CN117322507A - Polypeptide-rich feed and preparation method thereof - Google Patents

Polypeptide-rich feed and preparation method thereof Download PDF

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Publication number
CN117322507A
CN117322507A CN202311485311.7A CN202311485311A CN117322507A CN 117322507 A CN117322507 A CN 117322507A CN 202311485311 A CN202311485311 A CN 202311485311A CN 117322507 A CN117322507 A CN 117322507A
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enzymolysis
aqueous solution
fermentation
soybean
soybean meal
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胡竑邠
李宝林
高卫东
成廷水
张翼
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Jiangsu Zhihui Biotechnology Co ltd
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Jiangsu Zhihui Biotechnology Co ltd
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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
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Sustainable Development (AREA)
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  • Mycology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a polypeptide-rich feed and a preparation method thereof. According to the invention, the bean pulp is treated in advance by using non-starch polysaccharide degrading enzymes such as cellulase, alpha-galactosidase and the like, so that the viscosity of the feed liquid is obviously reduced, and the fluidity is increased; the mixed strain is inoculated after enzymolysis is carried out in the soybean concentrated protein aqueous solution, so that the fermentation time is shortened, and the fermentation efficiency is improved; the polypeptide-rich feed is prepared under the stirring state, so that the enzymolysis and fermentation speed is improved, the blockage of a pipeline and a die hole caused by caking is avoided, and the fermentation efficiency is greatly improved. Therefore, the technical scheme provided by the invention has the characteristics of short time, high efficiency and high protein small peptide content.

Description

Polypeptide-rich feed and preparation method thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a polypeptide-rich feed and a preparation method thereof.
Background
The soybean meal is used as a byproduct of soybean processing, and has low price and rich nutrients. For example, the soybean meal has a protein content of 40% to 50%, a fat of 1% to 2%, and a carbohydrate of about 10% to 15%, which is the most widely used vegetable protein feed material at present. However, "anti-nutritional factors" in soybean meal, such as soybean antigens (glycinin and conglycinin), trypsin inhibitors, and the like, affect the digestibility of animals, increasing the endogenous nitrogen loss of animals. Therefore, after untreated soybean meal is directly fed to animals, the problems of diarrhea, intestinal allergy and the like are easily caused, and the production performance and the feed utilization rate of livestock and poultry are seriously influenced.
The current method for eliminating the anti-nutritional factors in the soybean meal mainly comprises the following steps: physical, chemical, microbial fermentation, and enzyme preparation. Although the microbial fermentation method is relatively safe and healthy, the fermentation time is long and the turnover is slow; while the enzymolysis method can degrade non-starch polysaccharide and protein in the soybean meal, the bitter taste of the product can be increased, and the food calling property of the feed can be reduced.
Disclosure of Invention
The invention aims to provide a polypeptide-rich feed and a preparation method thereof, and the polypeptide-rich feed has the characteristics of short time, high efficiency, high protein small peptide content and good palatability.
The invention provides a method for preparing polypeptide-rich feed, which comprises the following steps:
mixing the soybean concentrated protein aqueous solution, alkaline protease and neutral protease for first enzymolysis, and inoculating a composite microbial inoculum into the soybean concentrated protein aqueous solution after the first enzymolysis for first fermentation to obtain a soybean concentrated protein aqueous solution after the first fermentation;
mixing the soybean meal aqueous solution, cellulase and alpha-galactosidase for second enzymolysis, mixing an enzymolysis solution obtained by the second enzymolysis, alkaline protease and neutral protease for third enzymolysis, and obtaining the soybean meal aqueous solution after enzymolysis;
performing second fermentation and drying on the first fermented soybean concentrated protein aqueous solution and the enzymatic hydrolysis soybean meal aqueous solution to obtain the polypeptide-rich feed;
the composite microbial inoculum comprises saccharomycetes, lactobacillus and bacillus;
the content of crude protein in the soybean meal is more than or equal to 46%;
the first enzymolysis, the first fermentation, the second enzymolysis, the third enzymolysis and the second fermentation are all carried out under the condition of stirring.
Preferably, the mass ratio of the soybean protein concentrate to water in the soybean protein concentrate aqueous solution is 10-15: 100;
in the first enzymolysis, the addition amount of the alkaline protease is 0.1wt.% to 0.3wt.% of the soybean protein concentrate; the addition amount of the neutral protease is 0.1wt.% to 0.15wt.% of the soybean protein concentrate;
the conditions of the first enzymolysis comprise: the time is 2-6 h, and the temperature is 50-55 ℃.
Preferably, the inoculation amount of the compound bacteria agent is 0.1wt.% to 0.2wt.% of the soybean protein concentrate in the soybean protein concentrate aqueous solution;
the viable count of the saccharomycetes in the composite microbial inoculum is more than or equal to 4 multiplied by 10 9 CFU/g, the viable count of lactobacillus is more than or equal to 6×10 9 CFU/g, the viable count of bacillus is more than or equal to 6 multiplied by 10 9 CFU/g。
Preferably, the conditions of the first fermentation include: the temperature is 35-38 ℃ and the time is 8-16 h.
