CN108925747B - Functional protein prepared from aquatic protein and yeast and preparation method thereof - Google Patents
Functional protein prepared from aquatic protein and yeast and preparation method thereof Download PDFInfo
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- 238000004806 packaging method and process Methods 0.000 claims abstract description 4
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- 238000000855 fermentation Methods 0.000 claims description 53
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- 229940088598 enzyme Drugs 0.000 claims description 18
- 239000002351 wastewater Substances 0.000 claims description 18
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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
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- 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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Abstract
The invention discloses a functional protein prepared by using aquatic protein and yeast and a method thereof, which comprises the following main steps: carrying out liquid culture on the saccharomycetes, and then centrifugally collecting saccharomycetes thalli; adding the collected saccharomycete thalli into sterile water for suspension to obtain saccharomycete suspension containing 200-500 g/L saccharomycete dry matter; adding an enzyme preparation and a high-temperature resistant yeast agent into the yeast suspension, and carrying out enzymolysis for 3-10 h at the temperature of 42-45 ℃ and the rotating speed of 50-100 rpm to obtain yeast hydrolysate; mixing yeast hydrolysate, aquatic protein and soybean meal according to a certain proportion, and adding a bacillus subtilis microbial inoculum for solid state fermentation; drying, crushing and packaging the fermented product to prepare a functional protein finished product. The functional protein prepared by the invention can be used as a fish meal replacement protein, so that the feed cost is reduced; but also can be used for preparing green feed so as to improve the quality of the feed, and the invention has good application prospect and practical significance.
Description
Technical Field
The invention relates to a method for preparing functional protein by using aquatic protein and yeast, belonging to the technical field of bioengineering.
Background
The yeast hydrolysate is prepared by using natural yeast as a raw material, utilizing endogenous enzyme and exogenous enzyme for directional hydrolysis, concentrating and drying, and fully releasing the content substances of yeast cells by a series of technological means so as to maximize the efficacy of functional substances of the yeast hydrolysate. A large number of studies at home and abroad show that the yeast hydrolysate is rich in functional components such as nucleotide, B vitamins, zymosan and the like, and the yeast-derived functional components can not only promote the growth of the raised animals, but also improve the immunity of the raised animals. However, the preparation of the yeast hydrolysate needs to be processed by the steps of enzymolysis, concentration, drying and the like, and the production cost is high, so that the yeast hydrolysate is difficult to be applied in the feed industry in a large amount.
The fermented soybean meal takes high-quality soybean meal as a main raw material, and by using protease generated by microorganisms under the fermentation of microbial strains, not only can antigen protein in the soybean meal be degraded, but also the digestion and absorption performance of prepared feed protein can be improved by being rich in small peptides. In recent years, the fermented soybean meal as a fish meal replacement protein is widely applied to the feed industry in China. However, compared with fish meal, the fermented soybean meal has defects in essential amino acids, growth promoting factors and the like, so that the fermented soybean meal is difficult to replace the fish meal in a large amount in aquatic feeds, lacks functional components and is only used as a fish meal replacement protein in the feed industry.
Disclosure of Invention
In order to solve the problems of the yeast hydrolysate and the fermented soybean meal, the invention aims to provide the method for preparing the functional protein by using the aquatic protein and the yeast, the process is simple and easy to operate, and the prepared functional protein can greatly replace imported fish meal and reduce the feed cost.
Another object of the present invention is to provide a functional protein prepared by the above method.
In order to solve the problems, the technical scheme adopted by the invention is as follows: a method for preparing functional protein by using aquatic protein and yeast is characterized by comprising the following steps:
(1) inoculating saccharomycete into liquid fermenting culture medium, fermenting at 28-32 deg.c to obtain fermented liquid with biomass of 20-70 g/L, and centrifuging to collect saccharomycete thallus;
(2) adding the yeast thallus collected in the step (1) into sterile water for suspension to obtain yeast suspension containing 200-500 g/L yeast dry matter;
(3) adding an enzyme preparation and a high-temperature resistant yeast agent into the yeast suspension obtained in the step (2), and carrying out enzymolysis for 3-10 h under the conditions that the temperature is 42-45 ℃ and the rotating speed is 50-100 rpm to obtain yeast hydrolysate;
(4) mincing low-value aquatic protein for later use or evaporating and concentrating the aquatic product processing wastewater until the protein content is 10-20% for later use; pulverizing soybean meal and sieving for later use;
(5) firstly, mixing the low-value aquatic protein ground in the step (4) or concentrated aquatic product processing wastewater and soybean meal according to a mass ratio of 0.2-0.5: 1, mixing; then adding 10-80% of the yeast hydrolysate obtained in the step (3) into the mixture, and uniformly mixing to obtain a solid fermentation culture medium;
(6) inoculating a bacillus subtilis microbial inoculum into the solid fermentation culture medium obtained in the step (5), and uniformly mixing;
(7) fermentation of raw materials: the fermentation mode is closed solid state fermentation, the fermentation temperature is controlled within the range of 30-50 ℃, and the fermentation time is 60-72 h;
(8) and (3) finished product treatment: and (4) drying, crushing and packaging the product obtained after the fermentation in the step (7) to obtain a functional protein finished product prepared from the aquatic protein and the yeast.
