CN110679783A

CN110679783A - Application of clostridium ethanolate protein in low-starch expanded floating feed for carnivorous fish

Info

Publication number: CN110679783A
Application number: CN201910955305.0A
Authority: CN
Inventors: 薛敏; 马世峰; 邹方起; 梁晓芳; 晁伟
Original assignee: Beijing Shoulang Biotechnology Co ltd; Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Beijing Shoulang Biotechnology Co ltd; Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2019-10-09
Filing date: 2019-10-09
Publication date: 2020-01-14

Abstract

The invention relates to a low-starch (starch level is less than 10%) puffed floating feed for carnivorous fishes. Part of protein raw materials of the feed use a novel microbial protein, namely clostridium ethanolate protein, and the feed is suitable for carnivorous fishes intolerant to starch. Contrast experiments prove that the feed can not only greatly reduce the using amount of fish meal and meet the nutrient requirements required by the growth and development of carnivorous fishes, but also obviously reduce the volume weight of the feed and improve the radial expansion rate, the particle hardness, the gelatinization degree and the stability in water of the low-starch feed; and the oil absorption rate is improved by improving the pore diameter structure in the particles, and the oil leakage rate is reduced. The invention also obviously improves the processing stable quality of the low-starch floating expanded feed.

Description

Application of clostridium ethanolate protein in low-starch expanded floating feed for carnivorous fish

Technical Field

The invention belongs to the technical field of agricultural aquaculture, and relates to application of a novel microbial protein raw material namely clostridium autoethanolate protein in production and culture of low-starch puffed floating feed for carnivorous fishes.

Background

"Low starch buoyancy feed" (i.e., a feed defined by the aquaculture feed industry to be within 10% starch) is suitable for use with carnivorous upper water habit fish. Since fish, especially carnivorous fish, have a low utilization of sugars, in particular starch. When the addition amount of starch in the feed exceeds a tolerance level, excessive accumulation of liver glycogen and fat and disturbance of glucose and fat metabolism are caused, so that chronic inflammation and apoptosis are caused, and liver injury is finally caused.

Carnivorous fishes, such as Micropterus salmoides, are important freshwater aquaculture species, but the traditional aquaculture is limited by the influence of feed preparation (such as depending on chilled baits, fish meal and the like), so that the production cost is too high and the diseases are frequent. After the low-starch expanded floating feed enters the market, the rapid development of the low-starch expanded floating feed is promoted. For example, the output of the largemouth black bass in 2018 breaks through 50 ten thousand tons.

In the production of low-starch expanded feed, the characteristics of the used protein raw materials have great influence on the quality of products, because the protein is denatured in the extrusion and expansion process, and hydrophobic bonds and disulfide bonds are bonded among denatured protein molecules to generate organization effect. In the production of the low-starch expanded floating feed, the selection of a protein raw material with higher nutritional value and good processing property is one of the key factors for solving the production problem of the low-starch expanded floating feed.

Fish meal is the most important protein source in aquatic feeds, but the price of the fish meal is continuously increased and adulterated due to the continuous deterioration of marine environment and the reduction of fishery resources in recent years. In addition, the fish meal with different freshness and different sources has great difference in processing performance, and the unstable quality of the fish meal is also one of the important reasons for the unstable production process and processing quality of the low-starch expanded floating feed. As the largest import country of fish meal, the problem of fish meal shortage is becoming more severe in China, and a new protein source is urgently needed to be searched for replacing the fish meal.

At present, three protein sources are mainly adopted to replace fish meal in aquaculture and are respectively an animal protein source, a plant protein source and a single-cell protein source. The animal protein has the problems of biological safety, while the plant protein has the problems of nutrient deficiency, high anti-nutritional factor content, low digestibility and the like. Meanwhile, vegetable proteins such as soybean and corn are staple food grain raw materials, and the problem of food safety exists when the vegetable proteins are used in a large amount in feed.

