoamylase, phytase, cellulase, pectinase and xylanase, wherein the weight ratio of amylase to glucoamylase to phytase to xylanase is 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.1-0.2; then adding neutral protease 1.8-2.5 times of total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase.

Preferably, neutral protease is added in an amount of 2 times of the total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase.

Preferably, the mixed enzyme preparation comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, xylanase, neutral protease, and neutral protease 0.2: 1: 0.4: 0.6: 0.4: 0.1: 5.4.

Specifically, the enzyme activity of the amylase is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand U/g, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the activity of the neutral protease is 5 ten thousand U/g.

The second technical problem to be solved by the present invention is to provide a bioprotein feed further comprising fermented peanut skin subjected to microbial fermentation and enzymatic treatment, the fermented peanut skin being hereinafter referred to as peanut skin protein.

The fermented peanut skin, also called peanut skin biological protein, is prepared by fermenting the following raw materials in parts by weight: 95-100 parts of peanut skin, 3-5 parts of corn flour, 0.1-0.2 part of liquid strain of bacillus subtilis, 0.3-0.4 part of solid strain of mixed yeast, 0.03-0.04 part of mixed enzyme preparation, 0.1-0.15 part of mixed nutrient salt and 50-70 parts of water.

Preferably, the fermented peanut skin, namely the peanut skin biological protein, is fermented from the following raw materials in parts by weight: 100 parts of peanut skin, 0.2 part of liquid strains of bacillus subtilis, 0.3 part of solid strains of mixed yeast, 0.15 part of mixed nutrient salt, 0.04 part of mixed enzyme preparation and 60 parts of water.

Further, in the fermentation of the peanut skin, the strains include: bacillus subtilis, Saccharomyces cerevisiae, Candida utilis, and Kluyveromyces marxianus.

Liquid strains of bacillus subtilis and liquid strains of beer yeast, candida utilis and kluyveromyces marxianus are prepared respectively. Then the liquid strains of the three yeasts are mixed and inoculated into a bran corn culture medium, and the mixed yeast solid strain is obtained after culture.

Specifically, the preparation method of the liquid strain of bacillus subtilis comprises the following steps: selecting 1 ring (inoculating ring) from slant preserved strains of bacillus subtilis, inoculating into 100mL beef extract peptone liquid culture medium, and performing shake culture (150-200 rpm) at 35-37 ℃ for 20-24h to obtain a culture solution of bacillus subtilis; wherein the concentration of bacteria is 2.0-3.2 hundred million/ml.

Preferably, the culture temperature of Bacillus subtilis is 36 ℃.

Specifically, the preparation method of the mixed yeast solid strain comprises the following steps: respectively selecting 1 ring (inoculating ring) from slant preserved strains of beer yeast, candida utilis and kluyveromyces marxianus, respectively inoculating into 100mLPDA (potato sucrose culture medium), and culturing for 2-3 d at 28-32 ℃ in a shake flask at 150-200 rpm to respectively obtain a beer yeast, candida utilis culture solution and kluyveromyces marxianus culture solution. Mixing the three culture solutions in sequence according to a proportion, and transferring into a bran corn culture medium; wherein, the cell concentration of the beer yeast culture solution is 2.0-3.2 hundred million/ml, the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/ml; preferably, the cell concentration of the beer yeast culture solution is 2.8 hundred million/ml, the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml.

In the preparation method, the solid strains of the mixed yeast comprise the following strains in parts by weight of the culture solution: the beer yeast is candida utilis and kluyveromyces marxianus in a ratio of 1: 2.8-3.5: 0.8-1.2.

Preferably, the solid strains of the mixed yeast comprise the following strains in parts by weight of the culture solution: the beer yeast is candida utilis and kluyveromyces marxianus at a ratio of 1: 2.8-3: 0.8-1.

Preferably, the solid strains of the mixed yeast comprise the following strains in parts by weight of the culture solution: the beer yeast, candida utilis and kluyveromyces marxianus is 1:3: 1.

The bran corn culture medium comprises the following components in percentage by weight: mixed yeast culture solution is 10: 0.1-0.2, uniformly mixing, and culturing at 28-32 ℃ for 2-3 days to obtain the mixed solid strain of the three yeasts.

Specifically, the preparation method of the mixed enzyme preparation comprises the following steps: according to the weight ratio, amylase: saccharifying enzyme: phytase cellulase, pectinase, xylanase, neutral protease, 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.04-0.1: 1-1.5, and mixing.

Preferably, the mixed enzyme preparation comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, xylanase, pectinase, neutral protease, and protease at 0.2: 1: 0.4: 0.6: 0.1: 0.4: 1.35.

The enzyme activity of the amylase is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand U/g, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the enzyme activity of the neutral protease is 5 ten thousand U/g.

Specifically, the preparation method of the mixed nutrient salt comprises the following steps: according to the weight ratio, 0.8-1.2 parts of KH₂PO₄And 0.2 part of MgSO₄Mixing uniformly; preferably, 1 part of KH is taken according to the weight ratio₂PO₄And 0.2 part of MgSO₄And (4) uniformly mixing.

The biological protein feed comprises fermented waste distiller's grains of the Jimo old wine, namely the biological protein of the Jimo old distiller's grains; or mixing the fermented waste distiller's grains of the Jimo wine (biological protein of the Jimo wine lees) and the fermented peanut red skin (biological protein of the peanut red skin) as raw materials.

Specifically, the raw material only comprises the bio-protein of the aged spent grain; or weighing the following raw materials in parts by weight of 100: 50-80 parts of Jimo vinasse biological protein and 20-50 parts of peanut red skin biological protein.

Further preferably, the raw materials are weighed according to the following total weight proportion of 100 parts: 80 parts of the biological protein of the aged black wine lees and 20 parts of the biological protein of the peanut red skin.

The third technical problem to be solved by the invention is to provide a method for preparing the biological protein feed by mixing the fermented waste vinasse of the Jimo rice wine (namely the biological protein of the Jimo rice wine lees) and the fermented peanut red skin (the biological protein of the peanut red skin).

The preparation method of the biological protein feed comprises two schemes of using the waste vinasse of the Jimo old wine alone or using the waste vinasse of the Jimo old wine and the peanut skin as raw materials in a mixing way.

The method comprises the following steps of:

A. preparing the bio-protein of the Jimo vinasse: mixing the raw materials according to the weight ratio, and fermenting for 40-48 h at the temperature of 30-35 ℃; wherein, the raw materials are as follows according to the weight ratio: 100 parts of waste vinasse of the Jimo old wine, 0.3-0.4 part of solid strain of mixed yeast and 0.02-0.04 part of mixed enzyme preparation;

B. and (3) fermenting again: and (3) fermenting the bio-protein of the Jimo old wine lees obtained in the step A1 for 40-48 hours at the temperature of 30-35 ℃ to obtain the Jimo old wine lees bio-protein.