The yeast in the step (1) is one of saccharomyces cerevisiae, candida utilis and rhodotorula mucilaginosa.
The enzyme preparation added in the step (3) is a mixture consisting of cellulase, beta-glucanase, beta-mannanase and protease.
The adding amount of the enzyme in the step (3) is respectively as follows according to the mass percentage of the added enzyme in the total mass of the yeast suspension: 0.001-2% of cellulase, 0.001-2% of beta-glucanase, 0.001-2% of beta-mannase and 0.001-2% of protease.
The adding amount of the high-temperature resistant yeast agent in the step (3) is 0.001-2% of the total mass percentage of the yeast suspension by the added mass.
The high-temperature resistant yeast agent in the step (3) is high-temperature resistant yeast which can resist the fermentation temperature of 42-45 ℃.
The low-value aquatic protein in the step (4) is one or a mixture of more than two of tilapia leftovers, antarctic krill, shrimp heads, scallop skirts, cod leftovers, squid leftovers, green scale fish leftovers, black carp leftovers, silver carp leftovers, grass carp leftovers or bonito leftovers.
The waste water from the water product processing in the step (4) is one or more of fish paste from fish meal processing, prawn cooking waste water, surimi processing waste water or alkali washing waste water from chitin processing.
In the step (6), the addition amount of the bacillus subtilis microbial inoculum accounts for 0.001-2% of the total mass percentage of the solid fermentation culture medium by the added mass.
The functional protein prepared by the method.
Compared with the prior art, the invention has the following advantages and characteristics:
(1) in the yeast hydrolysis process, the yeast dry matter content in the yeast suspension is high, and the hydrolyzed yeast hydrolysate does not need to be concentrated, so that the yeast hydrolysis efficiency can be improved, and the yeast hydrolysis cost can be reduced;
(2) in high concentration yeast suspension, the existing process simply depends on enzymolysis, and the wall breaking rate of yeast cells is very low. The invention discovers that the wall breaking rate of yeast cells in high-concentration yeast suspension can reach more than 90 percent by utilizing the synergistic action of enzymolysis and high-temperature resistant yeast fermentation, and obtains unexpected technical effects. For example, in yeast suspension containing 400g/L of yeast dry matter, the wall breaking rate of yeast by adopting an enzymolysis method is only 40%, but in an enzymolysis system with the same enzyme concentration, as long as 0.02% of high temperature resistant yeast agent is added, the wall breaking rate of yeast after enzymolysis can reach 91%, and an unexpected technical effect is obtained.
(3) The preparation of the yeast hydrolysate and the production of the fermented soybean meal are organically combined, and the combined technical characteristics are functionally supported by each other, so that a new technical effect is achieved: after the yeast hydrolysis is finished, the yeast can be directly mixed with the bean pulp and then fermented without enzyme deactivation, so that the cost can be reduced, and meanwhile, the residual enzyme preparation in the hydrolysate can carry out enzymolysis on the bean pulp, and the fermentation effect of the bean pulp is improved. And the yeast releases nutrient substances after hydrolysis, which is beneficial to the bacillus subtilis to produce enzyme, so that the fermentation effect of the soybean meal can be further improved. On the other hand, the yeast which is remained in the previous enzymolysis process and has not been subjected to wall breaking can be subjected to wall breaking under the fermentation of the bacillus subtilis, so that the wall breaking rate of the yeast in the whole process can be improved.
(4) The production of the yeast hydrolysate is organically combined with the preparation of the fermented feed protein, so that the prepared fermented feed protein is rich in yeast-derived functional components, and the post-treatment process of the yeast hydrolysate is integrated into the post-treatment process of the fermented feed protein, thereby greatly reducing the application cost of the yeast hydrolysate in the feed and promoting the application of the yeast hydrolysate in the feed industry.