Starch is an important component for ensuring that the material has enough viscosity and is expanded by heating, and the stable production of the bulking machine can be ensured only when the starch level in the feed reaches more than 20 percent in the traditional process for producing the bulked feed. Moreover, when the starch content in the feed formula is lower than 10%, the production stability of the feed is poor, and unplanned shutdown faults often occur in the production process, and meanwhile, due to the problems that the expansion rate of the particles is low, the volume weight is high, the particle feed partially sinks or even completely sinks in water, and the like, the feeding habit of water surface feeding of carnivorous fishes with the upper living habit cannot be met, and double waste is caused.

In application No. 201811049614.3, a low starch expanded compound feed for increasing the growth rate and survival rate of large yellow croaker is disclosed, comprising: russian white fish meal, peru fish meal, American chicken meal, wheat gluten, corn gluten meal, soybean meal, high gluten flour, cassava starch, enzymatic fish soluble paste, refined fish oil, soybean phospholipid oil, monocalcium phosphate, vitamin premix, mineral salt premix, choline chloride, lutein, an antioxidant and a mildew preventive. The feed in this patent has a starch content of 7.4% to 9.4% and is typically a low starch feed. But the feed expansion rate is low (only 1.4-1.6). The common knowledge in the field is that after the particles with the expansion ratio of less than 1.5 are sprayed with the grease, the volume weight is further increased, and the phenomenon of feed sinking is very likely to occur.

Also, patent application No. 20130044057.7 discloses a fish meal-free buoyant expanded feed for micropterus salmoides and a preparation method thereof. Comprises dephenolized cottonseed protein, globulin powder, soybean protein concentrate, rice protein powder, fish soluble protein, calcium dihydrogen phosphate, wheat gluten protein, wheat flour, phospholipid oil, etc. In the patent, fish meal is not used, but fish soluble protein (the using amount is 15-17 parts) contains a large amount of water-soluble fish protein, and in addition, the using amount of wheat meal in the feed reaches 22 parts, the starch content is at least more than 15 percent, and the feed does not belong to low-starch feed. Nevertheless, its post-processing expansion ratio is only 1.5, which may still cause the particles to sink.

And carnivorous fishes with water living habits are used to ingest floating pellet feed, and the feed cannot be ingested when sinking to the water bottom, thereby causing waste and pollution.

Therefore, two problems need to be solved in the production technology of the low-starch expanded floating feed for carnivorous fishes:

firstly, the cost of a protein source is reduced, and a novel low-starch expanded floating feed produced by using the protein source fish meal is found to replace fresh frozen feed;

and secondly, the problems of poor stability, low expansion rate, easy sedimentation, unstable production and the like of the low-starch feed in water are solved.

Clostridium ethanolate protein (ECM) is a novel mycoprotein produced by using clostridium ethanolate (clostridium autoethanogenum) as a fermentation strain, and is a single-cell protein. Clostridium ethanolate was first isolated from rabbit feces by Abrini et al (1994), carbon monoxide (CO) gas was used as the raw material in the production process, liquid fermentation was carried out, and the fermentation broth was centrifuged and dried to obtain high-protein biological feed raw material Clostridium ethanolate protein. The clostridium ethanolate protein has high protein content, complete amino acid types, better balance and no definite anti-nutritional factors. At present, except a small amount of literature research, the feed additive is not actually used as the raw material of aquatic feed.

Weihongcheng et al (2018) explored the effect of clostridium ethanolate protein substituting soybean meal on grass carp growth performance, plasma biochemical indexes and liver, pancreas and intestinal tract histopathology. However, the study is directed to grass carp, which is a vegetal fish, not a carnivorous fish. As is well known, grass carp can eat water grass and has strong tolerance to starch. In the above documents, the formulation is mainly based on vegetable protein suitable for producing float feed, and contains high level of starch (more than 20%, including 30% flour and 21.4% wheat middling), and has no processing difficulties. The document recommends that the amount of clostridium ethanolate protein used in grass carp feed is only 5% and is not used to replace fish meal.