Preferably, the mass ratio of the soybean meal to water in the soybean meal aqueous solution is 25-30: 100;
in the second enzymolysis, the addition amounts of the cellulase and the alpha-galactosidase are respectively 0.05wt.% to 0.1wt.% of the soybean meal in the soybean meal aqueous solution;
the conditions of the second enzymolysis comprise: the temperature is 50-55 ℃ and the time is 1-2 h.
Preferably, in the third enzymolysis, the addition amounts of the alkaline protease and the neutral protease are respectively 0.1wt.% to 0.3wt.% of the soybean meal in the enzymolysis liquid obtained by the second enzymolysis;
the conditions of the third enzymolysis comprise: the time is 3-5 h, and the temperature is 50-55 ℃.
Preferably, in the second fermentation, the volume ratio of the soybean concentrated protein aqueous solution after the first fermentation to the soybean meal aqueous solution after the enzymolysis is 1:5, a step of;
the conditions of the second fermentation include: the temperature is 35-38 ℃ and the time is 2-4 h.
Preferably, the stirring speed is 20-40 rpm.
Preferably, the drying conditions include: the air inlet temperature is 170-180 ℃, the air outlet temperature is 80-85 ℃ and the time is 7-10 s; during drying, the frequency of the atomizer frequency converter is 42-48 Hz.
The polypeptide-rich feed prepared by the method provided by the technical scheme is characterized in that the content of acid soluble protein in the polypeptide-rich feed is more than or equal to 56%, the content of crude protein is more than or equal to 50%, and the pH is less than or equal to 5.0.
The beneficial effects are that:
the invention provides a method for preparing polypeptide-rich feed, which comprises the following steps: mixing the soybean concentrated protein aqueous solution, alkaline protease and neutral protease for first enzymolysis, and inoculating a composite microbial inoculum into the soybean concentrated protein aqueous solution after the first enzymolysis for fermentation to obtain a soybean concentrated protein aqueous solution after the first fermentation; mixing the soybean meal aqueous solution, cellulase and alpha-galactosidase for second enzymolysis, mixing the second enzymolysis to obtain enzymolysis solution, alkaline protease and neutral protease for third enzymolysis, and obtaining the hydrolyzed soybean meal aqueous solution; performing second fermentation and drying on the first fermented soybean concentrated protein aqueous solution and the enzymatic hydrolysis soybean meal aqueous solution to obtain the polypeptide-rich feed; the composite microbial inoculum comprises saccharomycetes, lactobacillus and bacillus; the content of the crude protein of the soybean meal is more than or equal to 46 percent; the first enzymolysis, fermentation, second enzymolysis, third enzymolysis and second fermentation are all carried out under the condition of stirring. According to the invention, the bean pulp is treated in advance by using non-starch polysaccharide degrading enzymes such as cellulase, alpha-galactosidase and the like, so that the viscosity of the feed liquid is obviously reduced, and the fluidity is increased; the mixed strain is inoculated after enzymolysis is carried out in the soybean concentrated protein aqueous solution, so that the fermentation time is shortened, and the fermentation efficiency is improved; the polypeptide-rich feed is prepared under the stirring state, so that the enzymolysis and fermentation speed is improved, the blockage of a pipeline and a die hole caused by caking is avoided, and the fermentation efficiency is greatly improved.
Based on the technical advantages, the invention also provides the polypeptide-rich feed prepared by the technical scheme, wherein the acid soluble protein in the polypeptide-rich feed is more than or equal to 56%, the crude protein content is more than or equal to 50%, and the pH value is less than or equal to 5.0. Experiments prove that the soybean protein antigen and the anti-nutritional factor are thoroughly eliminated by adopting the technical scheme provided by the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a gel electrophoresis antigen detection chart of the final product obtained in example 9.
Detailed Description
The invention provides a method for preparing polypeptide-rich feed, which comprises the following steps: mixing the soybean concentrated protein aqueous solution, alkaline protease and neutral protease for first enzymolysis, and inoculating a composite microbial inoculum into the soybean concentrated protein aqueous solution after the first enzymolysis for fermentation to obtain a soybean concentrated protein aqueous solution after the first fermentation; mixing the soybean meal aqueous solution, cellulase and alpha-galactosidase for second enzymolysis, mixing the second enzymolysis to obtain enzymolysis solution, alkaline protease and neutral protease for third enzymolysis, and obtaining the hydrolyzed soybean meal aqueous solution; performing second fermentation and drying on the first fermented soybean concentrated protein aqueous solution and the enzymatic hydrolysis soybean meal aqueous solution to obtain the polypeptide-rich feed; the composite microbial inoculum comprises saccharomycetes, lactobacillus and bacillus; the content of crude protein in the soybean meal is more than or equal to 46%; the first enzymolysis, the first fermentation, the second enzymolysis, the third enzymolysis and the second fermentation are all carried out under the condition of stirring.