(5) The prepared product overcomes the defects of insufficient essential amino acid and growth promoting factors of the fermented soybean meal by the mixed fermentation of the aquatic protein and the soybean meal, and can replace a large amount of fish meal in feed.
The breeding test of the functional protein rich in the aquatic source functional components and the yeast source functional components in animals such as pigs, chickens, prawns, tilapia and the like in south China shows that the fish meal replacement protein prepared by the method can greatly replace imported fish meal and reduce the feed cost; but also can enhance the immunity and disease resistance of animals.
Detailed Description
The invention relates to a method for preparing functional protein by using aquatic protein and yeast, which comprises the following steps:
(1) activating saccharomycete with slant culture medium, seed expanding culture with seed culture medium, inoculating to liquid fermenting culture medium, fermenting at 28-32 deg.c to obtain fermented liquid with biomass of 20-70 g/L, and centrifuging to collect saccharomycete. Preferably, the yeast is selected from one of saccharomyces cerevisiae, candida utilis and rhodotorula mucilaginosa. For example, the saccharomyces cerevisiae used is saccharomyces cerevisiae CICC1540 strain of the chinese industrial microbial strain collection center; the used candida utilis is candida utilis CICC1314 strain of China industrial microorganism strain collection center; the Rhodotorula mucilaginosa is Rhodotorula mucilaginosa CICC31029 strain of China center for industrial microorganism culture collection. The slant culture medium and the seed culture medium are conventional YEPD culture media, and the liquid fermentation culture medium is as follows: molasses 8%, ammonium sulfate 1%, peptone 0.5%, potassium dihydrogen phosphate 0.5%.
(2) Adding the yeast thallus collected in the step (1) into sterile water for suspension to obtain yeast suspension containing 200-500 g/L of yeast dry matter, wherein the yeast suspension with high concentration ensures that the hydrolyzed yeast hydrolysate does not need to be concentrated.
(3) And (3) adding an enzyme preparation and a high-temperature resistant yeast agent into the yeast suspension obtained in the step (2), and carrying out enzymolysis for 3-10 h under the conditions that the temperature is 42-45 ℃ and the rotating speed is 50-100 rpm to obtain yeast hydrolysate. Preferably, the added enzyme preparation is a mixture of cellulase, beta-glucanase, beta-mannanase and protease. More preferably, the adding amount of the enzyme preparation is calculated by the mass of the added enzyme accounting for the total mass percentage of the yeast suspension, and respectively comprises the following steps: 0.001-2% of cellulase, 0.001-2% of beta-glucanase, 0.001-2% of beta-mannase and 0.001-2% of protease. Preferably, the addition amount of the high-temperature resistant yeast agent is 0.001-2% of the total mass percentage of the yeast suspension by the mass of the added microbial agent. The optimal fermentation temperature of the traditional saccharomyces cerevisiae is 28-33 ℃, and generally is not more than 36 ℃. In recent years, many high temperature resistant yeasts capable of fermenting at 42 ℃ to 45 ℃ have been commercially produced, and for example, the high temperature resistant yeast agent used in the embodiment of the present invention is a high temperature resistant yeast agent produced by Angel corporation in Hubei.
(4) Mincing low-value aquatic protein for later use or evaporating and concentrating the aquatic product processing wastewater until the protein content is 10-20% for later use; the soybean meal is crushed and sieved by a 60-mesh sieve for later use. Preferably, the low-value aquatic protein is one of tilapia leftovers, antarctic krill, shrimp heads, scallop skirts, cod leftovers, squid leftovers, green scale fish leftovers, black carp leftovers, silver carp leftovers, grass carp leftovers or bonito leftovers. Preferably, the aquatic product processing wastewater is one of fish paste in a fish meal processing process, prawn cooking wastewater, surimi processing wastewater or alkali washing wastewater in a chitin processing process.
(5) Firstly, mixing the low-value aquatic protein ground in the step (4) or concentrated aquatic product processing wastewater and soybean meal according to a mass ratio of 0.2-0.5: 1, mixing; and (3) adding 10-80% of the yeast hydrolysate obtained in the step (3) into the mixture, and uniformly mixing to obtain the solid fermentation medium.
(6) And (5) inoculating a bacillus subtilis microbial inoculum into the culture medium obtained in the step (5), and uniformly mixing. Preferably, the addition amount of the bacillus subtilis microbial inoculum is 0.001-2% of the total mass percentage of the added microbial inoculum in the fermentation medium.