And the scheme II takes the Jimo vinasse biological protein and the peanut red skin biological protein as raw materials, and comprises the following steps:

a1, preparing the Jimo vinasse biological protein: mixing the raw materials according to the weight ratio, and fermenting for 40-48 h at the temperature of 30-35 ℃; wherein, the raw materials are as follows according to the weight ratio: 100 parts of waste vinasse of the Jimo old wine, 0.2-0.4 part of solid strain of mixed yeast and 0.02-0.04 part of mixed enzyme preparation;

a2, preparing peanut red skin biological protein: mixing the raw materials according to the weight ratio, and fermenting for 48-72 hours at the temperature of 35-40 ℃; wherein, the raw materials are as follows according to the weight ratio: 95-100 parts of peanut skin, 3-5 parts of corn flour, 0.1-0.2 part of liquid strain of bacillus subtilis, 0.3-0.4 part of solid strain of mixed yeast, 0.03-0.04 part of mixed enzyme preparation, 0.1-0.15 part of mixed nutrient salt and 60-70 parts of water;

B. and (3) fermenting again: taking 50-80 parts of the Jimo rice wine lees biological protein obtained from A1 and 20-50 parts of the peanut red skin biological protein obtained from A2 according to the weight ratio, mixing, and fermenting for 40-48 hours at the temperature of 30-35 ℃.

The preparation method of the biological protein feed prepared by the method also comprises the following steps: and D, directly feeding the biological protein feed re-fermented in the step B or drying the biological protein feed until the water content is less than 14 percent and storing the biological protein feed.

The preparation method comprises the following steps:

the raw materials for preparing the Jimo rice wine lees biological protein in the step A1 are as follows by weight: 100 parts of waste vinasse of the Jimo old wine, 0.3 part of solid strain of mixed yeast and 0.03 part of mixed enzyme preparation;

the solid strains of the mixed yeast in the step A or the step A1, namely Candida utilis and Kluyveromyces marxianus;

preferably, in the solid strains of the mixed yeast of step a or step a 1: the weight ratio of the candida utilis to the kluyveromyces marxianus is 2.5-3.5: 1; preferably 2.5-3: 1; most preferably 3: 1;

in the step A or the step A1, the preparation method of the mixed yeast solid strain comprises the following steps: respectively inoculating 1 ring from slant preserved strains of candida utilis and kluyveromyces marxianus, respectively inoculating into 100mLPDA, shaking the bottles at the temperature of 28-32 ℃, and culturing for 2-3 d at the rotating speed of 150-200 rpm to respectively obtain a candida utilis culture solution and a kluyveromyces marxianus culture solution, wherein the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/ml; preferably, the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml;

the mixed enzyme preparation in the step A or the step A1 comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, pectinase and xylanase, wherein the ratio of amylase to glucoamylase to xylanase is 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.1-0.2; then adding neutral protease 1.8-2.5 times of total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase;

preferably, neutral protease is added in step A or step A1 according to 2 times of total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase;

preferably, the mixed enzyme preparation in the step A or the step A1 comprises the following components in parts by weight: amylase, diastase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at 0.2: 1: 0.4: 0.6: 0.4: 0.1: 5.4;

the enzyme activity of the amylase in the step A or the step A1 is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand units, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the enzyme activity of the neutral protease is 5 ten thousand U/g;

the raw materials for preparing the peanut red skin biological protein in the step A2 are as follows according to the weight ratio: 100 parts of peanut skin, 0.2 part of liquid strains of bacillus subtilis, 0.3 part of solid strains of mixed yeast, 0.04 part of mixed enzyme preparation, 0.15 part of mixed nutrient salt and 60 parts of water.

The preparation method of the liquid strain of the bacillus subtilis in the step A2 comprises the following steps: selecting 1 ring from the slant preservation strain by using an inoculating ring, inoculating the ring into 100mL beef extract peptone culture medium, and culturing for 20-24h at the rotating speed of 150-200 rpm in a shake flask at the temperature of 35-37 ℃ to obtain a culture solution of the bacillus subtilis, wherein the concentration of bacteria is 2.0-3.2 hundred million/mL.

The solid strains of the mixed yeast in the step A2 are Saccharomyces cerevisiae, Candida utilis (Candida utilis) and Kluyveromyces marxianus;

the solid strains of the mixed yeast in the step A2 comprise the following strains in percentage by weight: the beer yeast is candida utilis and kluyveromyces marxianus which is 1: 2.8-3.5: 0.8-1.2; preferably 1: 2.8-3: 0.8-1; optimally 1:3: 1;

the preparation method of the mixed yeast solid strain in the step A2 comprises the following steps: picking 1 ring from slant preserved strains of beer yeast, candida utilis and kluyveromyces marxianus by using an inoculating ring, respectively inoculating into 100mL of potato sucrose culture medium, and culturing for 2-3 d at a rotating speed of 150-200 rpm in a shake flask at a temperature of 28-32 ℃ to respectively obtain beer yeast, candida utilis culture solution and kluyveromyces marxianus culture solution, wherein the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/mL, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/mL; preferably, the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml;

the mixed enzyme preparation in the step A2 comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at a ratio of 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.04-0.1: 1-1.5;

preferably, the mixed enzyme preparation in the step A2 comprises the following components in parts by weight: amylase, diastase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at 0.2: 1: 0.4: 0.6: 0.4: 0.1: 1.35;

the enzyme activity of the amylase in the step A2 is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand U/g, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the enzyme activity of the neutral protease is 5 ten thousand U/g.

The mixed nutrient salt in the step A2 comprises the following components in parts by weight: KH (Perkin Elmer)₂PO₄0.8 to 1.2 parts of MgSO₄0.2 part; the mixed nutrient salt comprises the following components in parts by weight: KH (Perkin Elmer)₂PO₄1 part of MgSO (MgSO)₄0.2 part.

The invention has the beneficial effects that: the vinasse of the old wine and the peanut skin are fermented to improve the palatability, the protein digestion and utilization rate is improved (the content of acid soluble protein is improved), and the anti-nutritional factors are decomposed to a certain degree. And through compound fermentation, the amino acid composition is complemented and improved, and the feeding value is improved. And the problems of low digestion and absorption utilization rate and unbalanced nutrition caused by directly feeding vinasse are avoided, and the situation of mildewing is not easy to occur through fermentation treatment. The product of the invention can reduce the breeding cost and increase the income; after enzyme treatment and fermentation, the feed contains a large amount of probiotics, is rich in growth factors, peptides and the like, can improve the health level of animal organisms and intestinal tracts, enhances the disease resistance, can be used without antibiotics conventionally, and is beneficial to the promotion of antibiotic-free healthy culture.

