CN106071106B - Fermented cottonseed protein and method for preparing fermented cottonseed protein by solid-state aerobic fermentation - Google Patents
Fermented cottonseed protein and method for preparing fermented cottonseed protein by solid-state aerobic fermentation Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing fermented cottonseed protein by fermenting cottonseed protein and performing solid-state aerobic fermentation. The invention aims to solve the technical problem of providing fermented cottonseed protein. The fermented cottonseed protein is prepared by fermenting the following raw materials in parts by weight: 97-99 parts of cotton meal, 0-1.5 parts of corn flour, 0.7-1.5 parts of mixed yeast solid strain, 0.1-0.4 part of mixed nutrient salt, 0.03-0.1 part of mixed enzyme preparation and water; the total weight of the water accounts for 0.6-0.9 time of the total weight of the other five raw materials. The solid aerobic fermentation preparation method of the fermented cottonseed protein mainly comprises the following steps: the raw materials are uniformly mixed, fermented for 45-48 hours at 30-35 ℃ in an aerobic state, and dried. The method can effectively remove gossypol and anti-nutritional factors in the cottonseed meal, and provides a better way for the effective utilization of the cottonseed meal.
Description
Technical Field
the invention belongs to the technical field of microbial fermentation preparation, and particularly relates to fermented cottonseed protein and a preparation method thereof by preparing the fermented cottonseed protein through solid-state aerobic fermentation.
Background
At present, the breeding industry in China seriously depends on corn-soybean meal type daily ration, particularly the main protein raw material soybean meal is mostly imported and is limited by the international market, so that 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.
The cottonseed meal is a byproduct after oil extraction of the cottonseed, is rich in nutrient components, has more than 40% of protein, is relatively balanced in amino acid composition, and has higher arginine content than soybean meal. The annual cottonseed meal yield of China is about 600 million tons, the resource amount is the first worldwide, but the cottonseed meal contains more toxic gossypol and anti-nutritional factors, and the application of the cottonseed meal in animal daily ration is limited.
Gossypol is mainly present in cotton seed pigment gland, and is a tawny polyphenol pigment which is insoluble in water and soluble in organic solvent. In the process of oil preparation, due to the heat action of steaming, frying, squeezing and the like, most of gossypol is combined with protein and amino acid to become combined gossypol, and the combined gossypol is not absorbed by animals in the digestive tracts of the animals, so the toxicity is low. The other part of gossypol exists in free form, has high toxicity to animals, and especially can cause growth retardation, reproductive performance and production performance reduction and even death due to excessive intake or long intake time of monogastric animals. Young animals have a lower tolerance to gossypol. The content of free gossypol in the untreated cottonseed meal is 800-1100 mg/kg. Due to the existence of toxic gossypol, the use amount of the cottonseed meal in the livestock and poultry feed is 3-6%, and the cottonseed meal is not generally used by young livestock and poultry.
At present, commercial cottonseed protein products only adopt dephenolized cottonseed meal extracted by an organic solvent, the content of free gossypol can be below 400mg/kg, but the extraction method can cause the loss of partial nutrient substances.
The biological treatment method adopted in the prior art is mainly an anaerobic microbial fermentation method, but if the effects of detoxification, anti-nutritional factor elimination, digestion and absorption rate improvement, nutritional value improvement, feeding effect and the like are considered at the same time, the method is very difficult and is difficult to achieve the desired effect. In order to solve the problems, the prior experimental technology mainly adopts complex strain combinations, and different strains are respectively added to decompose corresponding substances, but the methods are usually against the principle of microbiology, and too many strains often mutually pollute each other, influence each other and have poor growth potential; or the strains have different optimal growth conditions and the growth in the same substrate is mutually interfered. In addition, in the fermentation method in the prior art, more bran and corn auxiliary materials (accounting for 10-20%) are often added, mixed meal fermentation is adopted, the content of cottonseed meal in the total raw materials is usually lower than 80%, the content of adverse factors such as gossypol and the like is diluted, the difficulty is far lower than that of single cottonseed meal fermentation, and the requirement of a single feed raw material cannot be met (the single component accounts for more than 95%).
from a microbiological point of view, for a raw material substrate, a unique strain is selected, and the corresponding culture conditions are applied. In the prior art, a general leaven is adopted, the process is simple, the general leaven is used for fermenting various raw materials (such as bean pulp, cottonseed meal, dregs and the like), and the general leaven cannot reach the best for each raw material in practice; if the industrial scale-up is carried out, the fermentation efficiency is low, and the method is only suitable for the agricultural production of the subsidiary industry.
At present, no better biological method capable of decomposing gossypol, anti-nutritional factors and the like in the cottonseed meal well and improving the solubility of protein under the condition of a single matrix is developed in the field.
Disclosure of Invention
Aiming at the defects of the existing chemical dephenolization method and microbial fermentation method, the invention provides fermented cottonseed protein and a preparation method thereof. The method adopts cottonseed meal accounting for more than 97% of the total raw materials, and is used for fermenting single raw material. The cottonseed meal is treated by adopting high aerobic fermentation and enzymolysis synchronously, so that toxic free gossypol can be reduced, anti-nutritional factors such as phytic acid in the raw materials can be obviously degraded, the content of acid soluble protein can be increased, and the nutritional value of the product can be improved.
The first technical problem to be solved by the invention is to provide a fermented cottonseed protein. The fermented cottonseed protein is prepared by fermenting the following raw materials in parts by weight: 97-99 parts of cotton meal, 0-1.5 parts of corn flour, 0.7-1.5 parts of mixed yeast solid strain, 0.1-0.4 part of mixed nutrient salt, 0.03-0.1 part of mixed enzyme preparation and water; the total weight of the water accounts for 0.6-0.9 time of the total weight of the other five raw materials.