In the present invention, the corresponding raw materials used are all conventionally purchased unless otherwise specified.
The invention preferably mixes the soy protein concentrate with water. In the invention, the mass ratio of the soybean protein concentrate to the water during mixing is preferably 10-15: 100, more preferably 12:100; the protein content of the soybean protein concentrate is preferably more than or equal to 65%. The addition of the soybean protein concentrate is beneficial to the enrichment and comprehensive nutrition of the solution after enzymolysis, and realizes rapid propagation. The temperature of the water during mixing is preferably 50-60 ℃, more preferably 55 ℃; the manner and apparatus of mixing is not particularly limited and may be employed in any manner and apparatus known in the art.
After the mixing, the soybean concentrated protein aqueous solution, alkaline protease and neutral protease are mixed for first enzymolysis. In the first enzymolysis, the alkaline protease of the invention is preferably added in an amount of 0.1wt.% to 0.3wt.%, more preferably 0.25wt.% of the soy protein concentrate; the enzyme activity of the alkaline protease is preferably 20-25 ten thousand U/g, more preferably 25 ten thousand U/g; the neutral protease is preferably added in an amount of 0.1wt.% to 0.15wt.%, more preferably 0.15wt.% of the soy protein concentrate; the enzyme activity of the neutral protease is preferably 4-5 ten thousand U/g, more preferably 5 ten thousand U/g. The efficiency of protein decomposition into polypeptides is facilitated by the addition of alkaline protease and neutral protease.
The first enzymolysis is carried out under the condition of stirring, and the stirring speed is preferably 20-40 rpm, more preferably 32rpm; the time of the first enzymolysis is preferably 2-6 hours, more preferably 4 hours, and the temperature is preferably 50-60 ℃, more preferably 55 ℃; the pH of the solution at the time of the first enzymolysis is preferably 6.5 to 7.0, more preferably 7.0. The condition of the first enzymolysis is reasonably regulated, so that the materials are uniformly mixed, and the enzymolysis reaction is more complete.
After the first enzymolysis, inoculating a composite microbial inoculum into the soybean concentrated protein aqueous solution after the first enzymolysis for first fermentation; the composite microbial inoculum comprises saccharomycetes, lactobacillus and bacillus. The invention is preferably carried out when the temperature of the soybean protein concentrate aqueous solution after the first enzymolysis is 35-38 ℃, and more preferably the temperature is 38 ℃; the inoculation amount of the composite microbial inoculum is preferably that the soybean concentrated protein aqueous solution after the first enzymolysis0.1wt.% to 0.2wt.%, more preferably 0.2wt.% of soy protein concentrate; the viable count of the saccharomycetes in the composite microbial inoculum is preferably more than or equal to 4 multiplied by 10 9 CFU/g, the viable count of lactobacillus is preferably not less than 6×10 9 CFU/g, the viable count of bacillus is preferably not less than 6×10 9 CFU/g. The addition of saccharomycetes, lactobacillus and bacillus is favorable for increasing the aromatic flavor of the product, improving the taste of the product, increasing the phagostimulant, and obviously degrading the anti-nutritional factors such as oligosaccharides in the soybean meal.
The first fermentation according to the present invention is carried out under stirring, preferably at a speed of 20 to 40rpm, more preferably 32rpm; the temperature of the first fermentation is preferably 35 to 38 ℃, more preferably 38 ℃, and the time is preferably 8 to 16 hours, more preferably 14 hours. The soybean concentrated protein aqueous solution after the first fermentation can be prepared through the first fermentation, has high concentration and large amount of amplified microbial flora, and is beneficial to quickly reaching the fermentation end point during the second fermentation.
The present invention preferably mixes the ground soybean meal with water while preparing the first fermented soybean concentrated protein aqueous solution. In the invention, the content of the crude protein of the soybean meal is more than or equal to 46%; the particle size of the crushed soybean meal is preferably 60-80 meshes, more preferably 80 meshes; the mass ratio of the crushed soybean meal to the water is preferably 25-30: 100, more preferably 25:100; the temperature of the water at the time of mixing is preferably 50 to 60 ℃, more preferably 55 ℃; the stirring speed of the mixing is preferably 20 to 40rpm, more preferably 32rpm; the stirring time is not particularly required, and the stirring is uniform.
