Fermented protein feed as well as preparation method and application thereof

Abstract

The invention provides a fermented protein feed as well as a preparation method and application thereof. The fermented feed is prepared in a mode of firstly performing enzymolysis and then performing fermentation, and tests prove that the obtained fermented protein feed is stable in acidic protein content and good in processing performance, the stability in water is remarkably improved, and the fermented protein feed has wide application value in aquatic feed.

Description

Technical Field

[0001] The present invention belongs to the field of biological fermented feed, and specifically relates to a fermented protein feed and a preparation method and application thereof. Background Art

[0002] In order to allow aquatic animals to eat as much feed as possible, and to reduce the pollution of the water environment by leftover bait and waste, aquaculture requires feed to have good stability in water. Feed solubility rate is a common indicator for evaluating the stability of aquatic feed in water. Affected by the feeding behavior and physiological characteristics of animals, different aquaculture species have different requirements for the stability of feed products in water. In the industry standard SC / T 1077-2004 General Technical Requirements for Fishery Formula Feeds, the stability indicators of compound pellet feeds for fish, crabs, and shrimps in water are: the solubility rate is less than 10% when soaked in water for 5 minutes, the solubility rate is less than 10% when soaked in water for 30 minutes, and the solubility rate is less than 12% when soaked in water for 120 minutes.

[0003] After nearly 20 years of development, there have been many studies on the stability of aquatic feed in water, and the loss rate has become one of the important indicators of the processing quality of aquatic feed products. For example, in GB / T 36205-2018 Grass Carp Formula Feed, NY / T 4126-2022 Prawn Juvenile Formula Feed, NY / T 4127-2022 Procambarus Clarkii Formula Feed, TK / Y0004-2020 River Crab Feed Standard and other product standards, the loss rate of the product has become an indispensable indicator of processing quality. In the group standard T / KYO004-2020, the stability index of river crab formula feed in water is that the loss rate is less than 10% when immersed in water for 120 minutes, which is higher than the requirement of SC / T 1077-2004.

[0004] It can be seen that in aquaculture production, the market's attention to the stability of feed products in water is increasing.

[0005] The water resistance of aquatic feed is the result of the interaction of feed raw materials, adhesives, processing technology and other factors. Relying on advanced equipment and technology, the selection of feed raw materials with good adhesive properties such as barley, flour, second meal, fish meal, and the addition of feed adhesive products such as sodium carboxymethyl cellulose and gelatin are the first choice for the design of aquatic formula.

[0006] In addition, processing technologies such as ultrafine grinding, multi-stage conditioning, puffing, high-ring die compression granulation, and post-ripening are the basis for achieving the best physical properties of feed and exerting the best nutritional effect. In actual production, when the ratio of large raw materials with good bonding properties such as flour and fish meal is good, the freshwater fish pellets are granulated with a ring die compression ratio of about 15, and a pellet feed product with good stability in water can be obtained (Liu Feng et al. "Factors affecting water resistance of aquatic feed." Feed Industry).

[0007] In addition, sodium carboxymethyl cellulose, polyurea formaldehyde, sodium alginate and guar gum are commonly used adhesives in aquatic feeds. Guar bean meal is a byproduct of guar gum extraction from guar beans. It is rich in protein. Compared with other plant proteins, guar bean byproducts can improve the density and durability of pellet feeds, but the content of residual anti-nutritional factor guar gum limits the amount of guar bean meal added in aquatic feeds. The main component of residual guar gum in guar meal is galactomannan, which is water-soluble and viscous. It has a positive growth-promoting effect when the addition ratio does not exceed 5%. A large proportion of addition will increase the viscosity of chyme, thereby reducing nutrient digestibility. When 0.6% sodium carboxymethyl cellulose and 0.3% of a new feed adhesive whose main components are lignin sulfonate and guar gum are added to crayfish feed, the stability of pellet feed in water after soaking for 5 minutes is significantly different. However, high-quality adhesive products are expensive and have strict process requirements during use (Xue Zhicheng. "Correct application and detection methods of aquatic feed adhesives in fish and shrimp feeds." Jiangxi Feed). The puffing, multi-layer conditioning, high ring die compression ratio and post-ripening processes have high requirements for equipment and facilities and process conditions. The bonding properties of pellet feed are improved mainly by increasing the gelatinization degree of starch, thereby improving the stability of pellet feed in water. However, most aquatic compound feeds are high-protein feed products. The application of raw materials with high starch content such as wheat bran, second flour, and flour competes with the proportion space of protein feed raw materials. Fish, shrimp and crabs are intolerant to starch, which increases the difficulty of formula design. The application of protein raw materials with good viscosity such as fish meal increases, and the cost increases accordingly.

[0008] In short, the ingredients of adhesive products on the market are diverse, many of which are high-molecular polymer ingredients with low nutritional value. Not only do they have high process requirements in production, but some products are also harmful to farmed animals and the environment. After adding guar gum to feed, the shape retention effect and stability in water are better, but when the guar gum content is high, it is not conducive to the growth and health of aquatic animals. The disadvantage of optimizing the stability of feed products in water through process is that it has high requirements for equipment and process. Although aquatic feed products with high fish meal content have high protein content and good stability in water, they are expensive and fish meal resources are limited.

[0009] Therefore, technologies and products that can improve the stability of aquatic pellet feed in water and pellet feed with good stability in water have good market value. Summary of the invention

[0010] As mentioned above, the market is paying more and more attention to the stability of feed products in water. At present, the main methods to improve the stability of feed products in water are: selecting feed raw materials with good bonding properties, adding feed adhesives, or improving the processing technology.

[0011] However, the above methods all have certain defects. For example, adding feed adhesives not only affects the nutritional value of feed, but also increases the difficulty of production; using process optimization to solve the problem of feed product stability in water has high equipment requirements, increased production costs and efficiency, etc.; using methods such as improving feed raw materials and increasing protein raw materials to improve the stability of feed products in water has a series of problems such as increased costs and increased difficulty in formula design.

[0012] In view of the problems existing in the prior art, the purpose of the present invention is to improve the process performance of feed raw materials, especially protein raw materials, through biotechnology, increase the stability of feed products in water, increase the nutrient content and feed absorption and utilization rate, reduce the cost of feed production, and simplify the production process, reducing the requirements for production equipment and feed product formulas.

[0013] In order to solve the above technical problems, the present invention comprises the following steps: (1) selecting guar soybean meal with good bonding performance and performing appropriate enzymolysis; (2) selecting an appropriate ratio of soybean meal and shrimp shell powder, adding enzymolysis liquid and then performing solid-state fermentation to obtain a composite biological protein feed with stable acidic protein content and good processing performance; and (3) adding an appropriate ratio into aquatic pellet feed, using the acidic protein and guar gum content as quality and process indicators, to obtain an aquatic feed product with a simple and easy preparation process and significantly improved stability in water.

[0014] Wherein, the fermented protein feed comprises the following components: water, guar gum, crude protein and acid-soluble protein.

[0015] According to the present invention, the moisture content of the fermented protein feed is 30.0%-65.0%, for example, it can be 30%, 32%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% and 65%, as well as point values ​​between the above values.

[0016] Preferably, the moisture content of the fermented protein feed is 34.0%-63.0%.

[0017] Further preferably, the moisture content of the fermented protein feed is 30.0%-50.0%.

[0018] More preferably, the moisture content of the fermented protein feed is 30.0%-40.0%.

[0019] According to some specific embodiments of the present invention, the moisture content of the fermented protein feed is 34%.

[0020] According to the present invention, the guar gum content in the fermented protein feed is 0.1%-1.5%, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% and 1.5%.

[0021] Preferably, the guar gum content is 0.1%-1.3%.

[0022] Further preferably, the guar gum content is 0.2%-1.2%.