Preferably, the fermented cottonseed protein is prepared by fermenting the following raw materials in parts by weight: 98 parts of cotton meal powder, 0.5 part of corn flour, 1.2 parts of mixed yeast solid strains, 0.25 part of mixed nutrient salt, 0.05 part of mixed enzyme preparation and water, wherein the total weight of the water accounts for 0.8 times of the total weight of the other five raw materials.
Wherein, the cotton meal powder in the fermented cottonseed protein is obtained by crushing cotton meal by a crusher with the aperture of 2.0mm and then screening by using a sieve with 20 meshes.
Further, in the above fermented cottonseed protein, the mixed yeast solid strains include the following strains: saccharomyces cerevisiae (Saccharomyces fibuligera), Candida utilis (Candida utilis), and Kluyveromyces marxianus (Kluyveromyces marxianus).
firstly, preparing mixed yeast culture solution of the saccharomycete composite saccharomycete solid strain, candida utilis and kluyveromyces marxianus respectively. Then the mixed liquid strain of the saccharomyces fibuligera solid strain, the candida utilis and the kluyveromyces marxianus is 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 saccharomyces fibuligerii solid strain in the fermented cottonseed protein comprises the following steps:
Selecting 1 ring (inoculating loop) from slant preservation strains of Saccharomycopsis fibuligera, inoculating into 100mL of potato sucrose culture solution, and culturing at 28-32 ℃ for 2-3 d in a shaking flask at 150-200 rpm to obtain Saccharomycopsis fibuligera culture solution. And then transferring the culture solution into a bran corn culture medium, and culturing at the temperature of 28-32 ℃ for 2-3 d to obtain the capsule-covering spore yeast solid strain, wherein the weight ratio of the capsule-covering spore yeast liquid to the bran corn culture medium is 0.5-1.5: 100.
Specifically, the preparation method of the mixed yeast culture solution in the fermented cottonseed protein comprises the following steps:
Respectively selecting 1 ring (inoculating ring) from slant preserved strains of Candida utilis and Kluyveromyces marxianus, respectively inoculating into 100mL of potato sucrose culture solution, performing shake flask culture (150-200 rpm) at 28-32 ℃ for 2-3 days, and respectively obtaining a Candida utilis culture solution and a Kluyveromyces marxianus culture solution; then, mixing the candida utilis culture solution and the kluyveromyces marxianus culture solution in a weight ratio of 0.75-0.9: 0.1-0.25, inoculating the mixture into a potato sucrose culture solution, and performing shake flask culture (150-200 rpm) at the temperature of 28-32 ℃ for 2-3 days to obtain a mixed yeast solution; wherein the weight ratio of the Candida utilis culture solution to the Kluyveromyces marxianus culture solution to the potato sucrose culture solution is 1: 100;
Specifically, the preparation method of the mixed yeast solid strain in the fermented cottonseed protein comprises the following steps:
According to the weight ratio, the solid strain of the saccharomycete Fungiella fibuligera: the mixed yeast liquid is mixed with bran corn culture medium, wherein the ratio of the bran corn culture medium to the mixed yeast liquid is 0.5-1.0: 0.2-0.5: 100, uniformly mixing, and culturing at the temperature of 28-32 ℃ for 2-3 d to obtain the mixed yeast solid strain.
Preferably, the culture solution of Candida utilis in the fermented cottonseed protein is 0.85: 0.15.
Specifically, the preparation method of the bran corn culture medium in the fermented cottonseed protein 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.
Specifically, the preparation method of the mixed nutrient salt in the fermented cottonseed protein comprises the following steps: according to the weight ratio, 1 part of KCl and 0.5 part of MgSO40.05 part of FeSO4And 0.025 parts of ZnSO4And (4) uniformly mixing.
Specifically, the preparation method of the mixed enzyme preparation in the fermented cottonseed protein comprises the following steps: and mixing phytase, cellulase, xylanase, pectinase and papain in a weight ratio of 10-20: 2-3: 0.8-1.5.
Preferably, the mixed enzyme preparation in the fermented cottonseed protein is phytase, cellulase, xylanase, pectinase, papain, 10: 2: 1.
Specifically, the enzyme activity of the phytase in the fermented cottonseed protein is 5000 units, the enzyme activity of the cellulase is 5000 units, the enzyme activity of the xylanase is 10 ten thousand units, the enzyme activity of the pectinase is 3 ten thousand units, and the enzyme activity of the papain is 80 ten thousand units.
The second technical problem to be solved by the invention is to provide a solid-state aerobic fermentation method for preparing the fermented cottonseed protein. The method comprises the following steps:
A. Crushing and screening: crushing the cotton dregs by a crusher with a 2.0 sieve. Then screening the cottonseed meal powder by using a sieve with the aperture of 20-30 meshes;
B. Mixing: uniformly mixing cotton meal powder, corn flour, mixed yeast solid strains, mixed nutrient salt, mixed enzyme preparation and water to obtain a fermentation material;
C. Fermentation: and (3) fermenting the fermentation material for 45-50 hours at 30-35 ℃ in an aerobic state, and drying to obtain the fermented cottonseed protein.
The common drying method is natural drying under the environment of not higher than 60 ℃.
it is preferable thatIn the preparation method, the thickness of the fermentation material is not more than 60mm during fermentation in the step B.
Preferably, in the above preparation method, the container used for fermentation in step B is made of any one of stainless steel material and bamboo material. For example, a stainless steel or bamboo fermentation plate may be used.