The research of using clostridium ethanolate protein as a novel protein source to replace fish meal and applying the clostridium ethanolate protein to the low-starch expanded floating feed of carnivorous fish is blank internationally in both feed processing technology and experimental research of culturing the carnivorous fish. Therefore, the research on the application of the clostridium ethanolate protein in the low-starch expanded floating feed of the carnivorous fish is very necessary.

Disclosure of Invention

The invention aims to solve the technical problems that a novel protein source is found, the problem of fish meal shortage is relieved, a special low-starch expanded floating feed formula with comprehensive and balanced nutritional ingredients is provided, all nutritional requirements required by growth and development of carnivorous fishes are met, and the feed has the requirements of good stability in water, no sedimentation, high expansion rate and stable production.

The invention achieves the above purpose through the following means.

1. The inventor finds that a novel microorganism protein raw material, namely clostridium ethanolate protein, is added into a raw material of a low-starch expanded buoyant feed for carnivorous fishes, and can partially replace fish meal.

The inventor evaluates various indexes of the low-starch feed added with the clostridium ethanolate protein, including experiments of volume weight, expansion rate, water absorption, water solubility, oil absorption rate, oil leakage rate and the like of the feed, also carries out breeding experiments by using the largemouth black perch, evaluates the growth performance, blood indexes and liver histological analysis of the largemouth black perch (in detail, see an embodiment), and obtains a proper addition ratio of the clostridium ethanolate protein according to comprehensive evaluation results of a plurality of indexes.

Therefore, the technical scheme adopted by the invention is as follows:

the low-starch expanded buoyancy feed for carnivorous fishes is characterized in that clostridium autoethanolate protein (ECM) for partially replacing fish meal is contained in raw materials for preparing the feed, and the weight percentage of the clostridium autoethanolate protein (ECM) accounts for 9-34.5 percent of the raw materials for preparing the feed.

The feed comprises two protein raw materials of fish meal and clostridium ethanolate protein in parts by weight:

FM 0-30；ECM 9-34.5；

wherein FM is low temperature dried fish meal, ECM is Clostridium ethanolicum protein.

According to the volume weight, the expansion rate, the water absorption, the water solubility, the growth performance of the largemouth black bass, the blood index and the comprehensive evaluation result of the liver histological analysis experiment of the feed, the optimal proportion is obtained as follows:

FM 10-20；ECM 18-27。

according to the volume weight, the expansion rate, the oil absorption rate, the oil leakage rate, the water absorption, the water solubility, the growth performance of the largemouth black bass, the blood index and the comprehensive evaluation result of the liver histological analysis experiment of the feed, the optimal proportion is obtained as follows:

FM 10；ECM 27。

the addition of clostridium ethanolate protein not only replaces part of fish meal, but also relieves the problem of fish meal shortage. And the clostridium ethanolate protein has high water absorption and high viscosity, plays a role in bonding in low-starch feed, and overcomes the defects of poor stability, low expansion rate, easy sedimentation, unstable production and the like of starch in water.

2. Selection of starch sources

The starch source is cassava flour and wheat flour, and the cassava flour and the wheat flour are matched in a proper proportion and are one of the key points for ensuring the quality of the low-starch expanded floating feed.

Wheat flour is the most digestible and absorbable starch source for fish, but has low swelling effect and low water floating rate in feed. Therefore, the cassava flour has the advantages of high amylopectin content, high viscosity after gelatinization and good processing characteristics. The invention discovers that the addition of the cassava flour can improve the feed quality and improve the expansion effect and the floating rate.

However, the feed digestion energy is reduced due to the excessive use of the cassava flour, so that the invention researches the proper proportion of the cassava flour and the wheat flour in the formula to obtain the feed; the preferable proportion of the dosage of the cassava flour and the wheat flour is as follows: 5 parts of cassava flour and 6 parts of wheat flour, wherein the parts are parts by weight.