[0023] According to the present invention, the acid-soluble protein content in the fermented protein feed is 2%-8.5%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 8.5%, and points between the above values.

[0024] Preferably, the acid-soluble protein content in the fermented protein feed is 3%-8%.

[0025] More preferably, the acid-soluble protein content in the fermented protein feed is 5%-8%.

[0026] According to some specific embodiments of the present invention, the acid-soluble protein in the fermented protein feed is 7.43%.

[0027] According to the present invention, the crude protein content of the fermented protein feed is about 14%-33%, for example, it can be 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, and points between the above values.

[0028] Preferably, the crude protein content of the fermented protein feed is about 15%-30%.

[0029] More preferably, the crude protein content of the fermented protein feed is about 20%-30%.

[0030] According to some specific embodiments of the present invention, the crude protein content of the fermented protein feed is 26.52%.

[0031] According to the present invention, in the fermented protein feed, the acid-soluble protein / crude protein ratio is 8-35%, for example, it can be 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% and 35%, as well as point values ​​between the above values.

[0032] Preferably, in the fermented protein feed, the ratio of acid-soluble protein to crude protein is 9%-32%.

[0033] More preferably, in the fermented protein feed, the ratio of acid-soluble protein to crude protein is 10%-30%.

[0034] More preferably, in the fermented protein feed, the ratio of acid-soluble protein to crude protein is 20%-30%.

[0035] Further preferably, in the fermented protein feed, the ratio of acid-soluble protein to crude protein is 28.02%.

[0036] According to the present invention, the fermented protein feed raw material comprises the following components: guar bean meal enzymatic hydrolysate, bacterial agent, soybean meal and shrimp shell powder.

[0037] According to the present invention, the guar bean meal enzymatic hydrolysate is prepared by the following method: Select guar soybean meal: water 1:6-1:15 (w / w), enzyme substrate concentration 800-3200U / g (guar soybean meal g), enzymolysis temperature 45-60℃, enzymolysis time 8-32h to obtain guar soybean meal enzymolysate.

[0038] According to the present invention, the enzymes are neutral proteases and alkaline proteases.

[0039] According to the present invention, the mass ratio of the neutral protease to the alkaline protease is 2:1-1:2, for example, it can be 2:1, 1:1, 1:2, and points between the above values.

[0040] According to some preferred embodiments of the present invention, the mass ratio of the neutral protease to the alkaline protease is 1:1.

[0041] According to the present invention, the protease is purchased from Xiasheng Group, and the guar soybean meal is purchased from Beijing Guarrun Technology Co., Ltd.

[0042] According to the present invention, the acid-soluble protein / crude protein ratio in the enzymatic hydrolyzate is 25%-75%, for example, it can be 25%, 26%, 27%, 28%, 29%, 30%, 32%, 33%, 34%, 36%, 38%, 40%, 41%, 42%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, and 75%.

[0043] Preferably, the ratio of acid-soluble protein to crude protein in the enzymatic hydrolysate is 30%-75%.

[0044] More preferably, the ratio of acid-soluble protein to crude protein in the enzymatic hydrolysate is 45%-75%.

[0045] More preferably, the ratio of acid-soluble protein to crude protein in the enzymatic hydrolysate is 60%-75%.

[0046] According to some preferred embodiments of the present invention, the ratio of acid-soluble protein to crude protein in the enzymatic hydrolysate is 68.92%.

[0047] According to the present invention, the fermented protein feed is prepared by a process of enzymatic hydrolysis followed by fermentation.

[0048] According to some specific embodiments of the present invention, the fermentation temperature is 25°C-35°C.

[0049] According to some specific embodiments of the present invention, the fermentation time is 48h-96h.

[0050] According to some specific embodiments of the present invention, the fermentation agent is: Bacillus / complex bacteria 1:4-4:1, and the fermentation agent inoculation amount is 0.15-0.60%.

[0051] According to the present invention, the composite bacterial agent is Bacillus pumilus, Enterococcus faecalis, Lactobacillus plantarum and Monascus.

[0052] The ratio of Bacillus pumilus: Enterococcus faecalis: Lactobacillus plantarum: Monascus purpureus was 7-12:1:7-12:1-4 in terms of CFU.

[0053] Specifically, Bacillus subtilis BFC1601 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 13131. The deposit number of Bacillus pumilus is CGMCC No. 16061, the deposit number of Enterococcus faecalis is CGMCC No. 16062, the deposit number of Lactobacillus plantarum is CGMCC No. 13132, and the deposit number of Monascus is CGMCC No. 15088.

[0054] According to the present invention, the mass ratio (w / w) of the fermented soybean meal to the shrimp shell powder is 3:0-0:3.

[0055] According to the present invention, the mass ratio (w / w) of the fermented soybean meal to the shrimp shell powder is 3:0-1:3.

[0056] Preferably, the mass ratio (w / w) of the fermented soybean meal to the shrimp shell powder is 2:1-1:2.

[0057] According to the present invention, during the fermentation process, the proportion of guar bean meal enzymatic hydrolyzate w / (w1 bean skin + w2 shrimp shell powder) is 30%-60%.

[0058] Preferably, the proportion of the guar bean meal enzymatic hydrolyzate is w / (w1 bean skin + w2 shrimp shell powder) 35%-55%.

[0059] More preferably, the proportion of the guar bean meal enzymatic hydrolyzate is w / (w1 bean skin + w2 shrimp shell powder) 40%-50%.

[0060] Another aspect of the present invention provides a method for preparing the guar meal enzymatic hydrolysate and fermented protein feed described in the first aspect.

[0061] The method for preparing the guar bean meal enzymatic hydrolysate and fermented protein feed of the present invention comprises the following steps: S1, preparation of guar meal enzymatic hydrolysate: Select guar soybean meal: water ratio of 1:6-1:15 (w / w), enzyme substrate concentration of 800-3200U / g (guar soybean meal g), enzymolysis temperature of 45-60℃, enzymolysis time of 8-32h to obtain guar soybean meal enzymolysate.

[0062] S2, preparation of fermented protein feed: The fermentation temperature is selected to be 25℃-35℃, the fermentation time is 48h-96h, the fermentation agent ratio is Bacillus / complex bacteria 2:3 (Bacillus:complex bacteria is 1-4:4-1), the fermentation agent inoculation amount is 0.15%-0.60%, the mass ratio of soybean meal to shrimp shell powder (w / w) is 3:0-0:3, and the proportion of guar meal enzymatic hydrolysate w / (w1+w2) is 30%-60%.

[0063] According to the present invention, in S1, the enzymes are neutral proteases and alkaline proteases.

[0064] According to the present invention, the mass ratio of the neutral protease to the alkaline protease is 2:1-1:2, for example, it can be 2:1, 1:1, 1:2, and points between the above values.

[0065] According to some preferred embodiments of the present invention, the mass ratio of the neutral protease to the alkaline protease is 1:1.

[0066] According to the present invention, the protease is purchased from Xiasheng Group, and the guar soybean meal is purchased from Beijing Guarrun Technology Co., Ltd.

[0067] According to the present invention, the acid-soluble protein / crude protein ratio in the enzymatic hydrolyzate is 25%-75%, for example, it can be 25%, 26%, 27%, 28%, 29%, 30%, 32%, 33%, 34%, 36%, 38%, 40%, 41%, 42%, 45%, 46%, 47%, 50%, 51%, 52%, 53%, 54%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, and 75%.

[0068] According to the present invention, in S2, the composite bacterial agent is Bacillus pumilus, Enterococcus faecalis, Lactobacillus plantarum and Monascus.

[0069] According to the present invention, the ratio of Bacillus pumilus: Enterococcus faecalis: Lactobacillus plantarum: Monascus is 7-12:1:7-12:1-4 in terms of CFU.