The invention has the beneficial effects that: compared with untreated cottonseed meal (about 44% of crude protein), the fermented cottonseed meal has the advantages that under the condition of the same water content, the crude protein is more than or equal to 50%, the total amino acid is more than or equal to 44%, the free gossypol removal rate is more than or equal to 60%, the phytic acid degradation rate is about 30%, the pectin degradation rate is 42%, the acid soluble protein content is increased by more than 100%, and the feeding value is greatly improved. The cottonseed meal in the fermentation raw material accounts for more than 97 percent, and compared with the prior art, the method can more fully and effectively treat the cottonseed meal. The product of the invention can reduce the breeding cost and increase the income; the product of the invention can improve the health level of animal organisms and intestinal tracts, enhance the disease resistance, can be used without antibiotics conventionally, and is beneficial to the implementation of antibiotic-free healthy culture; through enzyme treatment and fermentation, the feed is rich in growth factors, peptides and the like, so that the quality of meat, eggs and the like can be improved, and the quality of agricultural products can be improved; improving the breeding environment, being beneficial to harmlessness and reclamation of the excrement, and developing clean breeding and ecological cycle.
Detailed Description
In the prior art, a chemical solvent method and an anaerobic microorganism fermentation method are adopted, and the desired effect is difficult to achieve by simultaneously taking the effects of detoxification, anti-nutritional factor elimination, digestion and absorption rate improvement, nutritional value improvement and feeding effect improvement. In order to solve the difficulties, the prior art mainly adopts complex strain combinations, and strains are added to decompose corresponding substances, but the methods are usually against the principle of microbiology, and too many strains often pollute each other, influence each other and have poor growth potential; or the strains have different optimal growth conditions and the growth in the same substrate is mutually interfered. Therefore, the strains and the enzyme preparations must be reasonably selected to well process the gossypol and the anti-nutritional factors in the cottonseed meal under the condition of a single substrate.
In order to remove toxic and harmful substances well and select proper strains, hundreds of strains of three types of microorganisms including bacteria, saccharomycetes and filamentous fungi are respectively subjected to anaerobic culture and aerobic culture in a cottonseed meal culture medium, the growth amount, the gossypol decomposition rate, the crude protein content and the appearance odor of the strains are measured, and the filamentous fungi are eliminated; furthermore, three strains of Saccharopolyspora fibuligera, Candida utilis and Kluyveromyces marxianus are finally determined and selected for processing the cottonseed meal according to the growth amount, gossypol decomposition rate, crude protein content, appearance odor and other indexes of the strains measured by mixed culture of the strains, and the safety, the industrial production difficulty, the comprehensive fermentation effect and the like of the strains are comprehensively considered.
Experiments show that the main strain of the three strains is saccharomycete Fujianfecti, has high biological quantity and gossypol reducing capacity, the candida utilis as the secondary strain has weak gossypol reducing capacity, and the aroma-producing yeast of Kluyveromyces marxianus can produce a small amount of ester aroma and improve the palatability of fermented cottonseed protein; the three strains can inhibit gram-negative bacteria in the intestinal tract, improve the nonspecific immunity of the intestinal tract, and allow part of cells to pass through the intestinal tract and enter feces, thereby being beneficial to clean culture.
It has also been found that an excessively large inoculation amount of the secondary strain reduces the gossypol-reducing ability of the primary strain, and therefore, it is preferable to determine the inoculation ratio of the primary and secondary strains more accurately under industrial production conditions. A large number of experimental researches show that the gossypol can be well decomposed when the ratio of the mixed yeast culture solution to the solid saccharomycete, namely the candida utilis and the kluyveromyces marxianus, is controlled to be 0.2-0.5: 0.5-1.0. When preparing the mixed yeast solid strain, preferably 0.4 part of mixed yeast culture solution and 0.8 part of saccharomycete coated yeast solid strain.
It is further found that although the three strains can decompose gossypol by combined fermentation, the three strains have limited effects on degrading anti-nutritional factors and improving digestion and absorption. In order to solve the problems, phytase producing bacteria, pectinase producing bacteria and the like are selected by screening a large number of strains at first and are added into the strain combination, and the effect of reducing the anti-nutritional factors is expected.
In the face of the problems, further research shows that a complex enzyme preparation can be added to solve the problems, and experiments show that the combined action of cellulase, phytase, pectinase and xylanase not only can decompose main anti-nutritional factors in the rapeseed dregs, but also can improve the dephenolization capacity of microorganisms; further, the enzyme composite use has a certain synergistic effect, for example, the phytic acid content in the cottonseed meal is high, the phytase decomposes the phytic acid, the combination of the phytic acid on nutrient elements can be reduced, the phosphorus released from the decomposition of the phytic acid provides nutrients for the growth of microorganisms, the growth of the microorganisms is facilitated, and in addition, degradation products of pectin, xylan and the like also provide nutrients for the growth of the microorganisms.
It was also found that under the same conditions of use, papain has a degrading effect on several other enzyme proteins, and the activity of the enzyme decreases with time. Therefore, the experimental study is carried out on the dosage and the proportion of the papain, and the activity of other enzymes can be kept by more than 50 percent within the first 10 hours of the treatment process by controlling the addition of the papain. Industrial production experiments show that the mixed enzyme preparation can well treat anti-nutritional factors in cottonseed meal when the phytase, cellulase, xylanase, pectinase and papain are 10-20: 2-3: 0.8-1.5 in proportion. Preferably, phytase cellulase, xylanase, pectinase, papain and papain are selected, and as a result, the acid soluble protein of the fermented cottonseed protein is improved by about 100% compared with the original cottonseed meal, and the decomposition rate of the gossypol is improved to about 60%.
The invention discovers that: when the enzyme preparation is added, the strain inoculation amount should be controlled, and the enzymolysis process is also the strain amplification process. If the inoculation amount is too large, the cell proliferation is too fast, the fermentation time is too short, and the enzymolysis is not facilitated; on the contrary, the inoculation amount is too small, the fermentation time is prolonged, and the mixed bacteria are easy to proliferate.
The invention also finds that when the added water amount is 0.6-0.9 times of that of other solid raw materials, the requirement of enzymolysis on water can be well met, and good bacterium increasing and dephenolizing effects can be achieved.