After the mixing, the invention mixes the soybean meal aqueous solution, the cellulase and the alpha-galactosidase for the second enzymolysis. In the second enzymolysis, the addition amount of the cellulase of the invention is preferably 0.05wt.% to 0.1wt.%, more preferably 0.8wt.% of the soybean meal in the soybean meal aqueous solution; the enzyme activity of the cellulase is preferably 1.9-2.0 ten thousand U/g, more preferably 2 ten thousand U/g; the amount of the α -galactosidase added is preferably 0.05wt.% to 0.1wt.%, more preferably 0.1wt.% of the soybean meal in the aqueous solution of the soybean meal; the enzyme activity of the alpha-galactosidase is preferably 2000-3000 ten thousand U/g, more preferably 3000U/g. The addition of cellulase and alpha-galactosidase is favorable for degrading non-starch polysaccharide in soybean meal and reducing the anti-nutritional factors, and can also reduce the viscosity of feed liquid.
The second enzymolysis is carried out under stirring, and the stirring speed is preferably 20-40 rpm, more preferably 32rpm; the time of the second enzymolysis is preferably 1-2 hours, more preferably 2 hours, and the temperature is preferably 50-60 ℃, more preferably 55 ℃; the pH of the solution at the time of the second enzymatic hydrolysis is preferably 6.5 to 7.0, more preferably 7.0.
After the second enzymolysis, the invention mixes the enzymolysis liquid obtained by the second enzymolysis, alkaline protease and neutral protease for the third enzymolysis. In the third enzymolysis, the addition amount of the alkaline protease is preferably 0.1wt.% to 0.3wt.%, more preferably 0.25wt.% of the soybean meal in the soybean meal aqueous solution; the enzyme activity of the alkaline protease is preferably 20-25 ten thousand U/g, more preferably 25 ten thousand U/g; the addition amount of the neutral protease is preferably 0.1wt.% to 0.3wt.%, more preferably 0.25wt.% of the soybean meal in the soybean concentrated protein aqueous solution; the enzyme activity of the neutral protease is preferably 4-5 ten thousand U/g, more preferably 5 ten thousand U/g. After alkaline protease and neutral protease are added, the enzymolysis efficiency is improved, and the polypeptide content is increased.
The third enzymolysis is carried out under the condition of stirring, and the stirring speed is preferably 20-40 rpm, more preferably 32rpm; the time of the third enzymolysis is preferably 3-5 h, more preferably 5h, and the temperature is preferably 50-60 ℃ and more preferably 55 ℃; the pH of the solution at the time of the third enzymatic hydrolysis is preferably 6.5 to 7.0, more preferably 7.0. And the soybean meal aqueous solution after enzymolysis is prepared after the third enzymolysis, which is favorable for the rapid mass propagation of microorganisms and rapidly reaches the fermentation end point.
After the third enzymolysis, the soybean concentrated protein aqueous solution after the first fermentation is mixed with the soybean meal aqueous solution after the enzymolysis for the second fermentation. The mixing is preferably performed at a temperature of 35-38 ℃ of the aqueous solution of the soybean meal after enzymolysis, more preferably at a temperature of 38 ℃; the volume ratio of the soybean concentrated protein aqueous solution after the first fermentation to the soybean meal aqueous solution after the enzymolysis during the mixing is preferably 1:3 to 5, more preferably 1:5.
the second fermentation according to the invention is carried out under stirring, preferably at a speed of 20 to 40rpm, more preferably 32rpm; the second fermentation time is preferably 2 to 4 hours, more preferably 3 hours, and the temperature is preferably 35 to 38 ℃, more preferably 38 ℃. After the second fermentation, the soybean meal is favorable for thoroughly degrading anti-nutritional factors such as soybean antigen, oligosaccharide and the like in the soybean meal, increasing the fragrance of fermentation and improving the palatability.
The first enzymolysis and the first fermentation are preferably carried out in the same container; the second enzymolysis, the third enzymolysis and the second fermentation are preferably performed in the same container; the container is preferably a container having a breathing orifice. The invention is not limited to the source of the fermented container, and the fermented container can be purchased conventionally.
After the second fermentation, the present invention preferably dries the liquid after the second fermentation. The air inlet temperature during drying is preferably 170-180 ℃, more preferably 180 ℃, the air outlet temperature during drying is preferably 80-85 ℃, the drying time of the materials in the drying tower is preferably 7-10 s, and the frequency of the atomizer frequency converter during drying is preferably 42-48 Hz. The biological activity of the material is maintained to the maximum extent by reasonably regulating and controlling the drying condition, which is favorable for maintaining the color, the taste, the aroma and the solubility of the material.
The invention provides the polypeptide-rich feed prepared by the method in the technical scheme, wherein the acid soluble protein in the polypeptide-rich feed is more than or equal to 56%, the crude protein content is more than or equal to 50%, and the pH value is less than or equal to 5.0. Experiments prove that in the polypeptide-rich feed prepared by the invention, soybean protein antigen and anti-nutritional factors are thoroughly eliminated, so that the cost of the feed is reduced.