[0070] Specifically, Bacillus subtilis BFC1601 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 13131. The deposit number of Bacillus pumilus is CGMCC No. 16061, the deposit number of Enterococcus faecalis is CGMCC No. 16062, the deposit number of Lactobacillus plantarum is CGMCC No. 13132, and the deposit number of Monascus is CGMCC No. 15088.

[0071] Specifically, the number of viable Bacillus subtilis is ≥ 1.0×10 9 CFU / g, viable count of Bacillus pumilus ≥ 2.0×10 9 CFU / g, live Enterococcus faecalis count ≥3.0×10 8 CFU / g, viable Lactobacillus plantarum count ≥ 2.0×10 10 CFU / g, number of viable Monascus ≥5.0×10 8 CFU / g.

[0072] According to the present invention, the moisture content of the fermented protein feed prepared by the preparation method is 30.0%-65.0%.

[0073] According to the present invention, the acid-soluble protein content in the fermented protein feed prepared by the preparation method is 2%-8.5%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 8.5%, and points between the above values.

[0074] According to the present invention, the crude protein content of the obtained fermented protein feed is about 14%-33%, for example, it can be 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, and points between the above values.

[0075] Preferably, the crude protein content of the obtained fermented protein feed is about 15%-30%.

[0076] More preferably, the crude protein content of the obtained fermented protein feed is about 20%-30%.

[0077] According to some specific embodiments of the present invention, the crude protein content of the obtained fermented protein feed is 26.52%.

[0078] According to the present invention, in the obtained fermented protein feed, the acid-soluble protein / crude protein ratio is 8-35%, for example, it can be 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% and 35%, as well as point values ​​between the above values.

[0079] Preferably, in the obtained fermented protein feed, the ratio of acid-soluble protein to crude protein is 9%-32%.

[0080] More preferably, in the fermented protein feed, the ratio of acid-soluble protein to crude protein is 10%-30%.

[0081] Further preferably, in the obtained fermented protein feed, the ratio of acid-soluble protein to crude protein is 28.02%.

[0082] The invention discloses an application of the fermented protein feed in preparing aquatic feed with high stability.

[0083] The present invention obtains a composite protein feed product with high acid-soluble protein content and good water resistance through the process of enzymolysis followed by fermentation. After adding an appropriate amount to aquatic pellet feed, a pellet feed product with good stability in water is obtained, which is beneficial to aquatic ecological breeding and improves the yield of aquaculture products.

[0084] The fourth aspect of the present invention provides a product containing the fermented protein feed and the use of the product in feed.

[0085] According to the present invention, the feed is aquatic feed.

[0086] According to the present invention, the feed is a pellet feed, and the pellet feed is prepared from the fermented protein feed described in the first aspect or the protein feed prepared by the preparation method described in the second aspect.

[0087] According to some embodiments of the present invention, the pellet feed includes aquatic feed such as shrimp feed, river crab feed, grass carp feed, etc.

[0088] According to the present invention, the pellet feed comprises the fermented protein feed of the present invention, soybean meal and other auxiliary materials.

[0089] According to some specific embodiments of the present invention, the shrimp pellet feed includes the following ingredients: 220-250 parts of imported fish meal, 140-230 parts of soybean meal, 50-140 parts of peanut meal, 30 parts of brewer's yeast, 30-90 parts of the compound protein feed of the present invention, 255-265 parts of flour, 0-30 parts of shrimp shell powder, 25 parts of oil, 0-10 parts of sodium carboxymethyl cellulose and 30 parts of premixed feed.

[0090] According to some specific embodiments of the present invention, the river crab pellet feed includes the following ingredients: 50-100 parts of imported fish meal, 210-260 parts of soybean meal, 0-150 parts of rapeseed meal, 50-300 parts of the compound protein feed of the present invention, 140-200 parts of flour, 90 parts of wheat bran, 0-150 parts of rice bran, 20 parts of oil, 0-6 parts of sodium carboxymethyl cellulose and 30 parts of premixed feed.

[0091] According to some specific embodiments of the present invention, the grass carp pellet feed includes the following ingredients: 110-210 soybean meal, 30-180 rapeseed meal, 10-160 cottonseed meal, 50-150 of the compound protein feed of the present invention, 160-210 of second flour, 50-100 of wheat bran, 25-100 of rice bran, 10 of oil and fat, and 30 of premixed feed.

[0092] According to the present invention, the preparation of the aquatic pellet feed can be carried out through conventional procedures.

[0093] According to the present invention, the preparation process of the aquatic pellet feed may be, for example: S1 Except premixed feed, all raw materials are mixed (including enzymatically fermented feed).

[0094] S2 first coarsely grinds, then ultrafine grinds to achieve the grinding particle size of different aquatic feed products.

[0095] S3 tempering, the tempering temperature is above 90-95℃, the tempering time is above 45S, and the moisture content of the material after tempering is 12%~20%.

[0096] S4 pelleting, the diameters of pelleting die holes for shrimp, river crab and grass carp are 1.0mm, 2.50mm and 4.5mm respectively, the compression ratios of ring die (ring die particle diameter: ring die thickness ratio) are 1:10, 1:12 and 1:15 respectively, and the length of finished pellets is about 1-4 times of the diameter of the ring die.

[0097] S5 cooling process uses a countercurrent cooler with a cooling time of 10 min-30 min.

[0098] Complete the preparation of pellet feed for shrimp, river crab and grass carp.

[0099] According to the present invention, the superfine grinding of the shrimp, river crab and grass carp feed is respectively passed through 80 mesh, 60 mesh and 40 mesh sieves. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 It is an aquatic pellet feed product.

[0101] Figure 2 This is the dissolution rate detection picture.

[0102] Figure 3 This is a picture of the dissolution results of shrimp feed. Specific implementation plan In this specification, the numerical range expressed using "numerical value A - numerical value B" means a range including the endpoint numerical values ​​A and B.

[0103] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0104] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0105] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0106] In this specification, the aquatic compound premix feed was purchased from DSM Vitamins (Shandong) Co., Ltd. The product label is as follows: magnesium 3.2×10 4 -9.0×10 4 mg / kg, iron 4.0×10 3 -2.5×10 4mg / kg, zinc 700-2100mg / kg, manganese 250-750mg / kg, copper 60-180mg / kg, cobalt 5.0-15.0mg / kg, nicotinamide ≥1.7×10 3 mg / kg, L-ascorbic acid ester ≥3.2×10 3 mg / kg, moisture ≤10.0%.

[0107] 1. Preparation process of guar bean meal enzymatic hydrolysate The feed-water ratio, enzyme composition and enzyme substrate concentration, hydrolysis solution pH, hydrolysis temperature, hydrolysis time and hydrolysis conditions are important factors affecting the quality of enzymatic hydrolysis.

[0108] Adjusting pH during enzymatic hydrolysis will greatly increase the ash content of the product. The higher the enzyme substrate concentration, the higher the cost. Therefore, in the enzymatic hydrolysis process of this project, the guar bean meal is crushed through a 40-mesh sieve, the pH value is 8.0, and it is stirred at 45-60℃ to form a viscous slurry, and enzymatic hydrolysis is carried out at room temperature. A neutral protease:alkaline protease composition of 2-1:1-2 is used, NaOH is used to adjust the initial pH to 8.0, and the pH is not adjusted during the enzymatic hydrolysis process. The process parameters A material-water mass ratio, B enzyme substrate ratio (U / g), C enzymatic hydrolysis temperature, and D enzymatic hydrolysis time h are optimized.

[0109] The initial specifications of guar meal are shown in Table 1.