The inventor researches and discovers that the fermented material is too thin, so that water is easily lost, more containers are needed, the manufacturing cost is high, if the fermented material is too thick, anaerobic fermentation is realized, the effect is poor, so that the thickness of the fermented material is not more than 60mm, and preferably, the thickness of the fermented material is not more than 50mm during fermentation. Namely, the thin-layer solid aerobic fermentation is adopted, which is beneficial to improving the biomass.
Preferably, the container used for aerobic fermentation is made of any one of stainless steel materials or bamboo materials.
In the above method for producing fermented cottonseed protein, the container used for the fermentation in step B is preferably a stainless steel fermentation plate or a bamboo fermentation plate. For example, a stainless steel stainless tray having a length of not more than 2000mm, a width of not more than 1000mm and a height of not more than 60mm may be used as the fermentation tray.
Overall, compared to previously reported biological methods, which are primarily anaerobic fermentation alone. The method is based on an aerobic fermentation mode, and simultaneously adds an enzyme treatment process, so that the removal rate of gossypol is improved, the anti-nutritional factors are effectively reduced, the protein digestibility is improved, the feeding value of cottonseed meal is improved, a comprehensive optimization effect is achieved, the realization difficulty is very high, and very good comprehensive benefits can be obtained. A comparison of the process of the invention with conventional fermentation is shown in Table 1.
TABLE 1 comparison of the present Process with conventional fermentation Process for treating cottonseed meal
Different from other biological methods, the invention creatively uses a high aerobic solid state fermentation mode aiming at a single cottonseed meal raw material, can rapidly decompose toxic substances and obviously improve the contents of protein and amino acid, and can effectively degrade anti-nutritional factors through the synchronous enzyme hydrolysis.
In the following test examples and examples, the strain of Saccharomyces cerevisiae fibuligera (CGMCC) was deposited at 2016 (3.17.D.) in the general microbiological center of China Committee for culture Collection of microorganisms, with the accession number of CGMCC No. 12221. And (4) storage address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. The strain has strong detoxification capability on glucosinolate in rapeseed meal and gossypol in cottonseed meal, and is safe and nontoxic when being orally taken.
The candida utilis is stored in a microbiological laboratory of Sichuan university, originally comes from the institute of microbiology of Chinese academy of sciences (Beijing), and is stored in the China general microbiological culture Collection center (CGNCC), and the strain number is CGNCC 2.281;
Kluyveromyces marxianus is preserved in China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCCNo.9427;
Phytase is purchased from Beijing Xin ocean science and technology development Co., Ltd, and cellulase, pectinase, xylanase and papain are purchased from Sichuan Huade bioengineering Co., Ltd.
Example 1 cottonseed meal sieving pretreatment conditions
Crushing the cotton dregs raw material by an industrial crusher, wherein the sieve pore of the crusher is 2.0 mm. The crushed cotton dregs are respectively sieved by screens of 10 meshes, 20 meshes, 30 meshes, 40 meshes, 60 meshes and 80 meshes, and the weight of the materials above and below the screens is respectively weighed. Wherein the 10-mesh oversize is short fluff which accounts for about 2 percent of the total weight of the raw materials; the oversize material of 20 meshes is short villi and a small amount of cottonseed hull impurities, which account for about 8 percent of the total weight of the raw materials, and the undersize material has no obvious short villi visible to naked eyes; the screening of 30-80 meshes shows that the cotton seed in the oversize increases with the finer mesh. And in combination, 20-30 mesh sieve is selected as pretreatment before fermentation, so that the content of impurities such as fiber substances is reduced. Preferably 20 mesh, analytical results: the raw material crude protein before screening is 44%, and the free gossypol is 852 mg/kg; the crude protein content after sieving was 46.2% and free gossypol content was 833 mg/kg.
EXAMPLE 2 preparation of solid production strains
Selecting 1 ring (inoculating loop) from slant preservation strains of Saccharomycopsis fibuligera, inoculating into 100mL of potato culture solution, and shake-culturing at 30 deg.C (180rpm) for 52h to obtain Saccharomycopsis fibuligera culture solution.
Taking 50g of the saccharomycete complex yeast liquid, inoculating into 5000g of sterilized bran corn culture medium (calculated by dry materials), uniformly mixing, and culturing at the temperature of 30 ℃ for 48 hours to obtain the saccharomycete complex yeast solid strain.
Preparing a potato sucrose culture solution: peeling fresh potatoes, cutting the peeled fresh potatoes into blocks with the size of big toe, weighing 100g of the blocks, adding 500g of tap water, boiling for 20 minutes, filtering by using gauze, removing residues, taking supernate, fixing the volume to 500g by using the tap water, adding 10g of cane sugar, dissolving, and subpackaging into 250ml triangular bottles with 100g of each bottle. Sterilizing at 120 deg.C for 30 min.
Preparation of a bran corn culture medium: taking 750g of bran, 250g of corn and 800g of tap water, uniformly mixing, sterilizing at 120 ℃ for 30min, cooling and inoculating.
Respectively selecting 1 ring (inoculating ring) from slant preserved strains of Candida utilis and Kluyveromyces marxianus, respectively inoculating into 100g of potato sucrose culture solution, and culturing at 30 deg.C for 50h to obtain Candida utilis culture solution and Kluyveromyces marxianus culture solution; mixing Candida utilis culture solution 0.85 g: Kluyveromyces marxianus culture solution 0.15g, adding into potato sucrose culture solution 100g, and culturing at 30 deg.C for 48 hr to obtain mixed yeast solution;
Taking 8g of the saccharomycete composite saccharomycete solid strain and 4g of the mixed yeast liquid, mixing and inoculating into 1000g of sterilized bran corn culture medium (calculated by dry materials), uniformly mixing, and culturing at 30 ℃ for 52h to obtain the mixed yeast solid strain for fermentation production. Cell count was 6.45X 109Per gram of culture.