3. Research on moisture content of material after tempering in feed processing process

As the water absorption of the clostridium ethanolate protein is higher than that of fish meal, researches show that the water content of the modified material in the feed processing process is 27-30%, which is different from the water content of the modified material in the prior art which is 22-27%.

The improvement of the moisture content of the modified feed is beneficial to promoting sufficient water absorption and material viscosity increase of high-viscosity materials and simultaneously promoting starch gelatinization, thereby improving the swelling degree of the feed.

4. Oil adding mode in feed raw materials

The research of the invention finds that the grease can rapidly reduce the viscosity of the material and the detention time in the cavity of the bulking machine, so that the curing degree and the bulking rate of the material are reduced, and the floating feed production is not facilitated.

The invention also discovers that the adverse effect on the processing of the low-starch floating feed can be reduced to the maximum extent by adding various exogenous grease after the feed is granulated and formed.

Therefore, the scheme provided by the invention is that the feed particles are dried and then added with grease to obtain a finished product;

this is also different from the conventional method of the prior art in which the fats and oils are added together when the raw materials are mixed.

The addition mode of the oil is preferably spraying in a vacuum state, the variety of the oil used is selected according to the requirement of the feed, and the oil can be vegetable oil or animal oil as long as the physical properties of the oil can be sprayed.

In the raw materials of the low-starch expanded floating feed for carnivorous fishes, other required nutrient substances can be selected from any raw materials used by people in the technical field according to various requirements, and the raw materials comprise basic nutrient substances such as protein, fat, starch source, vitamins, mineral substances and the like.

In one example of the invention, the base nutrient formula for the other nutrients is: 14.8 parts of concentrated cottonseed protein, 16.3 parts of concentrated soybean protein, 6 parts of wheat flour, 5 parts of cassava flour, 6 parts of wheat gluten and 8.9-11.1 parts of 6 soybean oil, wherein the parts are parts by weight.

The basic nutrient formula can also contain the following components: 1-3.1 parts of cellulose, 1-1.8 parts of monocalcium phosphate and 1.4 parts of vitamin and mineral premix, wherein the parts are parts by weight.

See table 1 for details.

The preparation method of the low-starch expanded buoyant feed comprises the steps of crushing solid raw materials, mixing the raw materials of the feed, and then carrying out conditioning, extrusion expansion, granulation, air drying to obtain feed particles and the like. The method is characterized in that the moisture content of the material after tempering is higher than that of the prior art and is up to 27-30%; the oil raw materials required in the feed are added in a spraying mode after the feed particles are formed.

The invention carries out the following experiments on the prepared low-starch expanded floating feed for the carnivorous fish, which uses clostridium ethanolicum protein to replace fish meal (the specific data are shown in the embodiment in detail):

1. volume weight;

2. the radial expansion rate;

3. the rate of dissolution;

4. oil absorption and oil leakage;

5. the floating rate;

6. particle hardness;

7. water absorption and water solubility (water absorption (WAI) and Water Solubility (WSI) index).

Test results show that the volume weight, the dissolution rate and the oil leakage rate of the low-starch expanded floating feed are obviously reduced; the expansion rate, the particle hardness and the oil absorption rate are obviously improved; the cross section of the feed is enlarged, the pores are increased, the gelatinization degree of the feed is improved, and the capability of maintaining the integrity of the feed in water is improved (see the embodiment for specific data). The scheme of the invention is very excellent, and particularly, the processing performance of the clostridium acetobutylicum protein is found to be good for the first time.

The invention has the beneficial effects that:

1. the clostridium ethanolate protein is used for replacing fish meal in the carnivorous fish feed for the first time

The clostridium ethanolate protein is applied to the carnivorous fish feed instead of fish meal, so that the problems of high fish meal cost, poor sustainability and the like are solved, the culture economic benefit is increased, and the application of the novel single-cell protein in the aquatic feed is promoted.