Detailed Description

The Jimo old wine is famous wine in Qingdao, Shandong, China, and the vinasse is obviously different from other solid-state fermentation vinasse due to the uniqueness of the production process. The residue is more than 10 percent, the content of crude protein is up to 33 percent, but the amino acid composition is unbalanced, the lysine is low, and the methionine is high. At present, the method is not processed and treated, but directly used for cultivation. The peanut skin is a byproduct of peanut processing, the crude protein content is higher and is about 20%, and compared with the wine lees of the Jimo old wine, the peanut skin has higher lysine and lower methionine. The feed is usually prepared directly without treatment, and the feed is generally prepared only for cattle due to heavy bitter taste. At present, no treatment process aiming at the two raw materials is available and used as biological protein feed.

The two raw materials of the old wine vinasse and the peanut skin have certain complementarity in nutrition, but have larger property difference and cannot be processed under the same condition. Microbial fermentation and enzyme treatment are required to be carried out independently, and then compounding and after-fermentation are carried out.

The key of the invention is that the biological treatment is more biased to the enzyme decomposition, so as to improve the acid soluble protein and improve the protein utilization rate.

The peanut skin is a natural raw material and is not biologically decomposed. The key to the treatment is to eliminate the bitter taste, improve the palatability, eliminate various anti-nutritional factors and improve the feeding value. Microbial fermentation and enzymatic breakdown are also important in biological processes.

After the vinasse of the old wine and the peanut red skin are fermented independently, the vinasse of the old wine and the peanut red skin are compounded and fermented again, so that the palatability of the peanut red skin can be further improved by utilizing the unique fragrance of the vinasse of the old wine besides balanced amino acids.

After the fermentation of wine making, the vinasse also contains undegraded anti-nutritional factors. Can be partially degraded by enzyme action. The protease can increase the content of acid soluble protein. A small amount of amylase and saccharifying enzyme can release certain sugar to promote the growth of microorganisms. The peanut red skin contains anti-nutritional factors higher than that of distiller's grains of old wine, and crude fiber content is higher than that of the distiller's grains by more than 38%. It is necessary to select the appropriate combination of bacterial species and enzyme preparation. Because the peanut skin lacks a carbon source for microbial growth, certain corn flour needs to be supplemented. Experiments show that beer yeast, candida utilis and kluyveromyces marxianus are added in peanut skin fermentation, so that the bitter taste of the peanut skin can be eliminated, and the palatability is improved. The bacillus subtilis has no negative influence on the palatability, and can supplement probiotics in the fermented product. The phytase, cellulase, pectinase and xylanase are helpful for degrading anti-nutritional factors, and the addition of neutral protease can enhance the action of anti-nutritional factor clastic enzyme.

In the experiments and the embodiments, the bacillus subtilis is from China general microbiological culture Collection center (CGMCC) of the Committee for culture Collection of microorganisms, and the number of the bacillus subtilis is CGMCC 1.836.

The beer yeast (Saccharomyces cerevisiae) is from China general microbiological culture Collection center (CGMCC), and has a strain number of CGMCC 2.116.

Candida utilis (Candida utilis) is from China general microbiological culture Collection center (CGMCC) of the Committee for culture Collection of microorganisms, and the number of the strain is CGMCC 2.281.

Kluyveromyces marxianus is from China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 9427.

All enzyme preparations were purchased from knam bioengineering ltd. The enzyme activity of amylase is 1 ten thousand U/g, the enzyme activity of glucoamylase is 10 ten thousand U/g, the enzyme activity of phytase is 1 ten thousand U/g, the enzyme activity of cellulase is 1 ten thousand U/g, the enzyme activity of pectinase is 2 ten thousand U/g, the enzyme activity of xylanase is 20 ten thousand U/g, and the enzyme activity of neutral protease is 5 ten thousand U/g.

Example 1 screening of fermentation Strain of Ready-to-eat spent grains

Taking fresh Jimo old lees (waste lees), and subpackaging into 250ml triangular bottles, wherein each bottle contains 100 g.

Preparing 1000ml of PDA (potato sucrose) culture medium, and subpackaging into 250ml triangular bottles, wherein each bottle contains 50 ml. Steam sterilizing at 120 deg.C for 20 min after wrapping, and cooling.

Preparing 1000ml of beef extract peptone culture medium, and subpackaging 250ml triangular bottles with 50ml each. Steam sterilizing at 120 deg.C for 20 min after wrapping, and cooling.

Respectively picking 1 ring from different slant of strain to be preserved, and inoculating to the above culture medium. Inoculating the fungus into a PDA culture medium, and performing shake-flask culture at 30 ℃ for 3 d; the bacteria are inoculated into a beef extract peptone culture medium, and are cultured for 2d in a shaking way at the temperature of 33 ℃. As a seed culture.

According to the following table 1, 1ml of each seed culture solution was inoculated into a flask containing spent grains and cultured for 3 days at 30 ℃ (fungi) and 33 ℃ (bacteria). Cell number was determined and culture appearance and odor were observed. Tables 1 and 2 are partial culture results of bacteria and fungi, respectively.

TABLE 1 fermentation of spent grains by different bacteria

Bacterial Strain numbering

B2

B7

B8

B10

B13

B16

B21

B23

B24

Appearance of the product

Is not changed

Smell(s)

+

-

+

—

+

Bacteria count 10^8/g

0.05

0.03

0.06

0.04

0.06

0.055

0.05

0.06

Note: the number of bacteria is the plate count.

Appearance: the unchanged refers to the same color as the original vinasse.

Odor: - (no foreign odor), + (slight odor)

Table 1 the results show that: the bacterial strains used (mainly bacillus) grew poorly on the distillers grains. B7 is Bacillus licheniformis and B10 is Bacillus subtilis. The pH value of the vinasse is low, about 3.5-4, and the growth of the bacillus is not facilitated.

TABLE 2 fermentation of spent grains by different fungi

Fungal Strain numbering

F1

F4

F5

F11

F12

F14

F18

F22

F24

F25

Appearance of the product

Is not changed

Mold growth

Smell(s)

Wine aroma

Sour incense

Is not obvious

Musty taste

Cell number 10^8/g

0.15

0.32

0.24

0.12

0.1

Not counting

Note: the number of cells was counted under a microscope, as follows.

Appearance: and (5) carrying out (unchanged), and generating mildew spores after 2-3 days of mold growth.

Table 2 the results show: f1 is cerevisiae Fermentum, F4 is Candida utilis, and F5 is Kluyveromyces marxianus. Number F14 later is a filamentous fungus.