Example 3 Effect of the addition of nutrient salts on the results of the treatment
The cotton seed meal raw material was pulverized according to example 1 and sieved with a 20-mesh sieve to obtain cotton seed meal. Then, mixed yeast solid strains were prepared as in example 2.
Mixing KH with water2PO4、KCl、MgSO4、FeSO4、ZnSO4And adding phytase in groups according to the following table, uniformly mixing, culturing at 30 ℃ for 48h, and measuring the number of cells and the content of crude protein after fermentation.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| Cotton dregs powder (g) | 1000 | 1000 | 1000 | 1000 | 1000 | 1000 | 1000 | 1000 |
| Water (g) | 700 | 700 | 700 | 700 | 700 | 700 | 700 | 700 |
| Strain (g) | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| KH2PO4(g) | —— | 1 | —— | 1 | 1 | 1 | —— | —— |
| KCl(g) | —— | —— | 1 | —— | —— | —— | 1 | 1 |
| MgSO4(g) | —— | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| FeSO4(g) | —— | —— | —— | 0.05 | —— | 0.05 | 0.05 | 0.05 |
| ZnSO4(g) | —— | —— | —— | —— | 0.025 | 0.025 | 0.025 | 0.025 |
| Phytase (g) | —— | —— | —— | —— | —— | —— | —— | 0.2 |
| Number of cells (. times.10)9/g) | 1.45 | 1.75 | 1.56 | 1.73 | 1.76 | 1.81 | 1.55 | 1.83 |
| Crude protein (%) | 48.20 | 49.35 | 48.82 | 49.84 | 49.86 | 50.20 | 48.84 | 50.61 |
Nutrient salt additions were selected as group 8, i.e.: KCl 0.1%, MgSO40.05％、FeSO40.005％、ZnSO40.0025% and phytase 0.02%. The phytase can decompose phytic acid in the cottonseed meal to provide a phosphorus source for cell growth. The unit of the phytase activity is 5000 u/g.
Example 4 fermentation treatment of cottonseed meal
The cotton seed meal raw material is crushed according to the example 1 and screened by a 20-mesh sieve to obtain cotton seed meal, wherein the free gossypol is 833 mg/kg. Then, the mixed yeast solution and the solid saccharomycete strain were prepared as in example 2.
Preparing mixed nutrient salt: weighing KCl 100g and MgSO4 50g、FeSO45g and ZnSO42.5g of the mixture was pulverized by a pulverizer, and the whole was sieved with 100 mesh.
Preparing a mixed enzyme preparation: weighing 100g of phytase, 20g of cellulase, 10g of xylanase, 10g of pectinase and 10g of papain, and mixing and stirring uniformly.
980g of cottonseed meal powder, 5g of corn flour, 12g of mixed yeast solid strain, 2.5g of mixed nutrient salt, 0.5g of mixed enzyme preparation and 800g of water are mixed and mixed uniformly, the mixture is spread uniformly in a stainless steel plate of 700 multiplied by 350mm, the thickness of the mixture is 4cm, the mixture is fermented for 48 hours at the temperature of 30 ℃, and the mixture is dried for 24 hours at the temperature of 50 ℃ to obtain the fermented cottonseed protein.
The number of cells after fermentation was determined to be 2.2X 109Water content 12.2%, crude protein 51.7%, free gossypol 276 mg/kg.
Aspartic acid 4.41%, threonine 1.60%, serine 2.06%, glutamic acid 9.22%, glycine 1.97%, alanine 2.01%, cystine 0.56%, valine 2.04%, methionine 0.75%, isoleucine 1.53%, leucine 3.05%, tyrosine 1.33%, phenylalanine 2.72%, lysine 2.07%, histidine 1.33%, arginine 5.16%, proline 2.03%, and amino acids 43.83%. The small peptide content was 10.3% (control 4.4%, water 8.08%).
Example 5 fermentation treatment of cottonseed meal
The cotton seed meal raw material is crushed according to the example 1 and screened by a 20-mesh sieve to obtain cotton seed meal, wherein the free gossypol is 833 mg/kg. Then, the mixed yeast solution and the solid saccharomycete strain were prepared as in example 2.
Preparing mixed nutrient salt: weighing KCl 100g and MgSO4 50g、FeSO45g and ZnSO42.5g of the mixture was pulverized by a pulverizer, and the whole was sieved with 100 mesh.
974g of cottonseed meal powder, 15g of corn flour, 6.7g of mixed yeast solid strain, 4g of mixed nutrient salt, 0.3g of mixed enzyme preparation and 700g of water are mixed and mixed uniformly, the mixture is spread uniformly in a 700 x 350 stainless steel plate with the thickness of 6cm, the mixture is fermented for 48 hours at the temperature of 32 ℃, and the mixture is dried for 20 hours at the temperature of 50 ℃ to obtain the fermented cottonseed protein.
the number of cells after fermentation was determined to be 1.65X 109(ii) in terms of/g. 9.8% of water, 51.7% of crude protein, 1.62% of lactic acid and 329mg/kg of free gossypol. The content of acid soluble protein is 10.01% (4.94% of reference material), stachyose is less than 50.0mg/kg, and raffinose is less than 50.0 mg/kg.
Example 6 fermentation treatment of cottonseed meal
The cotton seed meal raw material is crushed according to the example 1 and screened by a 20-mesh sieve to obtain cotton seed meal, wherein the free gossypol is 833 mg/kg. Then, the mixed yeast solution and the solid saccharomycete strain were prepared as in example 2.
Preparing mixed nutrient salt: weighing KCl 100g and MgSO4 50g、FeSO45g and ZnSO42.5g of the mixture was pulverized by a pulverizer, and the whole was sieved with 100 mesh.
Preparing a mixed enzyme preparation: weighing 100g of phytase, 20g of cellulase, 10g of xylanase, 10g of pectinase and 10g of papain, and mixing and stirring uniformly.