[0110] Table 1 Guar soybean meal product indicators (%)

[0111] In the laboratory, a 500L mechanical stirring tank with a filling capacity of 70% was used. A 4-factor 4-level [L16(45)] orthogonal experimental method (Table 2) was adopted to determine the enzymatic hydrolysis process parameters of guar soybean meal with the y value (total acid-soluble protein / total crude protein of soybean meal*100%) as the indicator.

[0112] Table 2 Orthogonal design table for enzymatic hydrolysis of guar soybean meal

[0113] The experimental and analytical results show (Table 3) that the enzymatic hydrolysis process of guar meal is A4B3C2D4, that is, the feed-water ratio is 1:15, the enzyme substrate concentration is 2400U / g, the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis time is 32h, and the acid-soluble protein / crude protein ratio can reach 68.92%.

[0114] Table 3 Process plan and result analysis

[0115] The analysis results in Table 3 show that the sum of squares of deviations of factor D is less than that of the blank group. The combined errors give the analysis results in Table 4. The enzyme substrate concentration ( P <0.05) and enzymatic hydrolysis temperature ( P<0.05) had a significant difference in the content of enzymatic acidic protein, but the difference in feed-water ratio was not significant ( P >0.05). Although the highest acid protein / crude protein ratio can be obtained when the feed-water ratio is 1:15, the water content of the hydrolyzate has a direct impact on the subsequent solid-state fermentation ingredients, and the variance analysis is not significant. Therefore, taking the acid-soluble protein / crude protein ratio of 50% (w / w) as the indicator, A2B3C4D1 was selected as the hydrolysis condition, that is, feed-water ratio of 1:9, enzyme substrate concentration of 2400U / g, hydrolysis temperature of 60℃, and hydrolysis time of 8h, the hydrolyzate with a moisture content of 91.000%, crude protein of 5.000%, acid-soluble protein of 2.523%, and guar gum of 0.624% was obtained for the comparison of process parameters of composite protein solid-state fermentation.

[0116] Table 4 ANOVA of orthogonal experiment ANOVA

[0117] 2. Solid-state fermentation mixed protein feed preparation process After obtaining the guar soybean meal enzymatic hydrolysate, the enzyme is not inactivated, and the material is mixed with soybean meal and shrimp shell powder after being discharged, and a composite bacterial agent is selected to carry out solid-state fermentation.

[0118] The selection of fermentation agents, inoculation amount of the agents, substrate ratio, material-water ratio, fermentation temperature, fermentation time, etc. are important factors affecting the fermentation effect.

[0119] The present invention selects a composite bacterial agent for solid-state fermentation.

[0120] Among them, the composite bacterial agents include Bacillus subtilis strains and composite bacterial agents (Bacillus brevis, Enterococcus faecalis, Lactobacillus plantarum and Monascus).

[0121] Specifically, Bacillus subtilis BFC1601 has been deposited in the General Microbiology Center of China National Committee for the Administration of Microbiological Culture Collection, with a deposit number of CGMCC No. 13131. The composite bacterial agent contains Bacillus pumilus (with a deposit number of CGMCC No. 16061), Enterococcus faecalis (with a deposit number of CGMCC No. 16062), Lactobacillus plantarum (with a deposit number of CGMCC No. 13132) and Monascus purpureus (with a deposit number of CGMCC No. 15088), and the ratio of Bacillus pumilus: Enterococcus faecalis: Lactobacillus plantarum: Monascus purpureus is 7-12:1:7-12:1-4 in terms of CFU.

[0122] The number of viable Bacillus subtilis cells is ≥1.0×10 9 CFU / g, viable count of Bacillus pumilus ≥ 2.0×10 9 CFU / g, live Enterococcus faecalis count ≥3.0×10 8 CFU / g, viable Lactobacillus plantarum count ≥ 2.0×1010 CFU / g, number of viable Monascus ≥5.0×10 8 CFU / g.

[0123] In the present invention, the fermentation room temperature is 30°C±5°C, the fermentation time is 72h±24h, and the four process parameters A, the mass ratio of Bacillus subtilis to the composite bacterial agent (w / w), B, the inoculation amount of the fermentation bacterial agent (w / w mass ratio of the culture medium), C, the mass ratio of soybean meal to shrimp shell powder (w / w), and D, the proportion of guar soybean meal enzymatic hydrolyzate in the culture medium w / (w1+w2) are optimized.

[0124] The shrimp shell powder of the present invention is a powdery product made from inedible residues (shrimp heads, shells, tail brains, etc.) left during shrimp processing after drying, with a crude protein content of ≥32% and a moisture content of ≤13.5%.

[0125] In an embodiment of the present invention, the shrimp shell meal is a product of Qingdao Jinlong Feed Co., Ltd., and the soybean meal is 43% soybean meal purchased from Yihai Kerry on the market.

[0126] The composite protein culture medium substrate was prepared in the laboratory using 500 m 3 The ribbon mixer mixes evenly, with an installed capacity of 70%, using 4 factors and 4 levels [L16(4 5 )] The orthogonal experimental method (Table 5) was used to determine the solid-state fermentation process parameters of compound protein feed with y value (substrate acid-soluble protein content / substrate crude protein content*100%) as the index.

[0127] Table 5 Orthogonal design of solid-state fermentation of complex proteins (w / w)

[0128] The experimental and analysis results show (Table 6) that the factors affecting the acid-soluble protein / crude protein ratio of solid-state fermentation composite protein products are ranked as A>B>D>C, that is, bacteria ratio>microbial inoculation amount>enzyme hydrolysate ratio>soybean meal:shrimp shell.

[0129] Table 6 Solid-state fermentation process plan and result analysis

[0130] The results of variance analysis of K value showed (Table 7) that the factors affecting the ratio of acid-soluble protein / crude protein were ranked as A>B>D>C, that is, bacterial ratio>microbial inoculation amount>enzyme hydrolysate ratio>soybean meal:shrimp shell. Among them, the bacterial ratio had a significant effect on the ratio of acid-soluble protein / crude protein in composite protein fermentation (P<0.05), and the bacterial inoculation amount had a significant trend (P=0.061). When the process is A3B2C4D3, that is, the ratio of fermentation agent A is Bacillus subtilis / composite agent 3:2, B inoculation amount (w / w ratio of culture medium mass) fermentation agent inoculation amount 0.30%, C mass ratio of soybean meal to shrimp shell powder (w / w 0:3), D proportion of guar soybean meal enzymatic hydrolyzate w / (w1+w2) is 50%, the substrate acid-soluble protein / crude protein ratio can reach 31.99%, but due to the strong seasonality of shrimp shell powder supply and the soybean meal: shrimp shell ratio has no significant effect on the fermentation effect, the ratio of soybean meal to shrimp shell powder selected by the present invention is 3:0-0:3, more preferably 3:0-1:3.

[0131] Table 7 Variance analysis of solid-state fermentation process parameters

[0132] 3. Stability of pellet feed in water Feed formula and particle diameter directly affect the stability of pellet feed in water. The present invention selects prawn pellet feed with a diameter of 1.0 mm, river crab pellet feed with a diameter of 2.5 mm, and grass carp pellet feed with a diameter of 4.5 mm as representatives for the water stability experiment.

[0133] These three kinds of pellet feeds are produced in the factory through the process of coarse mixing → coarse grinding → ultrafine grinding → conditioning → granulation → cooling. The ultrafine grinding of shrimp, river crab and grass carp feeds are sieved through 80 mesh, 60 mesh and 40 mesh respectively. The conditioning temperature is above 90-95℃ and the conditioning time is above 45S. The moisture content of the material after conditioning is 12%~20%. The die hole diameters are 1.0mm, 2.50mm and 4.5mm respectively. The ring die compression ratio (ring die particle diameter: ring die thickness ratio) is 1:10, 1:12 and 1:15 respectively. The finished pellets are about 1-4 times the diameter of the ring die. A countercurrent cooler is used with a cooling time of 10 min-30 min. The pellets are produced according to the conventional production process of shrimp, river crab and grass carp pellet feeds. The prepared pellet feeds are shown in Figure 1 .