According to the results in tables 1 and 2, it is shown that bacillus and filamentous fungi are excluded. Considering that the vinasse is fermented by the beer yeast, Candida utilis and Kluyveromyces marxianus are selected as the vinasse fermenting strains.

Example 2 testing of enzyme preparation addition in fermentation of spent grains of distillers' grains

1g of amylase with the concentration of 1 ten thousand U/g is weighed, 100ml of water is added, and a 1% solution is prepared. 1g of 10 ten thousand U/g of glucoamylase was weighed and 100ml of water was added to prepare a 1% solution. 1g of 1 ten thousand U/g phytase is weighed, 100ml of water is added, and a 1% solution is prepared. 1g of 1 ten thousand U/g cellulase is weighed, 100ml of water is added, and a 1% solution is prepared. Weighing 1g of xylanase with 20 ten thousand U/g, and adding 100ml of water to prepare a 1% solution. Weighing 1g of 2 ten thousand U/g of pectinase, and adding 100ml of water to prepare a 1% solution. 1g of neutral protease with 5 ten thousand U/g is weighed, 100ml of water is added, and a 1% solution is prepared.

A PDA (potato sucrose) medium was prepared in the same manner as in example 1, and seed culture solutions of Candida utilis and Kluyveromyces marxianus were prepared, respectively. Then 20ml of culture solution are respectively taken and mixed according to the ratio of 1:1 to obtain the seed culture solution of the mixed yeast. Taking fresh Jimo old lees (waste lees), and subpackaging into 250ml triangular bottles, wherein each bottle contains 100 g. And respectively inoculating 1ml of mixed yeast seed culture solution into triangular flasks filled with old vinasse.

The enzyme preparations were added to the flasks according to Table 3 below. Incubate at 30 ℃ for 3 d. Cell number was determined and culture appearance and odor were observed.

TABLE 3 Effect of different enzyme preparations on fermentation of spent grains

Note: in the table, the enzyme preparations are abbreviated: sugar means saccharifying enzyme, phytase, cellulase, pectinase, xylanase, and neutral protease.

The added enzyme preparation can decompose the components of the vinasse, can promote the growth of yeast, has the most obvious amylase and has the functions of other enzymes. Further enzyme preparation combinations were tested, see table 4 below.

TABLE 4 Effect of different enzyme preparation combinations on fermentation of Ready-to-eat spent grains

Based on the results in Table 4, further combinations and addition of neutral proteases are shown in Table 5.

TABLE 5 Effect of different enzyme preparations and protease combinations on fermentation of Ready-to-eat spent grains

According to the results, the proportion of the enzyme preparation (based on 1 of the saccharifying enzyme) is selected: amylase, glucoamylase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at a ratio of 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.1-0.2: 1-1.5. Preferably: amylase 0.2, saccharifying enzyme 1, phytase 0.4, cellulase 0.6, xylanase 0.1, pectinase 0.4 and neutral protease 1.5.

Example 3 proportion and addition amount experiment of strains in fermentation of aged distillers' grains

Weighing 7 enzyme preparations according to the weight ratio: 0.2 part of amylase, 1 part of glucoamylase, 0.4 part of phytase, 0.6 part of cellulase, 0.1 part of xylanase, 0.4 part of pectinase and 1 part of neutral protease, and the total is 3.7 parts by weight. Then 10g of the mixed enzyme solution is weighed, 100ml of water is added, and 10 percent of the mixed enzyme solution is prepared.

PDA medium was prepared in the same manner as in example 1, and seed culture solutions of Candida utilis and Kluyveromyces marxianus were prepared, respectively.

Taking fresh Jimo old lees (waste lees), and subpackaging in 500ml triangular bottles, wherein each bottle contains 200 g. Seed culture solutions of different yeasts and enzyme preparations were inoculated, respectively, as shown in Table 6 below. Incubate at 30 ℃ for 3 d. Cell number and acid soluble protein.

TABLE 6 influence of different yeast ratios on fermentation of Ready-to-eat spent grain

As shown in Table 6, the ratio of the culture broth of Candida utilis to Kluyveromyces marxianus was 3: 1. For the number of growing cells, the total inoculum size was 0.5% < 1% < 2%.

Example 4 addition amount test of different enzyme preparations in fermentation of spent grains of Chinese ink

Weighing 6 enzyme preparations according to the weight ratio: 0.2 part of amylase, 1 part of glucoamylase, 0.4 part of phytase, 0.6 part of cellulase, 0.1 part of xylanase and 0.4 part of pectinase, wherein the total amount is 2.7 parts, and the xylanase is a mixed enzyme without protease. Then weighing 10g of the mixed enzyme solution, adding 100ml of water to prepare 10 percent of mixed enzyme solution without protease

PDA medium was prepared in the same manner as in example 1, and seed culture solutions of Candida utilis and Kluyveromyces marxianus were prepared, respectively. Then the culture solution is mixed according to the proportion of 3 parts of candida utilis culture solution and 1 part of kluyveromyces marxianus culture solution to form the mixed yeast culture solution.

Taking fresh Jimo old lees (waste lees), and subpackaging in 500ml triangular bottles, wherein each bottle contains 200 g. The mixed yeast cultures were inoculated and different enzyme preparations were added, respectively, as in Table 7 below. Incubate at 30 ℃ for 3 d. Cell number and acid soluble protein.

TABLE 7 Effect of different enzyme preparation additions and protease additions on fermentation of Ready-to-eat spent grain

Note: 1.82% of unfermented distillers soluble protein.

According to the results in Table 7, the amount of the 10% protease-free enzyme mixture was 0.3% to 0.4% based on the amount of the distiller's grains. The addition amount of 10% neutral protease is 0.2% -0.3%, which has little increase on the cell number, but can increase acid soluble protein. The ratio of the protease-free mixed enzyme to the neutral protease is 0.2-0.3: 0.6: 1-1.5: 3 (preferably 1: 2).

Example 5 screening of strains for peanut Red skin fermentation

Taking 960g of peanut skin and 40g of corn flour, adding 700ml of water, stirring uniformly, and subpackaging in 250ml triangular bottles with 100g (calculated according to dry materials) per bottle.

PDA (potato sucrose) medium and beef extract peptone medium were prepared separately as in example 1, and dispensed into Erlenmeyer flasks and sterilized. Respectively picking 1 ring from different slant of strain to be preserved, and inoculating to the above culture medium. Inoculating the fungus into a PDA culture medium, and performing shake-flask culture at 30 ℃ for 3 d; the bacteria are inoculated into a beef extract peptone culture medium, and are cultured for 2d in a shaking way at the temperature of 36 ℃. As a seed culture.