(7) Fermentation of raw materials: the fermentation mode is closed solid state fermentation, the fermentation temperature is controlled within the range of 30-50 ℃, and cooling measures such as turning and the like are adopted when the material temperature exceeds 50 ℃; the fermentation time is 60-72 h; the fermentation may be carried out in a fermentor, a fermentation tank, or a fermentation bag.
(8) And (3) finished product treatment: drying, crushing and packaging the product obtained after fermentation to obtain a functional protein finished product prepared from the aquatic protein and the yeast.
For a better understanding of the invention, the invention will be further described below with reference to examples regarding the selection of fermentation broths and enzymatic processes, but the scope of the invention is not limited thereto.
Example 1
The saccharomyces cerevisiae used is a saccharomyces cerevisiae CICC1540 strain, a conventional YEPD culture medium is used for activation and seed amplification culture, and a liquid fermentation culture medium for culturing the saccharomyces cerevisiae is as follows: molasses 8%, ammonium sulfate 1%, peptone 0.5%, potassium dihydrogen phosphate 0.5%. The saccharomyces cerevisiae is subjected to seed amplification culture by using a YEPD culture medium, then is cultured in a fermentation tank by using a liquid fermentation culture medium at 30 ℃ until the biomass is 30g/L, and then is centrifuged to collect saccharomycete thalli. And adding the collected yeast thalli into sterile water for suspension to obtain yeast suspension containing 300g/L of yeast dry matter (99% of yeast cells in the yeast suspension are complete and have no wall broken). Adding the yeast suspension into an enzymolysis tank, then adding 0.02% of cellulase, 0.03% of beta-glucanase, 0.03% of beta-mannase and 0.02% of protease into the enzymolysis tank, then adding 0.02% of high temperature resistant yeast agent (produced by Hubei Angel Yeast Co., Ltd.), and carrying out enzymolysis for 8h under the conditions that the temperature is 42 ℃ and the rotating speed is 50rpm, thus obtaining yeast hydrolysate (the yeast wall breaking rate is 92%). Concentrating fish slurry of fish meal processing factory to protein content of 23% for use, pulverizing soybean meal, and sieving with 60 mesh sieve for use. Mixing 600 kg of yeast hydrolysate, 200 kg of concentrated fish paste and 1000 kg of soybean meal, and then adding 3kg of bacillus subtilis microbial inoculum for uniformly mixing; adding a fermentation culture medium of a strain to build a pile, wherein the thickness of the material for building the pile is 130cm, covering and sealing the material for building the pile by using a plastic film, controlling the fermentation temperature within the range of 30-50 ℃, and adopting cooling measures such as turning when the material temperature exceeds 50 ℃, wherein the fermentation time is 65 hours; after the fermentation is finished, the fermentation product is dried and crushed to prepare a functional protein finished product. The detection results of all indexes of the functional protein are as follows: the content of protein is 53%, the content of nucleotide is 2.5%, the content of polysaccharide in yeast cell wall is 2.8%, and the content of acid-soluble protein (in protein) is 25%.
Example 2
The candida utilis is a candida utilis CICC1314 strain of China center for industrial microorganism strain preservation, and is activated and subjected to seed expansion culture by using a conventional YEPD culture medium, wherein the fermentation culture medium for culturing the candida utilis comprises 8 percent of molasses, 1 percent of ammonium sulfate, 0.5 percent of peptone and 0.5 percent of potassium dihydrogen phosphate. The Candida utilis was subjected to seed expansion culture using YEPD medium, followed by culture at 30 ℃ in a fermentation tank using a fermentation medium until the biomass became 40g/L, and then yeast cells were collected by centrifugation. And adding the collected yeast thalli into sterile water for suspension to obtain yeast suspension containing 400g/L of yeast dry matter (99% of yeast cells in the yeast suspension are complete and have no wall broken). Adding the yeast suspension into an enzymolysis tank, then adding 0.03% of cellulase, 0.04% of beta-glucanase, 0.05% of beta-mannase and 0.02% of protease into the enzymolysis tank, then adding 0.03% of high temperature resistant yeast agent (produced by Hubei Angel Yeast Co., Ltd.), and carrying out enzymolysis for 9h under the conditions that the temperature is 42 ℃ and the rotating speed is 60rpm, thus obtaining yeast hydrolysate (the yeast wall breaking rate is 91%). Mincing Antarctic krill for later use, and crushing the soybean meal into 60-mesh sieve for later use. Mixing 600 kg of yeast hydrolysate, 200 kg of antarctic krill and 1000 kg of soybean meal, and then inoculating 3kg of bacillus subtilis microbial inoculum and uniformly mixing; adding a fermentation culture medium of a strain to build a pile, wherein the thickness of the material for building the pile is 130cm, covering and sealing the material for building the pile by using a plastic film, controlling the fermentation temperature within the range of 30-50 ℃, and adopting cooling measures such as turning when the material temperature exceeds 50 ℃, wherein the fermentation time is 70 hours; after the fermentation is finished, the fermentation product is dried and crushed to prepare a functional protein finished product. The detection results of all indexes of the functional protein are as follows: the protein content is 52%, the nucleotide content is 3.2%, the yeast cell wall polysaccharide content is 3.8%, and the acid-soluble protein content (in protein) is 25%.