2. Can meet all the nutritional requirements required by the growth and development of carnivorous fishes

Experiments prove that the low-starch expanded buoyancy feed (the starch content is less than 10%) for the carnivorous fishes can meet all nutritional requirements required by the growth and development of the carnivorous fishes represented by largemouth bass, and solves the problems of liver damage, growth performance reduction and the like caused by overhigh starch content in a formula. Has no significant influence on the survival rate, the final average weight, the weight gain rate, the specific growth rate, the ingestion rate and the feed coefficient of the micropterus salmoides, and has no negative influence on the liver tissues of the micropterus salmoides

3. Good product quality and less waste

The feed can reduce the volume weight of the feed, improve the radial expansion rate, the granule hardness, the gelatinization degree and the stability in water of the feed, improve the oil absorption rate by improving the pore structure inside the granules, reduce the oil leakage rate, improve the production stability and reduce the waste and the unplanned shutdown caused by unqualified product quality.

4. Provides a preparation method and key process parameters of a low-starch expanded floating feed for carnivorous fishes

The invention provides a preparation method and key process parameters of a low-starch puffed floating feed for carnivorous fishes in the aquatic feed industry, and is beneficial to the progress of the industry.

Drawings

FIG. 1 is a cross-sectional scanning electron microscope image of a fish meal group feed and a low-starch expanded buoyancy feed for carnivorous fishes according to the present invention;

fig. 2 is a photograph of liver tissue sections of micropterus salmoides from the fish meal group, ECM10 group and ECM40 group.

Detailed Description

The present invention is further illustrated by the following specific examples.

Example 1 formulation, preparation and experimental verification of a low-starch expanded buoyant feed for carnivorous fish

Firstly, the purpose of experiment is as follows: the influence of the clostridium ethanolate protein on the quality of the low-starch expanded floating feed product of the carnivorous fish and the influence on the production stability after replacing fish meal are verified

Second, formula and preparation of experimental material-low starch puffed floating feed for carnivorous fish

1. The low-starch expanded floating feed formula for carnivorous fishes comprises two parts:

1) basic nutritional formula (weight percent): 14.8 percent of cottonseed protein (containing 61.51 percent of crude protein and 2.36 percent of crude fat), 16.3 percent of soybean protein concentrate (containing 64 percent of crude protein and 0.25 percent of crude fat), 6 percent of wheat flour, 5 percent of cassava flour, 6 percent of wheat gluten, 8.9 to 11.1 percent of 6 percent of soybean oil, 1 to 3.1 percent of cellulose, 1 to 1.8 percent of calcium dihydrogen phosphate and 1.4 percent of vitamin and mineral premix.

2) Two protein sources: the clostridium ethanolate protein (containing 83.2 percent of crude protein, 1.9 percent of crude fat, and produced by Beijing Hainan biological technology limited) is used for replacing the fish meal (containing 72 percent of crude protein, 8.84 percent of crude fat and 999 percent of Danish fish meal) in a gradient way;

3) grouping

4 groups of feeds formulated in different ratios of FM: ECM were named ECM10(FM: ECM ═ 30:9), ECM20(FM: ECM ═ 20:18), ECM30(FM: ECM ═ 10:27) and ECM40(FM: ECM ═ 0:34.5), respectively

In addition, only fish meal as a protein material was used as a Control (Control) without addition of clostridium ethanolate protein.

The feed formulation and nutrient levels are shown in table 1.

Table 1 feed formulation and nutrient levels (%)

2. Preparation method of low-starch puffed floating feed for carnivorous fishes

Crushing solid feed raw materials by using an ultrafine crusher, sieving 98 percent of the raw materials by using a 80-mesh sieve, weighing all the feed raw materials according to a formula proportion, and mixing according to a step-by-step enlargement principle.

The mixed material was fed from a volumetric feeder into a conditioner at a feed rate of 70kg/h and held constant during the test, the temperature of the material in the conditioner being maintained at 95 ℃. The parameters of the puffing process are adjusted according to the experimental design, including the moisture content of the material after tempering, the rotating speed of a screw of the puffing machine and the temperature of a die head, other parameters are kept constant, and the parameters of the puffing process are shown in table 2.