According to the following table 8, 1ml of each seed culture solution was inoculated into a flask containing peanut red skin, and cultured at 30 deg.C (fungi) for 3 days and at 36 deg.C (bacteria) for 1 day. Cell number was determined and culture appearance and odor were observed. Tables 8 and 9 are partial culture results of bacteria and fungi, respectively.

TABLE 8 fermentation of peanut skin by different bacteria

Bacterial Strain numbering

B2

B7

B8

B10

B13

B16

B17

B21

B23

B24

Appearance of the product

Is not changed

Smell(s)

+

-

+

-

+

-

+

Bacteria count 10^8/g

1.15

1.03

0.96

1.21

0.86

0.85

1.01

1.05

1.10

0.93

Note: the number of bacteria is the plate count.

Appearance: the unchanged color of the peanut skin is the same as the original color of the peanut skin.

Odor: - (no foreign odour), + (slight odour)

Table 8 shows: b7 is Bacillus licheniformis, B10 is Bacillus subtilis, and B21 is Bacillus laterosporus. In combination, bacillus licheniformis and bacillus subtilis (bacillus laterosporus is commonly used in organic fertilizers) are selected temporarily.

TABLE 9 fermentation of peanut skin by different fungi

Fungal Strain numbering

F1

F4

F5

F11

F12

F14

F18

F22

F24

F25

Appearance of the product

Is not changed

Mold growth

Smell(s)

Wine aroma

Sour incense

Is not obvious

Musty taste

Cell number 10^8/g

0.45

0.66

0.74

0.62

Not counting

Note: the number of cells was counted under a microscope, as follows.

Table 9 shows: f1 is cerevisiae Fermentum, F4 is Candida utilis, and F5 is Kluyveromyces marxianus. Number F14 later is a filamentous fungus. Beer yeast, candida utilis and kluyveromyces marxianus are selected as the fermentation strain of the peanut skin.

Example 6 testing of enzyme preparation addition to peanut Red skin fermentation

According to the method of example 2, 1% solutions of amylase, saccharifying enzyme, phytase, cellulase, xylanase, pectinase, and neutral protease were prepared, respectively.

PDA (potato sucrose) medium and beef extract peptone medium were prepared separately as in example 1, and dispensed into Erlenmeyer flasks and sterilized. Respectively picking 1 ring from different slant of strain to be preserved, and inoculating to the above culture medium. Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus are respectively inoculated into a PDA culture medium, and shake-flask culture is carried out for 3d at 30 ℃ to serve as seed culture solution. Inoculating Bacillus licheniformis and Bacillus subtilis into beef extract peptone medium, and culturing at 36 deg.C for 1 d. As a seed culture.

And mixing seed culture solutions of the bacillus licheniformis and the bacillus subtilis according to a ratio of 1:1 to obtain a mixed bacillus seed culture solution.

Beer yeast, candida utilis and kluyveromyces marxianus are mixed according to the ratio of 1:1:1 to be used as mixed yeast seed culture solution.

And mixing the mixed bacillus seed culture solution and the mixed yeast seed culture solution according to the ratio of 1:1 to obtain a mixed strain solution.

Taking 2850g of peanut skin, 150g of corn flour and 2100ml of water, stirring uniformly, and subpackaging into 250ml triangular bottles with 100g (calculated according to dry materials) per bottle.

The enzyme preparations and the mixed inoculum were added to each flask as shown in Table 10 below. Incubate at 30 ℃ for 3 d. Determination of cell number and observation of appearance and odor of culture

TABLE 10 Effect of different enzyme preparation combinations on the fermentation of peanut Red skin

Based on the results in Table 10, further combinations and addition of neutral proteases are shown in Table 11.

TABLE 11 Effect of different enzyme preparations and protease combinations on the fermentation of peanut Red skin

The growth promoting effect represents to some extent the decomposition of the material. According to the results, the complex enzyme has synergistic effect. Selecting the weight ratio of the enzyme preparation (based on 1 of saccharifying enzyme): amylase, glucoamylase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at a ratio of 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.04-0.1: 1-1.5. The preferable weight ratio is as follows: 0.2 part of amylase, 1 part of glucoamylase, 0.4 part of phytase, 0.6 part of cellulase, 0.1 part of xylanase, 0.4 part of pectinase and 1.5 parts of neutral protease.

Example 7 experiment of adding bacterial strains and mixed nutrient salt in peanut red skin fermentation

Weighing 7 enzyme preparations according to the weight ratio: 0.2 part of amylase, 1 part of glucoamylase, 0.4 part of phytase, 0.6 part of cellulase, 0.1 part of xylanase, 0.4 part of pectinase and 1.5 parts of neutral protease, and the components are uniformly mixed to obtain 4.2 parts in total. Then 10g of the mixed enzyme solution is taken, 100ml of water is added, and 10 percent of mixed enzyme solution is prepared.

PDA (potato sucrose) medium and beef extract peptone medium were prepared separately as in example 1, and dispensed into Erlenmeyer flasks and sterilized. Respectively picking 1 ring from different slant of strain to be preserved, and inoculating to the above culture medium. Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus are respectively inoculated into a PDA culture medium, and shake-flask culture is carried out at 30 ℃ for 2d to serve as seed culture solution. Inoculating Bacillus licheniformis and Bacillus subtilis into beef extract peptone medium, and culturing at 37 deg.C for 1 d. As a seed culture.

Taking 2280g of peanut skin, adding 120g of corn flour and 1680ml of water, and subpackaging in 500ml triangular bottles of 200g (calculated according to dry materials) per bottle. Different seed culture solutions and added enzyme preparations were inoculated, respectively, as in table 12 below. Incubate at 30 ℃ for 3 d. The number of cells was measured.

TABLE 12 Effect of different Strain ratios on the fermentation of peanut Red skin

Table 12 results: the average number of bacillus subtilis is higher than that of bacillus licheniformis, so the bacillus subtilis is preferably selected as a fermentation strain. The ratio of cerevisiae Fermentum to Candida utilis to Kluyveromyces marxianus is 1:3: 1. For cell growth, 2% of total yeast inoculum was higher than 1% of the number of cells.

The effect of the addition of nutritive salts on the fermentation of peanut skin is shown in table 13.

TABLE 13 Effect of nutrient salt addition on fermentation of peanut Red skin

	1	2	3	4	5
						Peanut red skin (g)	200	200	200	200	200
10% Mixed enzyme solution (ml)	0.4	0.4	0.4	0.4	0.4
						Bacillus subtilis culture solution (ml)	0.2	0.2	0.2	0.2	0.2
Mixed yeast culture solution (ml)	4	4	4	4	4
						10% mixed nutritive salt (ml)	——	1	2	3	4
Number of yeasts 10^8/g	1.52	1.55	1.57	1.57	1.58

Note: mixing yeast culture solution: mixing cerevisiae Fermentum, Candida utilis, and Kluyveromyces marxianus at ratio of 1:3: 1. The mixed nutrient salt is prepared according to KH₂PO₄︰MgSO₄Mixing the components in a ratio of 5: 1.