[0134] After cooling, samples of water-resistant pellets are intermittently sampled at the cooler outlet, mixed and divided into several samples for inspection. Referring to the determination of pellet feed stability in water in SC / T 1026 and SC / T1077, a 304 stainless steel round convenient filter ball with a diameter of 7cm and a mesh diameter of 0.83mm (20 mesh) (hereinafter referred to as the filter ball) is selected to detect the dissolution loss rate of the prepared aquatic pellet feed after soaking for different times. For stability in water, a 500mL beaker is filled with 400mL of laboratory grade 3 water. According to the test group, 10g of sample is accurately weighed and placed in the filter ball. It is soaked according to the test design. The experiment is repeated three times at each soaking time point to conduct a water resistance evaluation test for the feed, see. Figure 2 .

[0135] After accurately weighing the filter ball + sample, slowly put the loaded filter ball into the water in the beaker. After 5 minutes, slowly lift the filter ball to the water surface, and then slowly sink it into the water to make the feed leave the bottom of the filter ball. Repeat 3 times, and after soaking for a specified time, place the filter ball obliquely to drain the water, and place the filter ball + residual material in a 105℃ oven to dry to constant weight, and record the feed mass m2. The indoor temperature of the laboratory and the water temperature in the beaker are controlled at 25±2℃. At the same time, weigh a portion of feed to determine its dry matter mass m1, and the stability in water is calculated according to the formula: stability in water = (m1-m2) / m1*100%.

[0136] In SC / T 1077-2004, the stability index of shrimp pellet feed in water is that the dissolution rate is less than 12% when immersed in water for 120 minutes. In aquaculture, it is best for shrimp to finish eating within 1.5 hours after feeding (DB33 / T 2557-2022 Technical Specifications for Aquaculture of White Shrimp). Therefore, the water tolerance time intervals of shrimp in this experiment are designed to be 1h, 2h, 3h, 4h, 5h, and 6h.

[0137] The material-water ratio was 1:9, the enzyme substrate concentration was 2400U / g, the enzymolysis temperature was 60°C, and the enzymolysis time was 8h, and an enzymolysis solution with a moisture content of 91.000%, a crude protein of 5.000%, an acid-soluble protein of 2.523%, and a guar gum content of 0.624% was obtained.

[0138] The production process parameters were selected as Bacillus / composite bacteria 3:2, fermentation agent inoculation amount 0.45%, soybean meal and shrimp shell powder mass ratio (w / w) 3:0, and guar soybean meal enzymatic hydrolysate proportion w / (w1+w2) of 40%. The solid-state fermented protein feed with a moisture content of 43.900%, crude protein of 27.800%, acid-soluble protein of 7.822%, and guar gum of 0.250% was obtained, and the stability experiment of shrimp pellet feed in water was carried out.

[0139] There were 6 shrimp pellet feeds in this experiment. The shrimp pellet feed with 1% sodium carboxymethyl cellulose adhesive was selected as the control group C1, the shrimp pellet feed with 0.5% sodium carboxymethyl cellulose adhesive was selected as the control group C2, and the shrimp pellet feed with 0% sodium carboxymethyl cellulose adhesive was selected as the control group C3. In the C3 formula, the experimental T1 group (enzymatic fermentation compound protein product was added at 3%, replacing 1.0% soybean meal + 1.0% shrimp shell powder + 1.0% peanut meal in equal amounts), the experimental T2 group (enzymatic fermentation compound protein product was added at 6%, replacing 2.0% soybean meal + 2.0% shrimp shell powder + 2.0% peanut meal in equal amounts), and the experimental T3 group (enzymatic fermentation compound protein product was added at 9%, replacing 3.0% soybean meal + 3.0% shrimp shell powder + 3.0% peanut meal in equal amounts) were designed (Table 10). The solubility loss rate of the pellet feed was tested under laboratory conditions. The experimental data were subjected to one-way analysis of variance.

[0140] Table 10 Shrimp feed formula

[0141] *Aquaculture compound premix feed was purchased from DSM Vitamins (Shandong) Co., Ltd. The product label indicates as follows: Magnesium 3.2×10 4 -9.0×10 4 mg / kg, iron 4.0×10 3 -2.5×10 4 mg / kg, zinc 700-2100mg / kg, manganese 250-750mg / kg, copper 60-180mg / kg, cobalt 5.015.0mg / kg, nicotinamide ≥1.7×10 3 mg / kg, L-ascorbic acid ester ≥3.2×10 3 mg / kg, moisture ≤10.0%. Same as the following table.

[0142] The results of the dissolution loss test of shrimp pellets with a diameter of 1.0 mm (Table 11) show that all the shrimp pellets soaked in the test can maintain their complete shape and can be easily picked up ( Figure 3 ).

[0143] Taking 2h of soaking as an example, the order of the dissolution loss rate of shrimp pellet feed from low to high is T3 group < T2 group = C1 group < C2 group < T1 group < C3 group. The differences among C1 group, C2 group and C3 group are extremely significant (P < 0.001), and the differences among T1 group, T2 group and T3 group are extremely significant (P < 0.001). The differences between C3 group and T1 group, C2 group and T2 group, C1 group and T3 group are extremely significant (P < 0.001), while the difference between C1 group and T2 group is not significant (P > 0.05). The results show that the water resistance of the experimental T3 group is better than that of the control group C1 of shrimp pellet feed added with 1.0% sodium carboxymethylcellulose binder, and the water resistance of the experimental T2 group is better than that of the control group C2 of shrimp pellet feed added with 0.5% sodium carboxymethylcellulose binder. Without using binders and without increasing the production difficulty, the present invention can achieve an excellent effect of reducing the dissolution loss rate.

[0144] Table 11 Analysis of the dissolution loss rate results of shrimp feed (n = 3) project C1 C2 C3 T1 T2 T3 1h Mean <![CDATA[3.20% Ce ]]> <![CDATA[3.90% Bc ]]> <![CDATA[5.20% Aa ]]> <![CDATA[4.90% Ab ]]> <![CDATA[3.40% Bd ]]> <![CDATA[3.03% Cf ]]> Standard Deviation 0.26% 0.50% 0.17% 0.26% 0.44% 0.25% 2h Mean <![CDATA[10.33% D ]]> <![CDATA[12.27% B ]]> <![CDATA[13.40% A ]]> <![CDATA[11.67% C ]]> <![CDATA[10.33% D ]]> <![CDATA[9.50% E ]]> Standard Deviation 1.11% 1.15% 1.25% 0.90% 1.11% 0.69% 3h Mean <![CDATA[13.30% Ab ]]> <![CDATA[12.67% B ]]> <![CDATA[13.97% Aa ]]> <![CDATA[13.27% Ab ]]> <![CDATA[12.43% B ]]> <![CDATA[11.23% C ]]> Standard Deviation 2.19% 0.91% 0.42% 0.61% 0.58% 0.50% 4h Mean <![CDATA[20.57% Cd ]]> <![CDATA[19.30% E ]]> <![CDATA[22.77% Aa ]]> <![CDATA[21.77% Ab ]]> <![CDATA[20.97% Cc ]]> <![CDATA[19.77% E ]]> Standard Deviation 0.99% 1.00% 1.48% 1.69% 0.60% 0.74% 5h Mean <![CDATA[24.63% Aa ]]> <![CDATA[23.77% Ab ]]> <![CDATA[26.50% A ]]> <![CDATA[25.77% A ]]> <![CDATA[22.70% B ]]> <![CDATA[21.13% C ]]> Standard Deviation 2.25% 0.55% 1.01% 1.50% 0.90% 0.95% 6h Mean <![CDATA[29.17% B ]]> <![CDATA[29.97% B ]]> <![CDATA[34.00% A ]]> <![CDATA[21.43% D ]]> <![CDATA[30.57% B ]]> <![CDATA[27.87% C ]]> Standard Deviation 0.93% 0.46% 0.70% 18.59% 1.12% 0.29% Taking the data of C3, T1, T2 and T3 groups, the correlation between the dissolution loss rate of the experimental pellet feed and the acid-soluble protein content of the pellet feed (Table 10, acid-soluble protein value) was analyzed. The results showed that there was a certain linear negative correlation between the dissolution loss rate and the acid-soluble protein content (Table 12).