The puffing processing parameters are recorded through an automatic acquisition system and comprise conditioner water flow, conditioner steam flow, puffing machine screw rotating speed, puffing cavity temperature and puffing machine screw torque. And (3) collecting a sample after the processing parameters of the bulking machine reach a set value and are stable for 15min, drying the sample until the water content is lower than 10%, spraying soybean oil and fish oil by adopting a vacuum spraying method, and packaging for analysis and detection and culture tests.

TABLE 2 puffing process parameter table

Third, experiment project and experiment method of low-starch puffed floating feed for carnivorous fishes

1. Volume weight

Peeling a container with the volume of 1L, pouring a sample into a funnel, filling the container with the feed by using the funnel, scraping the opening of the container with an iron plate, measuring the weight, wherein the container cannot be vibrated or knocked before weighing, and the weighed weight is the volume weight of the feed. Each sample was repeated three times, and the mean value was taken as the volume weight (in g/L) of the sample.

2. Radial expansion ratio

20 specimens were randomly sampled, and the diameters thereof were measured with a vernier caliper, and the average value thereof was taken as the diameter of the specimen. The radial expansion of the sample is the particle diameter/orifice diameter.

3. Rate of dissolution

Weighing 10g of feed (accurate to 0.1g) and placing the feed into a prepared cylindrical mesh screen, then placing the mesh screen into a container with the water depth of 5.5cm and the water temperature of (25 +/-2) DEG C for soaking, then slowly lifting a mesh cage from the water to the water surface, slowly sinking the mesh cage into the water again to enable the feed to leave the screen bottom, soaking for 20 minutes, repeating the steps for three times, taking out the mesh screen, obliquely draining the attached water, placing the feed in the mesh screen into a 105 ℃ oven for drying to constant weight, meanwhile, weighing a sample (control material) which is not soaked in the water and has the same weight, placing the sample in the 105 ℃ oven for drying to constant weight, then weighing respectively, and calculating according to the following formula. Two parallel samples are taken for each sample to be measured, the arithmetic mean value is taken as a result, the numerical value is expressed to one decimal, and the allowable relative error is less than or equal to 4 percent.

In the formula:

s: the rate of dissolution, in percent (%);

m₁: the weight of the reference material after being dried is gram (g);

m₂: the mass of the soaked material after drying is gram (g).

4. Oil absorption and oil leakage

The oil absorption of the feed was studied using a laboratory scale vacuum sprayer. 100g of the particles which are not sprayed and 150g of excessive soybean oil are added into a spraying machine and vacuumized, and air is slowly released after uniform mixing. Gently press the particles using an oil absorbing paper to remove excess oil, then weigh M₁。

Weighing 100g of particles with oil absorption measured completely, placing the particles on oil absorption paper for 24h, and weighing M₂。

5. Rate of floating water

Buoyancy test for each formulation 10 pellets were placed in a 2L beaker with 75% water and 3 tests were run for each formulation. And (3) recording the number of the floating feeds after the floating feeds are placed for 30min, wherein the floating rate is as follows:

6. hardness of the particles

The hardness of the particles was measured by a texture analyzer (model: TA-XY2i, origin: British Stable Micro). And randomly taking 30 grains of each test sample for measurement, setting the speed of a probe to be 1mm/s, the force to be 25kg, lifting the probe after the grains deform under the stress of 40%, taking the peak value of the atlas at the stage as the measurement result of the hardness, and taking the average value of the hardness of the 30 grains as the hardness of the sample.