980g of cotton meal powder, 3g of corn flour, 15g of mixed yeast solid strain, 1g of mixed nutrient salt, 1g of mixed enzyme preparation and 600g of water are mixed and mixed uniformly, the mixture is spread uniformly in a stainless steel plate with the thickness of 700 multiplied by 350mm, the mixture is fermented for 50 hours at the temperature of 32 ℃ and dried for 20 hours at the temperature of 50 ℃, and then the fermented cottonseed protein is obtained.
The number of cells after fermentation was determined to be 1.8X 109The total content of the free gossypol is 318mg/kg, the water content is 11.12 percent, the crude protein content is 50.11 percent, the lactic acid content is 1.49 percent, the acid soluble protein content is 10.8 percent (the unfermented raw material is 4.4 percent, the water content is 8.08 percent), the water soluble protein content is 12.3 percent, the stachyose is less than 50.0mg/kg, and the raffinose is less than 50.0 mg/kg.
Example 7 Large Scale fermentation treatment of cottonseed meal
The cotton seed meal raw material is crushed according to the example 1 and screened by a 20-mesh sieve to obtain cotton seed meal, wherein the free gossypol is 833 mg/kg. Then, the mixed yeast solution and the solid saccharomycete strain were prepared as in example 2.
Preparing mixed nutrient salt: KCl 2000g, MgSO4 1000g、FeSO4100g and ZnSO450g of the mixture was mixed and pulverized by a pulverizer, and the whole was passed through a 100-mesh sieve.
Preparing a mixed enzyme preparation: weighing the raw materials according to the proportion that every 1000g of phytase is matched with 200g of cellulase, 100g of xylanase, 100g of pectinase and 100g of papain, and mixing and stirring uniformly.
The fermented cottonseed protein is prepared by taking 970kg of cottonseed meal powder, 15kg of corn flour, 11kg of mixed yeast solid strain, 3.2kg of mixed nutrient salt, 0.8kg of mixed enzyme preparation and 800kg of water, mixing uniformly, spreading uniformly in a 2000X 1000X 60mm stainless steel fermentation disc (the bottom is a 40-mesh screen) with the thickness of 5cm, charging 20kg (calculated by dry materials) in each disc, packaging 50 discs together, fermenting at the temperature of 33 ℃ for 48 hours, and drying at the temperature of 50 ℃ for 20 hours.
The number of cells after fermentation was determined to be 2.05X 109G, water content 12.5%, crude protein 50.8%, free gossypol 322 mg/kg.
Aspartic acid 4.52%, threonine 1.64%, serine 2.15%, glutamic acid 9.35%, glycine 1.99%, alanine 2.01%, cystine 0.68%, valine 2.06%, methionine 0.76%, isoleucine 1.51%, leucine 3.02%, tyrosine 1.38%, phenylalanine 2.76%, lysine 2.12%, histidine 1.38%, arginine 5.44%, proline 1.95%, and amino acids 44.76% in total. Acid soluble protein 11.5% (contrast material 4.45%, water content 8.9%), stachyose less than 50.0mg/kg, raffinose less than 50.0 mg/kg.
EXAMPLE 8 Large Scale fermentation treatment of cottonseed meal
The cottonseed meal raw material was pulverized according to example 1 and sieved with a 20 mesh sieve to obtain cottonseed meal, wherein the free gossypol content is 845 mg/kg. Then, the method of example 2 was followed to prepare the Zygosaccharomyces fibuligera solid strain and the mixed yeast liquid, respectively.
Preparing mixed nutrient salt: weighing KCl 10kg and MgSO4 5kg、FeSO40.5kg and ZnSO40.25kg of the mixture was pulverized by a pulverizer, and the whole was passed through a 100 mesh sieve.
Preparing a mixed enzyme preparation: weighing 10kg of phytase, 2kg of cellulase, 1kg of xylanase, 1kg of pectinase and 1kg of papain, and mixing and stirring uniformly.
990kg of cottonseed meal powder, 7kg of mixed yeast solid strain, 2.2kg of mixed nutrient salt, 0.8kg of mixed enzyme preparation and 900kg of water are mixed and mixed uniformly, the mixture is spread uniformly in a 2000 x 1000 x 60mm stainless steel fermentation plate (the bottom is a 40-mesh screen), the thickness is 4cm, 20kg of material (measured by dry material) is loaded in each plate, 50 plates are loaded together, the temperature of a fermentation chamber is controlled at 33 ℃, the fermentation is carried out for 48h, and the drying is carried out at 50 ℃ for 20h, so that the fermented cottonseed protein is obtained.
The number of cells after fermentation was determined to be 1.9X 109/g. 325mg/kg of free gossypol, 52.89% of crude protein, 9.96% of water, 7.25% of ash, 4.81% of aspartic acid, 2.29% of lysine, 1.84% of proline, 2.98% of leucine, 2.74% of phenylalanine, 2.24% of glycine, 2.14% of valine, 1.61% of histidine, 2.44% of serine, 1.53% of isoleucine, 0.61% of methionine, 2.08% of alanine, 9.35% of glutamic acid, 1.71% of threonine, 5.62% of arginine, 0.86% of cystine and 1.43% of free ammonia. Acid soluble protein 11.5% (contrast material 4.41%, water content 8.5%), stachyose less than 50.0mg/kg, raffinose less than 50.0 mg/kg.
example 9 Large-Scale fermentation treatment of cottonseed meal and fattening pig Breeding experiment
The cottonseed meal raw material was pulverized according to example 1 and sieved with a 20 mesh sieve to obtain cottonseed meal, wherein the free gossypol content is 845 mg/kg. Then, the method of example 2 was followed to prepare the Zygosaccharomyces fibuligera solid strain and the mixed yeast liquid, respectively.