[0145] For the enzyme hydrolysate, the sample before solid-state fermentation and the sample after solid-state fermentation, the change in the guar gum content was not significant. The guar gum content in the pellet feed changed with the addition amount of the composite protein feed fermented after enzymatic hydrolysis. To avoid the influence of other feed raw materials on the detection results of the guar gum content, during the subsequent analysis, the guar gum content in the pellet feed was only calculated using the value of the guar gum introduced by the enzymatically hydrolyzed guar bean meal. The calculated guar gum content data of C3, T1, T2 and T3 groups were 0.000%, 0.008%, 0.015% and 0.023% respectively. The correlation between the dissolution loss rate of the experimental pellet feed and the guar gum content of the pellet feed was analyzed. The results showed that there was a certain linear negative correlation between the dissolution loss rate and the guar gum content (Table 12). It can be seen from Table 12 that when the addition amount of the composite protein feed fermented after enzymatic hydrolysis in the shrimp feed is 3% - 9%, the acid-soluble protein content is 7.148 - 7.683%, and the guar gum content is 0.000 - 0.023%, the dissolution loss rate in water can be reduced and the water stability of the pellet feed can be improved.

[0146] Table 12 Correlation between the dissolution loss rate of shrimp pellet feed and acid-soluble protein and guar gum

[0147] Effect of the composite protein feed fermented after enzymatic hydrolysis on the water resistance of crab pellet feed In the industry standard SC / T 1077-2004, the stability index of crab pellet feed in water is that the loss rate is less than 10% when immersed in water for 30 minutes. In the group standard TK / Y 0004-2020 for river crab feed, the basic requirement for stability in water is that the loss rate is less than 10% when immersed in water for 120 minutes. In breeding, it is best for crabs to eat the feed about 60 minutes after feeding. Therefore, the water-resistant time intervals of the crab feed in this experiment are designed to be 0.5h, 1h, 1.5h, 2h, 3h, and 4h.

[0148] The material-water ratio was 1:9, the enzyme substrate concentration was 3200 U / g, the enzymolysis temperature was 55°C, and the enzymolysis time was 16 h, and an enzymolysis solution with a moisture content of 91.000%, a crude protein of 5.000%, an acid-soluble protein of 2.664%, and a guar gum content of 0.624% was obtained.

[0149] Bacillus / compound bacteria 3:2, fermentation agent inoculation amount 0.60%, soybean meal and shrimp shell powder mass ratio (w / w) 2:1, guar soybean meal enzymatic hydrolysate proportion w / (w1+w2) 30% were selected as production process parameters, and a solid-state fermented protein feed with a moisture content of 36.283%, crude protein of 29.034%, acid-soluble protein of 8.135%, and guar gum of 0.187% was obtained. The stability experiment of crab pellet feed in water was carried out.

[0150] There are 6 crab pellet feeds in this experiment. The test feed formula and grouping are shown in Table 13. The test feed formula design is as follows: C1 control group, no sodium carboxymethyl cellulose and no enzymatic fermentation compound protein feed; C2 group added 0.6% sodium carboxymethyl cellulose to the C1 group formula, and reduced 0.6% flour by the same amount; C3 group added 10% non-enzymatic non-fermented compound protein culture medium to the C1 group formula, and replaced 5% rapeseed meal + 4% soybean meal + 1% flour by the same amount; designed test group 1 (enzymatic fermentation compound protein product added 10% in the C1 group formula, equivalent to 5% rapeseed meal + 4% soybean meal + 1% flour), test group 2 (enzymatic fermentation compound protein product added 20% in the C1 group formula, equivalent to 10% rapeseed meal + 8% soybean meal + 2% flour), test group 3 (enzymatic fermentation compound protein product added 30% in the C1 group formula, equivalent to 15% rapeseed meal + 12% soybean meal + 3% flour), and tested the solubility loss rate of pellet feed under laboratory conditions. One-way analysis of variance was performed on the experimental data.

[0151] Table 13 Crab feed formula

[0152] *Same as Table 10.

[0153] The dissolution rate test of Eriocheir sinensis pellet feed with a diameter of 2.5 mm showed that although there was still residue in the filter ball after 4 hours of immersion in the C1 group pellet feed, it was almost completely disintegrated. However, most of the Eriocheir sinensis pellet feeds in the T1, T2, and T3 groups could maintain their complete pellet shapes, and the T1 and T2 groups could be carefully picked up with tweezers.

[0154] Taking the example of soaking for 30 minutes (0.5 h), the results of the dissolution rate test of Eriocheir sinensis pellet feed showed (Table 14) that the dissolution rates were ranked from low to high as follows: T2 group < T1 group < C2 group < T3 group < C3 group < C1 group. Among the C1, C2, and C3 groups, and between the T2 group and the T1 and T3 groups, the differences were extremely significant (P < 0.001). The difference between the C3 group and the T1, C2, and T3 groups was significant (P < 0.05), and the differences between the C2 group and the T1 and T3 groups were not significant (P > 0.05). The results showed that the dissolution rates of the experimental T1, T2, and T3 groups were significantly lower than those of the C3 raw material group without binder, and the dissolution rates of the experimental T1 and T2 groups were significantly lower than those of the C2 control group with 0.6% sodium carboxymethylcellulose binder. The dissolution rate of the T3 group was similar to that of the C2 group.

[0155] Table 14 Detection results of the dissolution rate of Eriocheir sinensis pellet feed (n = 3)

[0156] Taking the data of the T1, T2, and T3 groups, the correlation between the dissolution rate of the experimental pellet feed and the acid-soluble protein content of the pellet feed (Table 16, acid-soluble protein value) was analyzed. The results showed that when the addition amount of the fermented compound protein feed was 10% - 30%, the dissolution rate showed a certain linear positive correlation with the acid-soluble protein content (Table 15). The guar gum content data of the T1, T2, and T3 groups were calculated to be 0.019%, 0.040%, and 0.060% respectively. The correlation between the dissolution rate of the experimental pellet feed and the guar gum content of the pellet feed was analyzed. The results showed that the dissolution rate also showed a certain linear positive correlation with the guar gum content. When analyzing the reasons, it was found that during the production process of the pellet feed, when the addition amount of the enzymatically hydrolyzed and fermented compound protein feed was 30%, the moisture content of the material before conditioning was as high as 18.93%, which affected pelletizing. This indicated that when the addition amount of the enzymatically hydrolyzed and fermented compound protein feed was 30%, although the acid-soluble protein content and the guar gum content were high, due to the relatively high basic moisture content of the material, the conditioning and pelletizing performance would be reduced, affecting the water stability of the pellet feed.

[0157] Table 15 Correlation between the dissolution rate of Eriocheir sinensis pellet feed and acid-soluble protein and guar gum

[0158] 3.3 Influence of enzymatically hydrolyzed and then fermented compound protein feed on the water resistance of grass carp pellet feed In SC / T 1077-2004, the water stability index of compound granular fish feed is that the dissolution rate is less than 10% after soaking in water for 5 minutes. In GB / T 36205-2018 Grass Carp Compound Feed, the water stability index of grass carp granular feed is that the dissolution rate is less than 20% after soaking in water for 5 minutes. In aquaculture, it is appropriate for grass carp to finish eating basically within 5 - 15 minutes after feeding. Therefore, the water resistance time intervals of the grass carp feed in this experiment are designed as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, and 35 minutes.