7. Water absorption and solubility

Water Absorption (WAI) and Water Solubility (WSI) index

The sample is crushed and then screened by a 100-mesh sieve. 2.5g of the powdery puffed sample is put into a 60mL centrifuge tube, 30mL of distilled water is added, the mixture is placed in a constant temperature water tank at 30 ℃ for 30min, and then the mixture is centrifuged for 10min by 3000 g. The supernatant was slowly poured into a constant weight aluminum cup and dried in an oven at 135 ℃ for 2 h. Weigh the mass of gel in the centrifuge tube. Each sample was measured 3 times. Water Absorption (WAI) and water solubility (WAI) were calculated using the following equations:

in the formula, WAI: a water absorption index; w_g: gel weight (g)

In the formula, WSI: water solubility index (%);

W_ss: sample weight (g) after drying the suspension;

W_ds: weight (g) of sample after drying

8. Data analysis and statistics

The results are expressed as mean ± standard deviation, and the data were subjected to one-way analysis of variance (ANOVA) and multiple comparisons using the Duncan's method to test the significance of differences between treatments (P < 0.05).

Fourth, test results

1. The volume weight is obviously reduced

The volume weight of the feed is obviously reduced along with the increase of the replacement amount of the clostridium ethanolate protein (P < 0.05); the lower the volume weight, the lower the unit weight of the feed particles, and the feed particles are more suitable for floating on the water surface. The volume weight of the low-starch floating feed in the current market is generally about 400g/L, while the volume weight of the low-starch feed produced by using 20-40 parts of clostridium ethanolate protein is lower than 380g/L, so that the low-starch floating feed can still float on the water surface after absorbing water.

2. Improving the puffing effect

The addition of clostridium ethanolate protein can improve the puffing effect;

the radial expansion rate of the ECM20 group is not significantly different from that of the control group (P >0.05), and the radial expansion rate of the rest three groups is significantly higher than that of the control group (P < 0.05);

3. the dissolution rate is obviously reduced, and the stability of the feed in water is improved

The dissolution rate of each test group is obviously lower than that of a control group (P is less than 0.05), which indicates that the water stability of the feed can be improved by adding the clostridium ethanolate protein;

4. the oil absorption of the feed is improved, and the oil leakage rate is reduced

The ECM30 and ECM40 groups showed significantly greater oil absorption than the control group (P <0.05),

the oil leakage rate of the ECM20, ECM30 and ECM40 groups was significantly less than that of the control group (P <0.05), indicating that the addition of clostridium ethanolate protein can increase oil absorption of the feed; the feed has larger pores and increased amount of clostridium ethanolate protein (as shown in figure 1), which is helpful for improving oil absorption of the feed.

5. Ensure the floating rate

The addition of clostridium ethanolate protein reduces the volume weight, and can ensure that the particles do not sink after absorbing water;

6. the structure of the feed is more compact, and the feed is not easy to produce particles

The hardness of the particles of each test group is obviously higher than that of a control group (P is less than 0.05), which indicates that the structure of the feed is more compact after the clostridium ethanolate protein is added, and particles are not easy to generate;

7. improving the gelatinization degree of the feed

With the increase of the addition amount of clostridium ethanolate protein, the Water Absorption Index (WAI) is firstly obviously increased and then obviously decreased (P is less than 0.05); the WAI can reflect the gelatinization degree, and proves that the gelatinization degree of the feed can be improved by adding a proper amount of clostridium ethanolate protein;

8. improving the ability of feed to maintain integrity in water

With the increase of the addition amount of clostridium ethanolate protein, the Water Solubility Index (WSI) is firstly remarkably reduced and then remarkably increased (P < 0.05). WSI can reflect the ability of swollen gel particles to maintain integrity in water, and the addition of a proper amount of Clostridium ethanolate protein can be proved to improve the ability of feed to maintain integrity in water.

The data of the above indexes are shown in Table 3

TABLE 3 influence of Clostridium ethanolicum protein on the quality of expanded feed products in place of fish meal

Note: different shoulder letters in the same row indicate significant differences (P < 0.05).

Fifth, conclusion of experiment

After the clostridium ethanolate protein replaces fish meal, the low-starch expanded floating feed has an improvement effect on the quality of a low-starch expanded floating feed product for carnivorous fishes, and can improve the production stability.