The results in Table 13 show that the amount of the mixed nutrient salt added is 0.1-0.15 parts per 100 parts of peanut skin.

Example 8 experiment of the amount of different enzyme preparations added in peanut Red skin fermentation

Weighing 6 enzyme preparations according to the weight ratio: 0.2 part of amylase, 1 part of glucoamylase, 0.4 part of phytase, 0.6 part of cellulase, 0.1 part of xylanase and 0.4 part of pectinase, wherein the total amount is 2.7 parts, and the xylanase is a mixed enzyme without protease. Then 10g of the mixed enzyme solution is weighed, 100ml of water is added, and 10% of mixed enzyme solution without protease is prepared.

PDA medium was prepared in the same manner as in example 1, and seed culture solutions of three kinds of beer yeast, Candida utilis and Kluyveromyces marxianus were prepared, respectively. Then mixing 1 part of beer yeast, 3 parts of candida utilis culture solution and 1 part of kluyveromyces marxianus culture solution according to the proportion to obtain the mixed yeast culture solution.

A seed culture of Bacillus subtilis was prepared by preparing a beef extract peptone medium according to the method of example 1.

Preparing mixed nutrient salt, taking 1 part of KH₂PO₄And 0.2 part of MgSO₄And (4) mixing.

1900kg of peanut red skin is taken, 100g of corn flour and 1400ml of water are added, and the mixture is subpackaged into 500ml triangular bottles, wherein 200g of the mixture is taken (calculated according to dry materials). The different seed cultures and the added enzyme preparations were inoculated separately as in Table 14 below. Incubate at 30 ℃ for 3 d.

TABLE 14 Effect of different enzyme preparation additions and protease additions on peanut skin fermentation

	1	2	3	4	5	6	7	8	9
										Peanut red skin (g)	200	200	200	200	200	200	200	200	200
Mixed nutritive salt (g)	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
										Bacillus subtilis culture solution (ml)	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Mixed yeast culture solution (ml)	4	4	4	4	4	4	4	4	4
										10% protease-free enzyme mixture (ml)	0.2	0.3	0.4	0.2	0.3	0.4	0.2	0.3	0.4
10% neutral protease (g)	0.1	0.1	0.1	0.15	0.15	0.15	0.2	0.2	0.2
										Number of yeasts 10^8/g	1.49	1.53	1.54	1.52	1.58	1.57	1.55	1.56	1.56
Acid soluble protein (% dry basis)	1.02	1.05	1.06	1.33	1.34	1.34	1.36	1.36	1.37

Note: 0.72 percent of red-dressing acid-soluble protein of the unfermented peanut.

According to the results in Table 14, the amount of 10% protease-free enzyme mixture was 0.15% to 0.2% based on the amount of peanut red coats. The addition amount of 10% neutral protease is 0.075-0.1%, which has little increase to cell number, but can increase acid soluble protein. The ratio of the protease-free mixed enzyme to the neutral protease was 2: 1.

Example 9 preparation of bioprotein feed by mixing spent grains bioprotein with peanut skin bioprotein

According to the results of example 4 and example 8, 0.2 parts of amylase, 1 part of saccharifying enzyme, 0.4 parts of phytase, 0.6 parts of cellulase, 0.1 part of xylanase and 0.4 part of pectinase are weighed according to the weight ratio, and the total weight is 2.7 parts, and the amylase is a protease-free mixed enzyme. Adding 1.5 parts of neutral protease (the ratio of protease-free mixed enzyme to neutral protease is 1:2), and mixing to obtain mixed enzyme preparation (mixed enzyme preparation A) for fermenting distiller's grains; adding 1.35 parts of neutral protease (the ratio of protease-free mixed enzyme to neutral protease is 2:1), and mixing to obtain mixed enzyme preparation (mixed enzyme preparation B) for fermenting peanut red skin.

PDA medium was prepared in the same manner as in example 1, and seed culture solutions of three kinds of beer yeast, Candida utilis and Kluyveromyces marxianus were prepared, respectively.

Mixing 3 parts of Candida utilis culture solution and 1 part of Kluyveromyces marxianus culture solution to obtain mixed yeast culture solution (mixed yeast solution A) for fermenting distiller's grains. Weighing 750g of bran and 250g of corn flour, adding 600ml of water, uniformly stirring, sterilizing at 120 ℃ for 25 minutes, cooling, adding 50ml of mixed yeast liquid A, and culturing at 30 ℃ for 3d to obtain the mixed yeast solid strain (solid strain A) for vinasse fermentation.

Then mixing 1 part of beer yeast, 3 parts of candida utilis culture solution and 1 part of kluyveromyces marxianus culture solution according to the proportion to obtain the mixed yeast culture solution (mixed yeast solution B) for peanut skin fermentation. Weighing 750g of bran and 250g of corn flour, adding 600ml of water, uniformly stirring, sterilizing at 120 ℃ for 25 minutes, cooling, adding 50ml of mixed yeast liquid B, and culturing at 30 ℃ for 3 days to obtain the mixed yeast solid strain (solid strain B) for peanut skin fermentation.

Weighing 10kg of the aged wine lees (with water content of 60 percent), and adding 40g of solid strain A and 4g of mixed enzyme preparation A. Mixing, placing into plastic bag with breather valve, and fermenting at 30 deg.C for 3 d.

Weighing 9.5kg of peanut skin, 500g of corn flour, 7kg of water, 10ml of liquid strain of bacillus subtilis, 40g of solid strain B, 4g of mixed enzyme preparation B and 10g of mixed nutrient salt. Firstly adding the solid strain B, the mixed enzyme preparation B and the mixed nutrient salt into water for dissolving, then adding the liquid strain of the bacillus subtilis, and then uniformly mixing with the peanut skin and the corn flour. Placing into plastic bag with breather valve, and fermenting at 30 deg.C for 3 d.

Mixing the fermented distiller's grains and peanut skin according to the following table 15, respectively packaging into plastic bags, continuously fermenting at 30 deg.C for 48 hr, and determining the nutritional index of the feed.

TABLE 15 mixing of fermented distiller's grains and fermented peanut skin at different ratios

	1	2	3	4
					Fermenting vinasse: fermented red skin (g)	400:600	500:500	600:400	800:200
Measurement of moisture (%)	49.5％	50.5％	51.5％	53.5％
					CP(％)	25.86	27.07	28.54	31.46
Total amino acids (%)	22.16	23.53	25.11	28.47
					Lys(％)	0.690	0.602	0.520	0.357
Met(％)	0.393	0.474	0.553	0.722

Note: 1. the CP, total amino acids, Lys and Met contents are expressed in terms of 10% aqueous material.