[0159] Select a feed - to - water ratio of 1:9, an enzyme - substrate concentration of 1600 U / g, an enzymatic hydrolysis temperature of 45 °C, and an enzymatic hydrolysis time of 32 h to obtain an enzymatic hydrolysate with a moisture content of 91.000%, crude protein of 5.000%, acid - soluble protein of 2.345%, and guar gum of 0.624%.

[0160] Select Bacillus spp. / compound bacteria at a ratio of 2:3, an inoculation amount of fermentation bacteria agent of 0.45%, a mass ratio (w / w) of soybean meal to shrimp shell powder of 0:3, and the proportion of guar meal enzymatic hydrolysate w / (w1 + w2) of 30% as production process parameters to obtain a solid - state fermented protein feed with a moisture content of 36.750%, crude protein of 23.900%, acid - soluble protein of 7.522%, and guar gum of 0.187%, and conduct a water stability experiment on grass carp granular feed.

[0161] Take the grass carp granular feed without added binder as the control group, and design experimental group 1 with an addition amount of enzymatic hydrolysis and fermentation compound protein product of 5% (equivalent substitution of 2.5% rapeseed meal + 2.5% rice bran), experimental group 2 with 10% (equivalent substitution of 5% rapeseed meal + 5% rice bran), and experimental group 3 with 15% (equivalent substitution of 7.5% rapeseed meal + 7.5% rice bran) (Table 16). Detect the dissolution rate of the granular feed under laboratory conditions. Conduct a one - way analysis of variance on the experimental data.

[0162] Table 16 Grass Carp Feed Formulation

[0163] *Same as Table 10.

[0164] Taking soaking for 5 minutes as an example, the experimental results of the dissolution rate of the grass carp granular feed show (Table 17) that the dissolution rates are ranked from low to high as T3 group < T2 group < T1 group < C group. The inter - group differences between T2 and T3 groups and T1 group, C group are extremely significant (P < 0.001), and the inter - group difference between T2 group and T3 group is not significant (P > 0.05). The results show that compared with the control group, after adding 5 - 15% of the enzymatic hydrolysis and fermentation protein feed, the dissolution rate of the grass carp granular feed after immersion in water is extremely significantly reduced, and the water resistance is extremely significantly improved.

[0165] Table 17 Effect of enzymatic fermentation feed products on the dissolution rate of grass carp pellet feed (n=3)

[0166] The data of groups C, T1, T2, and T3 were taken to analyze the correlation between the solubility loss rate of grass carp pellets and the acid-soluble protein content of pellets (Table 18, acid-soluble protein value). The results showed that when the addition amount of fermented compound protein feed was 5%-15%, the solubility loss rate and the acid-soluble protein content showed a good linear negative correlation (Table 18). The guar gum content data of groups C, T1, T2, and T3 were calculated to be 0.000%, 0.009%, 0.019%, and 0.029%, respectively. The correlation between the solubility loss rate of pellets in the experimental group and the guar gum content of pellets was analyzed. The results showed that the solubility loss rate and the guar gum content also showed a good linear negative correlation. This shows that when the addition amount of enzymatic fermented compound protein feed in grass carp pellets is 5%-15%, the acid-soluble protein content is 1.210-1.979%, and the guar gum content is 0.009-0.029%, which can improve the water stability of grass carp pellets.

[0167] Table 18 Correlation between the solubility loss rate of group T and acid-soluble protein and guar gum

[0168] In summary, 3%-30% of the enzymatically hydrolyzed and then fermented composite protein feed of the present invention is added to aquatic pellet feed to replace or partially replace feed raw materials such as rapeseed meal, rice bran, fish meal, soybean meal, peanut meal, shrimp shell powder, etc. in the feed formula, and the dissolution rate of the pellet feed in water is reduced and the stability in water is improved. The enzymatically hydrolyzed and fermented composite protein feed of the present invention has a simple process, is easy to use, has obvious effects, and has a wide range of application prospects.

[0169] The material-water ratio was 1:15, the enzyme substrate concentration was 3200 U / g, the enzymolysis temperature was 60°C, and the enzymolysis time was 32 h, and an enzymolysis solution with a moisture content of 94.375%, a crude protein content of 3.125%, an acid-soluble protein content of 2.295%, and a guar gum content of 0.390% was obtained.

[0170] The Bacillus / composite bacteria ratio was 4:1, the fermentation agent inoculation amount was 0.45%, the mass ratio of soybean meal to shrimp shell powder (w / w) was 2:1, the proportion of guar meal enzymatic hydrolysate w / (w1+w2) was 60%, and the solid fermentation feed with a moisture content of 61.758%, crude protein of 17.609%, acid-soluble protein of 5.106%, and guar gum of 0.234% was obtained.

[0171] The shrimp pellet feed added with 1% sodium carboxymethylcellulose binder was selected as the control group C (Table 19). In the test group formulations, test group T1 (enzymatically hydrolyzed and fermented compound protein product was added at 3%, replacing 1.0% shrimp shell powder + 1.0% soybean meal + 1.0% peanut meal), test group T2 (enzymatically hydrolyzed and fermented compound protein product was added at 6%, replacing 2.0% shrimp shell powder + 2.0% soybean meal + 2.0% peanut meal), and test group T3 (enzymatically hydrolyzed and fermented compound protein product was added at 9%, replacing 3.0% shrimp shell powder + 3.0% soybean meal + 3.0% peanut meal) were designed. The dissolution rate of the pellet feed was detected under laboratory conditions. One-way analysis of variance was performed on the test data.

[0172] Table 19 Formulation of Shrimp Feed

[0173] *Same as Table 10.

[0174] Taking 2h of soaking as an example, the dissolution rate of the shrimp pellet feed ranked from low to high as T3 group < T2 group < T1 group < C group. The T2 group and T3 group were significantly lower than the T1 group and C group (P < 0.001), and there was no significant difference between the C group and the T1 group (P > 0.05). The results showed that adding 3 - 9% of the enzymatically hydrolyzed and fermented protein feed to the shrimp pellet feed had a lower dissolution rate than the control group C added with the binder (Table 20).

[0175] Table 20 Analysis of the Results of the Dissolution Rate of Shrimp Feed (n = 3)

[0176] Example 2 Preparation of Enzymatically Hydrolyzed and Then Fermented Compound Protein Feed and Its Application in Improving the Water Resistance Performance of Crab Pellet Feed A liquid of enzymolysis with a water content of 93.077%, crude protein of 3.846%, acid-soluble protein of 2.591%, and guar gum of 0.480% was obtained by selecting a material-to-water ratio of 1:12, an enzyme-substrate concentration of 2400U / g, an enzymolysis temperature of 45°C, and an enzymolysis time of 16h.

[0177] A solid-state fermented feed with a water content of 45.13%, crude protein of 22.938%, acid-soluble protein of 4.966%, and guar gum of 0.192% was obtained by selecting Bacillus spp. / compound bacteria at a ratio of 2:3, an inoculation amount of the fermentation agent of 0.60%, a mass ratio (w / w) of soybean meal to shrimp shell powder of 1:2, and the proportion of the enzymolysis liquid of guar soybean meal w / (w1 + w2) of 40%.