For further explanation of the present invention, a polypeptide-rich feed and a preparation method thereof according to the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Experimental example 1
The enzyme preparation has the effect on the enzymolysis effect of the soybean meal, and comprises the following steps:
1) 100g of 80-mesh soybean meal is taken and added with 400g of water for dissolution, so as to obtain an aqueous solution of the soybean meal. Wherein the physical and chemical indexes of the bean pulp are as follows: the content of crude protein in the soybean meal is more than or equal to 46%, the content of crude ash is less than or equal to 7% and the content of water is less than or equal to 13%, and the manufacturer is Henan sunlight oil group limited company;
2) Adding 1.74g of compound protease No. 1 (the addition amount of the compound protease No. 1 is 1.74wt.% of the soybean meal in the step 1) into the soybean meal aqueous solution in the step 1), and adjusting the pH of the soybean meal aqueous solution to 8.0 by using a sodium hydroxide solution to obtain a soybean meal aqueous solution containing an enzyme preparation; wherein the mass ratio of alkaline protease to neutral protease in the composite protease No. 1 is 7:3, i.e. consisting of 1.218g of alkaline protease and 0.522g of neutral protease; the enzyme activity of the alkaline protease is 25 ten thousand U/g, and the enzyme activity of the medium alkaline egg is 5 ten thousand U/g; both alkaline and neutral proteases were purchased from nanning Pang Bo bioengineering limited;
3) Placing the soybean meal aqueous solution containing the enzyme preparation in the step 2) into a water bath kettle with the temperature of 55 ℃, stirring by a stirrer, observing the enzymolysis speed, placing the soybean meal aqueous solution into a constant-temperature drying oven for drying overnight with the temperature of 65 ℃ after enzymolysis for 6 hours, and measuring crude protein, ash, moisture, acid-soluble protein and HPLC after crushing.
Experimental example 2
The difference from example 1 is that 1.74g of composite protease No. 2 (the addition amount of composite protease No. 2 is 1.74wt.% of the soybean meal in step 1)) was added in step 2), and the pH of the aqueous soybean meal solution was adjusted to 8.0 with sodium hydroxide solution to obtain an aqueous soybean meal solution containing an enzyme preparation; wherein the mass ratio of alkaline protease to neutral protease in the composite protease No. 2 is 7:3, i.e. consisting of 1.218g of alkaline protease and 0.522g of neutral protease; the enzyme activity of the alkaline protease is 25 ten thousand U/g, and the enzyme activity of the medium alkaline egg is 5 ten thousand U/g; both alkaline protease and neutral protease were purchased from the wuhan new warrior company limited.
Experimental example 3
The difference from example 1 is that in step 2), 1.74g of hydrolase is added, and the aqueous solution of soybean meal containing the enzyme preparation is obtained without adjusting the pH of the aqueous solution of soybean meal. Wherein the enzyme activity of the alkaline protease purchased from Norou technology development Co., tianjin is 25 ten thousand U/g, and the enzyme activity of the medium alkaline egg is 5 ten thousand U/g.
Experimental example 4
The difference from example 1 is that 1.74g of the complex protease 3 (the amount of the complex protease 3 added is 1.74wt.% of the soybean meal in step 1)) was added in step 2), and the pH of the aqueous soybean meal solution was not required to be adjusted, to obtain an aqueous soybean meal solution containing an enzyme preparation; wherein the mass ratio of the alkaline protease to the neutral protease in the composite protease No. 3 is 1:1, namely, the composite protease consists of 0.87g of alkaline protease and 0.87g of neutral protease; the enzyme activity of the alkaline protease is 25 ten thousand U/g, and the enzyme activity of the neutral egg is 5 ten thousand U/g; both alkaline protease and neutral protease were purchased from the company of biosciences, limited biosciences, henghua, east, nanning.
Experimental example 5
The difference from example 1 is that 1.74g of the complex protease No. 4 was added in step 2) (the amount of the complex protease No. 4 added was 1.74wt.% of the soybean meal in step 1)), and the pH of the aqueous soybean meal solution was not required to be adjusted to obtain an aqueous soybean meal solution containing an enzyme preparation; wherein the mass ratio of the alkaline protease to the neutral protease in the composite protease No. 4 is 1:1, namely, the composite protease consists of 0.87g of alkaline protease and 0.87g of neutral protease; the enzyme activity of the alkaline protease is 25 ten thousand U/g, and the enzyme activity of the medium alkaline egg is 5 ten thousand U/g; alkaline protease is purchased from Henan Yangshao Biochemical engineering Co., ltd, and neutral protease is purchased from Nanning Donghenghua Biotechnology Co., ltd.
Results and analysis
1) The enzymatic fluidity of experimental examples 1 to 5 was analyzed, and the viscosity of the material was measured using a calibrated viscometer having a viscosity of 2mpa.s for water measured at 25℃and the results are shown in Table 1 (0 h in Table 1 means the moment of adding the enzyme preparation).