2. The water content of the fermented vinasse is 55 percent, and the water content of the fermented red skin is 45 percent

3. Before fermentation, the vinasse CP is 33.9%, the total AA is 30.3%, the Lys0.186 and the Met0.89 are added;

before fermentation, red skin CP is 19.7%, total AA 15.4%, lys0.99, met0.065;

after fermentation and matching, lysine and methionine are complementary, and the nutrition is more balanced. 50-80 parts of fermented vinasse, 20-50 parts of fermented red skin and 100 parts of total weight can be selected to prepare the biological protein.

Example 10 preparation of bioprotein feed by mixing spent grains bioprotein with peanut skin bioprotein

Seed culture solutions of solid seed species A, solid seed species B, Bacillus subtilis, mixed enzyme preparation A, mixed enzyme preparation B and mixed nutrient salts were prepared, respectively, as in example 9.

100kg of the aged wine lees (with the water content of 60 percent) is weighed, and 400g of solid strain A and 40g of mixed enzyme preparation A are added. Mixing, placing into plastic bag with breather valve, and fermenting at 30 deg.C for 3 d.

Weighing 95kg of peanut skin, 5kg of corn flour, 70kg of water, 100ml of liquid strain of bacillus subtilis, 400g of solid strain B, 40g of mixed enzyme preparation B and 100g of mixed nutrient salt. Firstly adding the solid strain B, the mixed enzyme preparation B and the mixed nutrient salt into water for dissolving, then adding the liquid strain of the bacillus subtilis, and then uniformly mixing with the peanut skin and the corn flour. Placing into plastic bag with breather valve, and fermenting at 30 deg.C for 3 d.

Mixing the fermented distiller's grains and peanut skin at a ratio of 6:4, spreading out, placing in a metal plate with a material layer thickness of 5cm, and fermenting at 30 deg.C for 40 h.

CP was determined 28.9% and acid soluble protein 2.296%.

In conclusion, the invention provides a biological protein feed prepared by mixing the waste vinasse of the Jimo old wine and the peanut skin as raw materials and a preparation method thereof. The vinasse of the old wine and the peanut skin are fermented to improve the palatability, the content of acid-soluble protein is improved, the digestion and utilization rate of the protein is improved, and anti-nutritional factors are decomposed to a certain degree. The two raw materials are subjected to compound fermentation, the amino acid composition is complementary and improved, the contents of crude protein, lysine, methionine and crude fiber can be complementary, and the nutritional value and the feeding value are improved. The product of the invention can reduce the breeding cost and increase the income; after enzyme treatment and fermentation, the feed contains a large amount of probiotics, is rich in growth factors, peptides and the like, can improve the health level of animal organisms and intestinal tracts, enhances the disease resistance, can be used without antibiotics conventionally, and is beneficial to the promotion of antibiotic-free healthy culture.

Claims

1. A biological protein feed is characterized in that: the raw material of the wine comprises the biological protein of the Jimo old wine lees, which is prepared by fermenting the waste wine lees of the Jimo old wine; the fermented waste distiller's grains of the Jimo old wine are prepared by fermenting the following raw materials in parts by weight:

100 parts of waste vinasse of the Jimo old wine, 0.2-0.4 part of solid strain of mixed yeast and 0.02-0.04 part of mixed enzyme preparation;

the water content of the waste vinasse of the Jimo old wine is 55-65%;

preferably, the water content of the spent grains of the Jimo old wine is 60%.

2. The bioprotein feed of claim 1, wherein: at least one of the following is satisfied:

the Jimo vinasse bioprotein is prepared by fermenting the following raw materials in parts by weight: 100 parts of waste vinasse of the Jimo old wine, 0.3 part of solid strain of mixed yeast and 0.03 part of mixed enzyme preparation;

the solid strains of the mixed yeast, namely Candida utilis and Kluyveromyces marxianus;

preferably, in the solid strains of the mixed yeast: the weight ratio of the candida utilis to the kluyveromyces marxianus is 2.5-3.5: 1; preferably 2.5-3: 1; most preferably 3: 1;

the preparation method of the mixed yeast solid strain comprises the following steps: respectively inoculating 1 ring from slant preserved strains of candida utilis and kluyveromyces marxianus, respectively inoculating into 100ml of PDA culture medium, and culturing at a temperature of 28-32 ℃ in a shake flask at a rotating speed of 150-200 rpm for 2-3 d to respectively obtain a candida utilis culture solution and a kluyveromyces marxianus culture solution, wherein the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/ml; preferably, the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml;

the mixed enzyme preparation comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, pectinase and xylanase, wherein the ratio of amylase to glucoamylase to xylanase is 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.1-0.2; then adding neutral protease 1-2.5 times of total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase;

preferably, neutral protease is added according to 2 times of the total amount of amylase, saccharifying enzyme, phytase, cellulase, pectinase and xylanase;

preferably, the mixed enzyme preparation comprises the following components in parts by weight: amylase, diastase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at 0.2: 1: 0.4: 0.6: 0.4: 0.1: 5.4;

the enzyme activity of the amylase is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand U/g, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the enzyme activity of the neutral protease is 5 ten thousand U/g;

the water content of the waste vinasse of the Jimo old wine is 60%.

3. The bioprotein feed of claim 1 or 2, wherein: the raw materials also comprise peanut red skin biological protein which is prepared by fermenting peanut red skin; the peanut red skin biological protein is fermented peanut red skin which is prepared by fermenting the following raw materials in parts by weight:

95-100 parts of peanut skin, 3-5 parts of corn flour, 0.1-0.2 part of liquid strain of bacillus subtilis, 0.3-0.4 part of solid strain of mixed yeast, 0.03-0.04 part of mixed enzyme preparation, 0.1-0.15 part of mixed nutrient salt and 60-70 parts of water.