[0178] The river crab pellet feed added with 0.6% sodium carboxymethylcellulose binder was selected as the control group C. In the C formula, experimental groups T1 (enzymatically hydrolyzed and fermented compound protein product was added at 6%, replacing 1% fish meal + 1% flour + 3% rice bran), T2 (enzymatically hydrolyzed and fermented compound protein product was added at 10%, replacing 2% fish meal + 2% flour + 6% rice bran), and T3 (enzymatically hydrolyzed and fermented compound protein product was added at 15%, replacing 3% fish meal + 3% flour + 9% rice bran) were designed (Table 21). The dissolution rate of the pellet feed was detected under laboratory conditions. One-way analysis of variance was performed on the experimental data.

[0179] Table 21 River Crab Feed Formulas

[0180] *Same as Table 10.

[0181] Taking 30 minutes of soaking as an example, the dissolution rates of the river crab pellet feeds from low to high were ranked as T3 < T2 < T1 < C. The differences between groups C, T1, T2, and T3 were extremely significant (P < 0.001), and the differences between groups C, T1, and T2 were significant (P < 0.05). The results showed that adding 5%, 10%, and 15% of the enzymatically hydrolyzed and fermented protein feed to the river crab feed could obtain river crab pellet feeds with better water resistance than the control group C added with 0.6% sodium carboxymethylcellulose binder (Table 22).

[0182] Table 22 Analysis of the Dissolution Rate Results of River Crab Feed (%,n = 3)

[0183] Example 3 Preparation of Enzymatically Hydrolyzed and Then Fermented Compound Protein Feed and Its Application in Improving the Water Resistance of Freshwater Fish Pellet Feed A liquid hydrolyzate with a water content of 87.143%, crude protein of 7.143%, acid-soluble protein of 1.846%, and guar gum of 0.891% was obtained by selecting a material-to-water ratio of 1:6, an enzyme-substrate concentration of 800 U / g, an enzymatic hydrolysis temperature of 45°C, and an enzymatic hydrolysis time of 8 h.

[0184] A solid-state fermented feed with a water content of 34.893%, crude protein of 32.243%, acid-soluble protein of 3.102%, and guar gum of 0.267% was obtained by selecting Bacillus spp. / compound bacteria at a ratio of 1:4, an inoculation amount of the fermentation inoculant of 0.15%, a mass ratio (w / w) of soybean meal to shrimp shell powder of 3:0, and a proportion of the guar bean meal hydrolyzate of w / (w1 + w2) of 30%.

[0185] Taking the grass carp pellet feed C in Table 23 as the control group, Experiment Group 1 was designed with an addition amount of 5% of the enzymatically hydrolyzed and fermented compound protein product (equivalent substitution of 2.5% rapeseed meal + 2.5% cottonseed meal), Experiment Group 2 with 10% (equivalent substitution of 5% rapeseed meal + 5% cottonseed meal), and Experiment Group 3 with 15% (equivalent substitution of 7.5% rapeseed meal + 7.5% cottonseed meal) (Table 23). The dissolution rate of the pellet feed was detected under laboratory conditions. One-way analysis of variance was performed on the experimental data.

[0186] Table 23 Grass Carp Feed Formulation

[0187] *Same as Table 10.

[0188] Taking the example of soaking the pellet feed for 5 minutes, the dissolution rates of the grass carp pellet feeds ranked from low to high as Group T3 < Group T2 < Group T1 < Group C. The differences between Group C and Group T1, Group T2, and Group T3 were extremely significant ( P <0.001), and the difference between Group T1 and Group T2 was not significant ( P >0.05). The results showed that in grass carp pellet feeds, when 5 - 15% of the enzymatically hydrolyzed and fermented protein feed was used to replace rapeseed meal and cottonseed meal, the dissolution rate of the pellet feed decreased and the water resistance performance improved (Table 22).

[0189] Table 24 Analysis of Grass Carp Feed Dissolution Rate Results (%,n = 3)

Claims

1. A fermented protein feed, characterized in that: The fermented protein feed comprises the following components: 30.0%-65.0% moisture, 0.1%-1.5% guar gum, 14%-33% crude protein, and 2%-8.5% acid-soluble protein.

2. A fermented protein feed, characterized in that: The fermented protein feed comprises the following components: guar bean meal enzymatic hydrolysate, bacterial agent, soybean meal and shrimp shell powder.

3. The fermented protein feed according to claim 1 or 2, characterized in that: The obtained fermented protein feed is prepared using enzymatically hydrolyzed guar meal, bacterial agent, soybean meal and shrimp shell powder as raw materials. Preferably, the feed has a moisture content of 34.0%-63.0%, a guar gum content of 0.1%-1.3%, an acid-soluble protein content of 3%-8.5%, and a crude protein content of 15%-30%.

4. The fermented protein feed according to any one of claims 1 to 3, characterized in that: The fermented protein feed is prepared by the following method: guar bean meal: water 1:6-1:15 (w / w), enzyme substrate concentration 800-3200 U / g, enzymolysis temperature 45-60° C., enzymolysis time 8-32h, to obtain guar bean meal enzymolysis liquid; Preferably, the enzyme is a neutral protease and an alkaline protease, Preferably, the mass ratio of the neutral protease to the alkaline protease is 2:1-1:

2.

5. The fermented protein feed according to claim 4, characterized in that: The obtained guar meal enzymatic hydrolysate is further fermented, the fermentation temperature is 25°C-35°C, the fermentation time is 48h-96h, the fermentation agent is: Bacillus / complex bacteria 1:4-4:1, and the fermentation agent inoculation amount is 0.15-0.60%.

6. The fermented protein feed according to claim 5, characterized in that: The composite bacterial agent is Bacillus pumilus, Enterococcus faecalis, Lactobacillus plantarum and Monascus. Preferably, the ratio of Bacillus pumilus: Enterococcus faecalis: Lactobacillus plantarum: Monascus is 7-12:1:7-12:1-4 in terms of CFU.

7. The fermented protein feed according to any one of claims 2 to 6, characterized in that: The mass ratio of the soybean meal to the shrimp shell powder is 3:0-0:

3. Preferably, the proportion of the guar bean meal enzymatic hydrolysate w / (w1 soybean meal+w2 shrimp shell powder) is 30%-60%; Preferably, the proportion of the guar soybean meal enzymatic hydrolysate is w / (w1 soybean meal + w2 shrimp shell powder) 35%-55%; More preferably, the proportion of the guar soybean meal enzymatic hydrolyzate is w / (w1 soybean meal+w2 shrimp shell powder) 40%-50%.

8. A method for preparing a fermented protein feed, characterized in that: The steps include: S1, preparation of guar meal enzymatic hydrolysate: Guar soybean meal: water is 1:6-1:15 (w / w), enzyme substrate concentration is 800-3200U / g, enzymatic hydrolysis temperature is 45-60℃, enzymatic hydrolysis time is 8-32h, and guar soybean meal enzymatic hydrolysate is obtained; S2, preparing fermented protein feed: fermentation temperature 25°C-35°C, fermentation time 48h-96h, fermentation agent inoculation amount 0.15%-0.60%, soybean meal and shrimp shell powder mass ratio 3:0-0:3, guar meal enzymatic hydrolyzate proportion w / (w1+w2) 30%-60%; Preferably, in S1, the enzyme is a neutral protease and an alkaline protease, Preferably, the mass ratio of the neutral protease to the alkaline protease is 2:1-1:

2.

9. The preparation method according to claim 8, characterized in that: The fermentation bacteria agent has a ratio of Bacillus:complex bacteria of 1-4:4-1. Preferably, the complex bacteria agent is Bacillus pumilus, Enterococcus faecalis, Lactobacillus plantarum and Monascus; preferably, the ratio of Bacillus pumilus: Enterococcus faecalis: Lactobacillus plantarum: Monascus is 7-12:1:7-12:1-4 in terms of CFU.

10. Use of the fermented protein feed according to claims 1-7 or the fermented protein feed prepared by the method according to claims 8-9 in preparing feed products, preferably, the feed product is aquatic feed.

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