TABLE 1 flowability observations during enzymolysis
As can be seen from table 1, the viscosity of the feed liquid gradually becomes thinner as the addition amount of alkaline protease in the formula increases; in the same enzyme preparation formula experiment, the viscosity of the feed liquid is slightly reduced and the fluidity is increased after the PH is adjusted to 8.0 by sodium hydroxide.
Crude proteins, crude ash, moisture, acid soluble proteins and HPLC analysis were measured for the products after enzymolysis pulverization in experimental examples 1 to 5, see standard NYT2218-2012, and specific results are shown in Table 2.
TABLE 2 influence of different enzymatic hydrolysis on the conversion of products
As can be seen from Table 2, the pH adjusted acid soluble eggs of groups 1 and 2 were higher than those of the control groups 4 and 5, and the corresponding molecular weight of group pH8.0 was smaller in the molecular weight distribution of HPLC. Meanwhile, the alkaline enzyme has high action speed, and the addition amount of the alkaline enzyme and the neutral enzyme is close to 7:3, the enzymolysis effect and speed can be obviously improved, but the difference of enzymes of different factories is not obvious.
Example 1
A method for preparing polypeptide-rich feed comprises the following steps:
1) Preparing strain propagation materials by proportioning: weighing 400kg of soybean protein concentrate (the mass content of protein is more than or equal to 65%, shandong's Wang Shiye Co., ltd.), adding 55 ℃ water into a reaction tank, mixing to prepare a feed liquid with a mass ratio of 15%, adding 0.3wt.% alkaline protease (25 ten thousand units of enzyme activity) and 0.15wt.% neutral protease (5 ten thousand units of enzyme activity) of the feed liquid soybean protein concentrate, and stirring at 55 ℃ and 32rpm for enzymolysis for 2 hours (both the alkaline protease and the neutral protease are purchased from Henan Yangshao bioengineering Co., ltd.);
2) Inoculating strain for propagation: after enzymolysis, cooling the material obtained in the step 1) to 38 ℃, and inoculating a composite microbial inoculum consisting of saccharomycetes, lactobacillus and bacillus: the inoculation amount of the composite microbial inoculum is 0.1wt.% of the soybean protein concentrate in the step 1); composite microbial inoculumThe number of viable bacteria of the medium yeast is more than or equal to 4 multiplied by 10 9 CFU/g, the viable count of lactobacillus is more than or equal to 6×10 9 CFU/g, the viable count of bacillus is more than or equal to 6 multiplied by 10 9 CFU/g, yeast, lactobacillus and bacillus are purchased from Qingdao blue organism Co., ltd, and are subjected to stirring fermentation culture at 32rpm (the fermentation temperature is 38 ℃ C., the fermentation time is 12 hours) to obtain a fermented soybean concentrated protein aqueous solution for later use;
3) Taking 2000kg of crushed 60-mesh soybean meal (the soybean meal is purchased from Jiangsu Hui Fu protein technology Co., ltd.; 46% of crude protein of soybean meal), preparing a feed liquid with the mass ratio of the soybean meal of 25% in a reaction tank by using water with the temperature of 55 ℃ until the feed liquid is uniformly mixed and stirred;
4) Adding 0.1wt.% cellulase (enzyme activity of 2 ten thousand units) and 0.1wt.% alpha-galactosidase (enzyme activity of 250U/mg) into the feed liquid obtained in the step 3), and stirring and hydrolyzing at 55 ℃ for 1h at 32rpm to obtain an hydrolyzed soybean meal aqueous solution; (cellulase and alpha-galactosidase were purchased from Jiangsu Yi agricultural organisms Co., ltd.)
5) Adding 0.3wt.% alkaline protease (25 ten thousand units of enzyme activity) and 0.15wt.% neutral protease (5 ten thousand units of enzyme activity) of the soybean meal into the aqueous solution of the soybean meal obtained after the enzymolysis in the step 4), and stirring at 55 ℃ for enzymolysis for 2 hours at 32rpm; obtaining an aqueous solution of the soybean meal after enzymolysis;
6) Cooling the soybean meal aqueous solution in the step 5) to 35-38 ℃ for standby;
7) Mixing the soybean meal aqueous solution obtained in the step 6) and the fermented soybean concentrated protein aqueous solution obtained in the step 2) according to a volume ratio of 5:1, stirring and fermenting for 5 hours at 32rpm, wherein the fermentation temperature is 38 ℃;
8) Spray-drying the fermented product of step 7) (specific conditions during drying are: the air inlet temperature is 170-180 ℃, the air outlet temperature is 80-85 ℃, the frequency of an atomizer is 44 Hz), and the finished product is obtained after passing through a 60-mesh sieve and removing impurities.
Example 2
The difference from example 1 is that in step 5) the enzyme is digested for 4 hours; fermenting for 3h in the step 7).