4. The bioprotein feed of claim 3, wherein: at least one of the following is satisfied:

the peanut red skin biological protein is prepared by fermenting the following raw materials in parts by weight: 100 parts of peanut skin, 4 parts of corn flour, 0.2 part of liquid strain of bacillus subtilis, 0.3 part of solid strain of mixed yeast, 0.04 part of mixed enzyme preparation, 0.15 part of mixed nutrient salt and 60 parts of water;

the preparation method of the liquid strain of the bacillus subtilis comprises the following steps: picking 1 ring from the slant preserved strain by using an inoculating loop, inoculating the ring into 100mL beef extract peptone culture medium, and culturing for 20-24h at the rotating speed of 150-200 rpm in a shake flask at the temperature of 35-37 ℃ to obtain a culture solution of bacillus subtilis, wherein the concentration of bacteria is 2.0-3.2 hundred million/mL;

the solid strain of the mixed yeast is beer yeast Saccharomyces ceremony, Candida utilis (Candida utilis) and Kluyveromyces marxianus;

the solid strains of the mixed yeast comprise the following strains in parts by weight: the beer yeast is candida utilis and kluyveromyces marxianus which is 1: 2.8-3.5: 0.8-1.2; preferably 1: 2.8-3: 0.8-1; optimally 1:3: 1;

the preparation method of the mixed yeast solid strain comprises the following steps: picking 1 ring from slant preserved strains of beer yeast, candida utilis and kluyveromyces marxianus by using an inoculating ring, respectively inoculating the 1 ring into a 100mLPDA culture medium, and culturing for 2-3 d at a rotating speed of 150-200 rpm in a shake flask at a temperature of 28-32 ℃ to respectively obtain beer yeast, a candida utilis culture solution and a kluyveromyces marxianus culture solution, wherein the cell concentration of the beer yeast culture solution is 2.0-3.2 hundred million/ml, the cell concentration of the candida utilis culture solution is 6.5-7.0 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.5-5.0 hundred million/ml; preferably, the cell concentration of the beer yeast culture solution is 2.8 hundred million/ml, the cell concentration of the candida utilis culture solution is 6.7 hundred million/ml, and the cell concentration of the kluyveromyces marxianus culture solution is 4.7 hundred million/ml;

the mixed enzyme preparation comprises the following components in parts by weight: amylase, glucoamylase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at a ratio of 0.2: 1: 0.4-0.6: 0.6-0.8: 0.2-0.4: 0.04-0.1: 1-1.5;

preferably, the mixed enzyme preparation comprises the following components in parts by weight: amylase, diastase, phytase, cellulase, pectinase, xylanase, neutral protease, and protease at 0.2: 1: 0.4: 0.6: 0.4: 0.1: 1.35;

the mixed nutrient salt comprises the following components in parts by weight: KH (Perkin Elmer)₂PO₄0.8 to 1.2 parts of MgSO₄0.2 part; the mixed nutrient salt comprises the following componentsThe components in weight ratio are as follows: KH (Perkin Elmer)₂PO₄1 part of MgSO (MgSO)₄0.2 part;

5. The bioprotein feed of claim 3 or 4, wherein: the raw materials are weighed according to the following weight proportion of 100 parts: 50-80 parts of Jimo vinasse biological protein and 20-50 parts of peanut red skin biological protein.

6. The bioprotein feed of claim 5, wherein: the raw materials are weighed according to the following weight proportion of 100 parts: 80 parts of the biological protein of the aged black wine lees and 20 parts of the biological protein of the peanut red skin.

7. A process for preparing a bioprotein feed of claim 1 or 5, characterized by: the raw material of the biological protein feed is the Jimo vinasse biological protein, and the method comprises the following steps:

A. preparing the bio-protein of the Jimo vinasse: mixing the raw materials according to the weight ratio, and fermenting for 40-48 h at the temperature of 30-35 ℃; wherein, the raw materials are as follows according to the weight ratio: 100 parts of waste vinasse of the Jimo old wine, 0.2-0.4 part of solid strain of mixed yeast and 0.02-0.04 part of mixed enzyme preparation;

B. and (3) fermenting again: and C, taking the bio-protein of the aged wine lees obtained in the step A, and fermenting for 40-48 hours at the temperature of 30-35 ℃ to obtain the bio-protein of the aged wine lees.

8. The method for preparing a bioprotein feed according to claim 3 or 6, comprising: the raw materials of the biological protein feed are the biological protein of the Jimo vinasse and the biological protein of the peanut skin, and the biological protein feed comprises the following steps:

a2, preparing peanut red skin protein: mixing the raw materials according to the weight ratio, and fermenting for 48-72 hours at the temperature of 35-40 ℃; wherein, the raw materials are as follows according to the weight ratio: 95-100 parts of peanut skin, 3-5 parts of corn flour, 0.1-0.2 part of liquid strain of bacillus subtilis, 0.3-0.4 part of solid strain of mixed yeast, 0.03-0.04 part of mixed enzyme preparation, 0.1-0.15 part of mixed nutrient salt and 60-70 parts of water;

B. and (3) fermenting again: weighing the following raw materials in parts by weight of 100: 50-80 parts of the Jimo vinasse biological protein obtained from A1 and 20-50 parts of the peanut red skin biological protein obtained from A2 are mixed and fermented for 40-48 hours at the temperature of 30-35 ℃.

9. The method for preparing a bioprotein feed according to claim 7 or 8, wherein: comprises the following steps: and D, directly feeding the biological protein feed re-fermented in the step B or drying the biological protein feed until the water content is less than 14 percent and storing the biological protein feed.

10. The method for preparing a bioprotein feed according to claim 7 or 8, wherein: at least one of the following is satisfied:

the raw materials for preparing the Jimo rice wine lees biological protein in the step A or the step A1 are as follows according to the weight ratio: 100 parts of waste vinasse of the Jimo old wine, 0.3 part of solid strain of mixed yeast and 0.03 part of mixed enzyme preparation;

the enzyme activity of the amylase in the step A or the step A1 is 1 ten thousand U/g, the enzyme activity of the saccharifying enzyme is 10 ten thousand U/g, the enzyme activity of the phytase is 1 ten thousand U/g, the enzyme activity of the cellulase is 1 ten thousand U/g, the enzyme activity of the pectinase is 2 ten thousand U/g, the enzyme activity of the xylanase is 20 ten thousand U/g, and the enzyme activity of the neutral protease is 5 ten thousand U/g;

the raw materials for preparing the peanut red skin biological protein in the step A2 are as follows according to the weight ratio: 100 parts of peanut skin, 4 parts of corn flour, 0.2 part of liquid strain of bacillus subtilis, 0.3 part of solid strain of mixed yeast, 0.04 part of mixed enzyme preparation, 0.15 part of mixed nutrient salt and 60 parts of water;

the preparation method of the liquid strain of the bacillus subtilis in the step A2 comprises the following steps: picking 1 ring from the slant preserved strain by using an inoculating loop, inoculating the ring into 100mL beef extract peptone culture medium, and culturing for 20-24h at the rotating speed of 150-200 rpm in a shake flask at the temperature of 35-37 ℃ to obtain a culture solution of bacillus subtilis, wherein the concentration of bacteria is 2.0-3.2 hundred million/mL;

the mixed nutrient salt in the step A2 comprises the following components in parts by weight: KH (Perkin Elmer)₂PO₄0.8 to 1.2 parts of MgSO₄0.2 part; the mixed nutrient salt comprises the following components in parts by weight: KH (Perkin Elmer)₂PO₄1 part of MgSO (MgSO)₄0.2 part;