Claims (6)
1. A method for preparing functional protein by using aquatic protein and yeast is characterized by comprising the following steps:
(1) inoculating saccharomycete into liquid fermenting culture medium, fermenting at 28-32 deg.c to obtain fermented liquid with biomass of 20-70 g/L, and centrifuging to collect saccharomycete thallus; the yeast is one of saccharomyces cerevisiae, candida utilis and rhodotorula mucilaginosa;
(2) adding the yeast thallus collected in the step (1) into sterile water for suspension to obtain yeast suspension containing 200-500 g/L yeast dry matter;
(3) adding an enzyme preparation and a high-temperature resistant yeast agent into the yeast suspension obtained in the step (2), and carrying out enzymolysis for 3-10 h under the conditions that the temperature is 42-45 ℃ and the rotating speed is 50-100 rpm to obtain yeast hydrolysate; the enzyme preparation is a mixture consisting of cellulase, beta-glucanase, beta-mannase and protease, and the added mass of the enzyme preparation accounts for the total mass percent of the yeast suspension, and the enzyme preparation respectively comprises the following components: 0.001-2% of cellulase, 0.001-2% of beta-glucanase, 0.001-2% of beta-mannase and 0.001-2% of protease; the adding amount of the high-temperature resistant yeast agent accounts for 0.001-2% of the total mass percentage of the yeast suspension by the added mass;
(4) mincing low-value aquatic protein for later use or evaporating and concentrating the aquatic product processing wastewater until the protein content is 10-20% for later use; pulverizing soybean meal and sieving for later use;
(5) firstly, mixing the low-value aquatic protein ground in the step (4) or concentrated aquatic product processing wastewater and soybean meal according to a mass ratio of 0.2-0.5: 1, mixing; then adding 10-80% of the yeast hydrolysate obtained in the step (3) into the mixture, and uniformly mixing to obtain a solid fermentation culture medium;
(6) inoculating a bacillus subtilis microbial inoculum into the solid fermentation culture medium obtained in the step (5), and uniformly mixing;
(7) fermentation of raw materials: the fermentation mode is closed solid state fermentation, the fermentation temperature is controlled within the range of 30-50 ℃, and the fermentation time is 60-72 h;
(8) and (3) finished product treatment: and (4) drying, crushing and packaging the product obtained after the fermentation in the step (7) to obtain a functional protein finished product prepared from the aquatic protein and the yeast.
2. The method of claim 1, wherein the functional protein derived from yeast is selected from the group consisting of: the high-temperature resistant yeast agent in the step (3) is high-temperature resistant yeast which can resist the fermentation temperature of 42-45 ℃.
3. The method for preparing functional protein according to claim 1, wherein the method comprises the following steps: the low-value aquatic protein in the step (4) is one or a mixture of more than two of tilapia leftovers, antarctic krill, shrimp heads, scallop skirts, cod leftovers, squid leftovers, green scale fish leftovers, black carp leftovers, silver carp leftovers, grass carp leftovers or bonito leftovers.
4. The method for preparing functional protein according to claim 1, wherein the method comprises the following steps: the waste water from the water product processing in the step (4) is one or more of fish paste from fish meal processing, prawn cooking waste water, surimi processing waste water or alkali washing waste water from chitin processing.
5. The method for preparing functional protein according to claim 1, wherein the method comprises the following steps: in the step (6), the addition amount of the bacillus subtilis microbial inoculum accounts for 0.001-2% of the total mass percentage of the solid fermentation culture medium by the added mass.
6. A functional protein produced by the method of any one of claims 1 to 5.
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