Example 3
The difference from example 1 is that in step 5) the enzymatic hydrolysis is carried out for 5h; fermenting for 4h in the step 7).
Comparative example 1
The difference from example 1 is that only lactic acid bacteria were inoculated in step 2) in an amount of 50 g/ton of material; the viable count of lactobacillus is more than or equal to 6 multiplied by 10 9 CFU/g。
Comparative example 2
The difference with the example 1 is that in the step 2), only the composite microbial inoculum composed of saccharomycetes and bacillus is inoculated, and the inoculation amount is 200 g/ton of material; the viable count of the saccharomycetes in the composite microbial inoculum is more than or equal to 4 multiplied by 10 9 CFU/g, the viable count of bacillus is more than or equal to 6 multiplied by 10 9 CFU/g。
Comparative example 3
The difference from example 1 is that the fermentation temperature in step 7) is 36 ℃.
Comparative example 4
The difference from example 1 is that the temperature of the fermentation in step 7) is 42 ℃.
Comparative example 5
The difference from example 1 is that step 4) is omitted, i.e. no cellulase and no alpha-galactosidase are added.
Comparative example 6
The difference from example 1 is that the soybean protein concentrate aqueous solution obtained after fermentation of the aqueous soybean meal solution obtained in step 7) was used in a volume ratio of 3:1
Comparative example 7
The difference from example 1 is that no stirring process is involved in steps 1) to 8).
Comparative example 8
The difference from example 1 is that the experiment was carried out starting from step 3), the seed of step 2) was not added in step 7), but the seed was directly inoculated in the amount of step 1), and fermentation was carried out for 12 hours.
Results and analysis:
1) The finished products prepared in examples 1 to 3 and comparative examples 1 to 8 were examined, and the detection indexes are shown in Table 3.
TABLE 3 nutritional index for preparing finished products by different treatment methods
Project Moisture content Coarse ash Crude protein Acid soluble proteins Protein solubility pH
Detection method GB/T6435 GB/T6438 GB/T6432 GB/T22492 GB/T19541 GB/T8884
Example 1 5.58 7.90 50.94 45.50 75.30 5.38
Example 2 5.49 7.72 51.17 54.80 77.40 5.80
Example 3 5.67 7.87 50.46 56.21 76.10 4.89
Comparative example 1 5.78 7.49 50.19 47.22 74.3 5.22
Comparative example 2 5.46 7.34 50.46 46.93 77.3 5.31
Comparative example 3 5.80 7.45 50.29 54.19 72.99 4.91
Comparative example 4 5.24 7.59 51.09 53.94 73.24 5.24
Comparative example 5 5.10 7.98 50.11 48.77 78.91 5.53
Comparative example 6 5.59 8.10 53.81 56.41 75.23 4.45
Comparative example 7 5.33 7.69 50.77 34.47 68.26 5.87
Comparative example 8 5.68 7.73 50.69 53.28 77.64 6.27
As can be seen from Table 3, in comparative examples 1 to 2, soybean antigen was not completely eliminated when only 1 to 2 strains were used; the fermentation temperature in comparative examples 3 to 4 was 36 to 42℃and the pH of the product was slightly higher than that in example 3; in comparative examples 5 to 6, no cellulase or galactosidase was added, and the acid soluble protein of the product was significantly lower than in example 3. In the step of 2 hours of enzymolysis in example 1, even if the post fermentation step is prolonged, the acid soluble protein of the final product is lower than in examples 2 and 3, indicating that the degree of hydrolysis is insufficient; in the embodiment 2, the enzymolysis time is 4 hours, the fermentation time is shortened to 3 hours, the pH value of the product is higher, the effect of the fermentation process is not achieved, and the electrophoresis detection result also shows that the fermentation time needs to be prolonged; the acid soluble protein of example 3 exceeds 56% and the pH is less than 5.0, which is a preferred embodiment of the present invention.
2) The finished test stachyose and raffinose contents (test by the company limited by the European analysis service (Suzhou)) prepared in example 3 were compared with the average stachyose and raffinose contents in the known fermented soybean meal, and the results are shown in Table 4. (wherein, the average content sources of stachyose and raffinose in the fermented soybean meal are shown in the known documents of Li Ying, han Yunsheng, zhao Qingyu, and the like. The soybean meal is compared with the main nutrition components, anti-nutritional factors and in-vitro digestibility in the fermented soybean meal for analysis, DOI 10.15906/j.cnki.cn11-2975/s.20192218).
TABLE 4 determination of stachyose and raffinose content in example 3
As can be seen from Table 4, the degradation effect of the zymolytic protein on stachyose and raffinose is good and reaches a level lower than the limit level of HPLC detection.
Example 9
The results of the enzymatic fermentation of peeled Thuja occidentalis using the protocol of example 3 for 5 batches (numbered 20200604, 20200605, 20200606, 20200607, 20200608 respectively) and gel electrophoresis antigen detection of the 5 batches (and comparison of Thuja occidentalis raw material) are shown in FIG. 1.