Preparing mixed nutrient salt: weighing KCl 10kg and MgSO4 5kg、FeSO40.5kg and ZnSO40.25kg of the mixture was pulverized by a pulverizer, and the whole was passed through a 100 mesh sieve.
Preparing a mixed enzyme preparation: weighing 10kg of phytase, 2kg of cellulase, 1kg of xylanase, 1kg of pectinase and 1kg of papain, and mixing and stirring uniformly.
4900kg of cottonseed meal powder, 30kg of corn flour, 50kg of mixed yeast solid strain, 15kg of mixed nutrient salt, 5kg of mixed enzyme preparation and 4000kg of water are mixed uniformly, the mixture is spread uniformly in a 2000X 1000X 60mm stainless steel fermentation plate (the bottom is a 40-mesh screen), the thickness is 5cm, 20kg (measured by dry materials) of the mixture is loaded in each plate, 250 plates are loaded in total, the temperature of a fermentation chamber is 34.5 ℃, the mixture is fermented for 46h, and the mixture is dried for 20h at the temperature of 50 ℃ to obtain the fermented cottonseed protein.
The number of cells after fermentation was determined to be 1.7X 109/g. 322mg/kg of free gossypol. Crude protein 53.47%, moisture 10.12%, ash 6.89%, aspartic acid 4.83%, lysine 2.18%, proline 1.77%, leucine 2.95%, phenylalanine 2.99%, glycine 2.26%, valine 2.05%, histidine 1.64%, serine 2.45%, isoleucine 1.45%, methionine 0.60%, alanine 2.07%, glutamic acid 9.49%, threonine 1.69%, arginine 5.64%, cystine 0.89%, free ammonia 1.68%. 11.8% of acid soluble protein (4.47% of reference raw material and 8.65% of water), less than 50.0mg/kg of stachyose and less than 50.0mg/kg of raffinose.
120 Neijiang pigs and Duroc binary hybrid pigs are selected and randomly divided into 2 groups, each group comprises 60 pigs, 1 group is fed with complete feed of traditional raw materials, and the other 1 group is fed with biological feed added with fermented cottonseed protein. The average weight of the traditional feed group is 65.4kg, and the average weight of the biological feed group is 64.85 kg.
The traditional feed formula comprises: 69% of corn, 16% of bean pulp (crude protein 43%), 6% of cottonseed meal (crude protein 44%), 5% of bran and 4% of micro-regulator. The formula of the biological feed comprises: 69% of corn, 7% of soybean meal, 13% of fermented cottonseed protein (crude protein 53.47%), 7% of bran and 4% of micro-regulator.
The biological feed replaces 13% of fermented cottonseed protein with cottonseed meal and partial soybean meal in the traditional feed group, keeps consistency of two groups of crude proteins, has equivalent metabolic energy, and supplements the balance with 2% of bran.
Two sets of formulations are monovalent: the traditional feed group is 36 yuan/ton higher than the biological feed group.
Feeding under the same feeding condition for 82 days. Traditional feed group: 685g of daily gain, 3.572 of feed conversion ratio and 7.86 of money conversion ratio; biological feed group: the daily gain is 736g, the feed conversion ratio is 3.410, the money conversion ratio is 7.38, and the cost is reduced by 0.48 yuan/kg of weight gain.
the 4 parts of the micro-regulator comprises the following components: 10g of calcium hydrogen phosphate, 2g of lysine, 8.0mg of copper, 80.0mg of iron, 60.0mg of manganese, 40.0mg of zinc, 0.15mg of selenium Se, 0.35mg of iodine, 50000IU of vitamin A, 310000IU of vitamin D, 25IU of vitamin E, 35mg of vitamin K, 1210mg of vitamin B, 120mg of vitamin B, 216mg of vitamin B, 66 mg of vitamin B, 35mg of nicotinamide, 25mg of pantothenic acid and 0.5mg of folic acid. IU in the micro-regulator is international unit.
Example 10 Large-Scale fermentation treatment of cottonseed meal and broiler Chicken raising experiment
The cottonseed meal raw material was pulverized according to example 1 and sieved with a 20 mesh sieve to obtain cottonseed meal, wherein the free gossypol content is 845 mg/kg. Then, the method of example 2 was followed to prepare the Zygosaccharomyces fibuligera solid strain and the mixed yeast liquid, respectively.
Preparing mixed nutrient salt: weighing KCl 10kg and MgSO4 5kg、FeSO40.5kg and ZnSO40.25kg of the mixture was pulverized by a pulverizer, and the whole was passed through a 100 mesh sieve.
Preparing a mixed enzyme preparation: weighing 10kg of phytase, 2kg of cellulase, 1kg of xylanase, 1kg of pectinase and 1kg of papain, and mixing and stirring uniformly.
980kg of cotton meal powder, 7kg of corn flour, 10kg of mixed yeast solid strain, 2.2kg of mixed nutrient salt, 0.8kg of mixed enzyme preparation and 800kg of water are mixed and mixed uniformly, the mixture is spread uniformly in a 2000 x 1000 stainless steel fermentation tray (the bottom of the fermentation tray is a 40-mesh screen), the thickness of the mixture is 4cm, 20kg of materials (counted by dry materials) are loaded in each tray, 50 trays are loaded together, the temperature of a fermentation chamber is controlled at 34.8 ℃, the mixture is fermented for 45 hours, and the fermented cottonseed protein is obtained after drying for 20 hours at the temperature of 50 ℃.
Measuring the cell number of the fermented cottonseed protein finished product to be 2.15 multiplied by 109(ii) in terms of/g. 49.4 percent of crude protein, 13.3 percent of water and 317mg/kg of free gossypol.