As can be seen from FIG. 1, the color of the antigen protein bands of each batch of products is very light and the outline thereof can only be distinguished in a fuzzy manner compared with the unfermented soybean meal, which indicates that the inhibition factors such as beta-conglycinin, alpha-globulin, trypsin and the like in the soybean meal are completely degraded after the technical scheme provided by the invention is adopted.
In conclusion, after the technical scheme provided by the invention is adopted to carry out enzymolysis and fermentation on the soybean meal, the contents of crude protein and acid soluble protein in the product are more than 50%, and substances such as stachyose, raffinose and the like are thoroughly eliminated. Therefore, the polypeptide-rich feed prepared by the invention is simple and convenient to operate and has high polypeptide content.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A method of preparing a polypeptide-rich feed comprising the steps of:
mixing the soybean concentrated protein aqueous solution, alkaline protease and neutral protease for first enzymolysis, and inoculating a composite microbial inoculum into the soybean concentrated protein aqueous solution after the first enzymolysis for first fermentation to obtain a soybean concentrated protein aqueous solution after the first fermentation;
mixing the soybean meal aqueous solution, cellulase and alpha-galactosidase for second enzymolysis, mixing an enzymolysis solution obtained by the second enzymolysis, alkaline protease and neutral protease for third enzymolysis, and obtaining the soybean meal aqueous solution after enzymolysis;
performing second fermentation and drying on the first fermented soybean concentrated protein aqueous solution and the enzymatic hydrolysis soybean meal aqueous solution to obtain the polypeptide-rich feed;
the composite microbial inoculum comprises saccharomycetes, lactobacillus and bacillus;
the content of crude protein in the soybean meal is more than or equal to 46%;
the first enzymolysis, the first fermentation, the second enzymolysis, the third enzymolysis and the second fermentation are all carried out under the condition of stirring.
2. The method according to claim 1, wherein the mass ratio of the soy protein concentrate to water in the soy protein concentrate aqueous solution is 10 to 15:100;
in the first enzymolysis, the addition amount of the alkaline protease is 0.1wt.% to 0.3wt.% of the soybean protein concentrate; the addition amount of the neutral protease is 0.1wt.% to 0.15wt.% of the soybean protein concentrate;
the conditions of the first enzymolysis comprise: the time is 2-6 h, and the temperature is 50-55 ℃.
3. The method of claim 1, wherein the amount of inoculation of the composite microbial inoculum is 0.1wt.% to 0.2wt.% of soy protein concentrate in the soy protein concentrate aqueous solution;
the viable count of the saccharomycetes in the composite microbial inoculum is more than or equal to 4 multiplied by 10 9 CFU/g, the viable count of lactobacillus is more than or equal to 6×10 9 CFU/g, the viable count of bacillus is more than or equal to 6 multiplied by 10 9 CFU/g。
4. A method according to any one of claims 1 to 3, wherein the conditions of the first fermentation comprise: the temperature is 35-38 ℃ and the time is 8-16 h.
5. The method according to claim 1, wherein the mass ratio of the soybean meal to water in the aqueous solution of the soybean meal is 25-30: 100;
in the second enzymolysis, the addition amount of the cellulase and the alpha-galactosidase is 0.05wt.% to 0.1wt.% of the soybean meal respectively;
the conditions of the second enzymolysis comprise: the temperature is 50-55 ℃ and the time is 1-2 h.
6. The method according to claim 1, wherein the alkaline protease and the neutral protease are added in an amount of 0.1wt.% to 0.3wt.% of the soybean meal, respectively, in the third enzymatic hydrolysis;
the conditions of the third enzymolysis comprise: the time is 3-5 h, and the temperature is 50-55 ℃.
7. The method according to claim 1, wherein in the second fermentation, the volume ratio of the first fermented soybean protein concentrate aqueous solution to the enzymatic soybean meal aqueous solution is 1:3 to 5;
the conditions of the second fermentation include: the temperature is 35-38 ℃ and the time is 2-4 h.
8. The method of claim 1, wherein the stirring speed is 20 to 40rpm.
9. The method of claim 1, wherein the drying conditions comprise: the air inlet temperature is 170-180 ℃, the air outlet temperature is 80-85 ℃ and the time is 7-10 s; during drying, the frequency of the atomizer frequency converter is 42-48 Hz.
10. The polypeptide-rich feed prepared by the method of any one of claims 1 to 9, which is characterized in that the content of acid soluble protein in the polypeptide-rich feed is more than or equal to 56%, the content of crude protein is more than or equal to 50%, and the pH is less than or equal to 5.0.
CN202311485311.7A 2023-11-08 2023-11-08 Polypeptide-rich feed and preparation method thereof Pending CN117322507A (en)

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