Aspartic acid 4.52%, threonine 1.64%, serine 2.17%, glutamic acid 9.77%, glycine 2.03%, alanine 1.98%, cystine 0.60%, valine 2.02%, methionine 0.76%, isoleucine 1.53%, leucine 3.04%, tyrosine 1.38%, phenylalanine 2.62%, lysine 2.19%, histidine 1.40%, arginine 5.58%, proline 2.00%, and amino acids 45.21% in total. The degradation rate of phytic acid is about 30 percent, the degradation rate of pectin is 42 percent, and the acid soluble protein is 11.2 percent (the contrast raw material is 4.43 percent, and the water content is 8.5 percent).
Experiment of broiler feeding
About 600 white-foot white-bark pockmarked-feather chickens with about 1.6kg are selected and randomly divided into 2 groups, each group comprises 300 chickens, the average initial weight of the control group is 1.645 kg/chicken, and the average initial weight of the biological group is 1.618 kg/chicken.
The formula of the control group is as follows: 68% of corn, 18% of bean pulp (crude protein 43%), 6% of cottonseed meal (crude protein 44%), 3% of bran, 1% of soybean oil and 4% of micro-regulator; the formula of the biological feed group is as follows: 68% of corn, 10% of soybean meal (46% of crude protein), 13% of fermented cottonseed protein (51.3% of crude protein and 10% of water content), 4% of bran, 1% of soybean oil and 4% of micro-regulator. The biological feed group replaces 8% of soybean meal and 6% of cottonseed meal with 13% of fermented cottonseed protein, and the insufficient 1% of fermented cottonseed protein is complemented with bran. The energy and crude protein levels were essentially identical for both groups. The cost unit price of the biological protein group formula is reduced by 30 yuan/ton feed.
feeding under the same feeding condition for 31 days. Control group: the total weight gain is 110.27kg, the feed conversion ratio is 2.72, the feed price is 2.2 yuan/kg, the qian meat ratio is 5.98: 1, biological feed group: the total weight gain is 120.08kg, the feed conversion ratio is 2.59, the feed price is 2.17 yuan/kg, the qian meat ratio is 5.62: 1, namely, the weight gain per kilogram can reduce the cost by 0.36 yuan. The number of the biological group is 0, and the number of the control group is 1. The biological group feed group did not contain any antibiotics.
The micro-regulator is prepared from the following components in percentage by weight per kg of feed: 10g of calcium hydrogen phosphate, 1g of choline, 1g of baking soda, 2g of lysine, 1g of methionine, 0.2g of phytase, 8.0mg of copper, 80.0mg of iron, 60.0mg of manganese, 40.0mg of zinc, 0.15mg of selenium, 0.35mg of iodine, 50000IU of vitamin A, and vitamin D310000IU, vitamin E25 IU, vitamin K35mg, vitamin B1210mg of vitamin B120mg of vitamin B216mg of vitamin B66mg, nicotinamide 35mg, pantothenic acid 25mg, folic acid 0.5 mg. The feed micro-regulator of the control group also contains per kilogram: virginiamycin 20mg, salinomycin 60 mg. The micro-regulator for the biological group feed supplements more than one kilogram: lysine 0.4 g. IU in micro-regulator-international unit.
Claims (7)
1. The fermented cottonseed protein is characterized in that: is prepared by fermenting the following raw materials in percentage by weight: 97-99 parts of cotton meal, 0-1.5 parts of corn flour, 0.7-1.5 parts of mixed yeast solid strain, 0.1-0.4 part of mixed nutrient salt, 0.03-0.1 part of mixed enzyme preparation and water; the total weight of the water accounts for 0.6-0.9 time of the total weight of the other five raw materials;
The mixed yeast solid strains comprise the following strains: saccharomyces cerevisiae (Saccharomyces fibuligera), Candida utilis (Candida utilis), and Kluyveromyces marxianus (Kluyveromyces marxianus);
The mixed enzyme preparation is prepared by mixing phytase, cellulase, xylanase, pectinase and papain in a weight ratio of 10-20: 2-3: 0.8-1.5.
2. The fermented cottonseed protein of claim 1, wherein: is prepared by fermenting the following raw materials in percentage by weight: 98 parts of cotton meal powder, 0.5 part of corn flour, 1.2 parts of mixed yeast solid strain, 0.25 part of nutrient salt, 0.05 part of mixed enzyme preparation and 80 parts of water.
3. The fermented cottonseed protein of claim 1, wherein: the cotton meal powder is obtained by pressing oil from cotton seeds, and then crushing and screening the oil to remove impurities.
4. The fermented cottonseed protein of claim 1, wherein: the preparation method of the nutrient salt comprises the following steps: according to the weight ratio, 1 part of KCl and 0.5 part of MgSO40.05 part of FeSO4And 0.025 parts of ZnSO4And (4) uniformly mixing.
5. The fermented cottonseed protein as claimed in any one of claims 1 to 4, wherein: the enzyme activity of the phytase in the mixed enzyme preparation is 5000 units, the enzyme activity of the cellulase is 5000 units, the enzyme activity of the xylanase is 10 ten thousand units, the enzyme activity of the pectinase is 3 ten thousand units, and the enzyme activity of the papain is 80 ten thousand units.
6. The solid-state aerobic fermentation method for preparing the fermented cottonseed protein as claimed in any one of claims 1 to 5, wherein: the method comprises the following steps:
A. Crushing and screening: crushing the cottonseed meal by using a crusher with a 2.0-mesh sieve, and then pulverizing the cottonseed meal by using a sieve with the aperture of 20-30 meshes;
B. mixing: uniformly mixing cotton meal powder, corn flour, mixed yeast solid strain, nutrient salt, mixed enzyme preparation and water to obtain a fermentation material;
C. Fermentation: and (3) fermenting the fermentation material for 45-50 hours at 30-35 ℃ in an aerobic state, and drying to obtain the fermented cottonseed protein.
7. The method of claim 6, wherein the step of fermenting the cottonseed protein comprises: in the step B, the thickness of the fermentation material is not more than 60mm during aerobic fermentation.
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