Example 2 Low-starch expanded buoyant feed for carnivorous fish Lateolabrax micropterus culture test

First, experiment purpose

The influence of the clostridium ethanolate protein on the growth performance and the liver tissue of the carnivorous fish represented by the largemouth bass is verified after the clostridium ethanolate protein is used for replacing fish meal.

Second, Experimental methods

1. Experiment feed

The feed of example 1 was used for the breeding test.

2. Place of implementation

The method is carried out in an indoor circulating water culture system in a national aquaculture feed safety evaluation base (Beijing, Nankou).

3. Aquatic target animal for test

Perch, purchased from Guangdong mountain Foshan.

Randomly selecting healthy and uniform micropterus salmoides (average weight of 47.99 + -0.01 g) and distributing to a volume of 0.26m³In the conical cultivation bucket.

This experiment designed 5 treatment groups, 4 replicates per treatment group, with 25 fish per barrel.

4. Method of producing a composite material

Feeding for 2 times per day with feeding time of 8:00 and 16: 00.

Periodically detecting water quality, wherein the water quality condition is kept at dissolved oxygen DO more than 7.0 mg/L; the ammonia nitrogen level is less than 0.5 mg/L; pH 7.5-8.5; the water temperature is 20 +/-2 ℃.

The culture test is carried out from 8 months and 15 days in 2018 to 10 months and 8 days in 2018 for 8 weeks.

5. Test results

1) The effect on growth performance of micropterus salmoides is shown in Table 4.

TABLE 4 influence of Clostridia ethanolata protein on growth performance of Lateolabrax

n＝4)

As can be seen from the data in Table 4, the survival rate of the largemouth bass reaches 100% after the clostridium ethanolicum protein replaces the fish meal. The average weight of powder, the weight gain rate, the specific growth rate, the ingestion rate and the feed coefficient have no obvious difference.

2) Influence of different clostridium ethanolate protein levels in feed on micropterus salmoides plasma biochemical indexes

See table 5.

TABLE 5 influence of Clostridia ethanolata protein on the biochemical indicators of Perolax micropterus blood instead of fish meal: (n＝8)

The plasma Glucose (Glucose), alanine Aminotransferase (ALT) activity and Triglyceride (TG) of each test group were significantly lower than the control group (P <0.05) after addition of clostridium ethanolicum protein;

glutamate-oxaloacetate transaminase (AST) activity of ECM30 group was significantly lower than control and ECM10 group (P < 0.05);

the Total Cholesterol (TC) of ECM10 group was significantly higher than that of each of the other groups (P <0.05), while the Total Cholesterol (TC) of each of the other groups was not significantly different (P > 0.05);

the high density lipoprotein (HDL-C) of ECM10 group was significantly higher than the control group (P < 0.05);

the low density lipoprotein (LDL-C) was significantly lower in the ECM30 group than in the control group (P < 0.05).

The high density lipoprotein/total cholesterol (HDL-C/TC) of the test group has no significant difference with the control group (P > 0.05).

3) Influence of different clostridium ethanolate protein levels in feed on liver tissues of micropterus salmoides

The results of liver histological sections of micropterus salmoides from Control group, ECM10 group and ECM40 group are shown in fig. 2.

The liver cells of the three groups of micropterus salmoides are arranged neatly, the cell boundary is obvious, the cell nucleus is clear and visible, and no obvious abnormality is seen.

After the clostridium ethanolate protein replaces fish meal, the growth performance of the largemouth bass is not obviously influenced, the activity of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in blood plasma is reduced, Triglyceride (TG) is obviously reduced, and high density lipoprotein/total cholesterol (HDL-C/TC) has no obvious difference.

The clostridium ethanolate protein has the function of improving liver function and lipid metabolism, and has no negative influence on the liver tissue structure of the micropterus salmoides. The clostridium ethanolate protein has good breeding effect after replacing fish meal as a novel protein source, can replace the fish meal, and improves the economic benefit of breeding.

The above are only a few specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and its concept within the technical scope of the present invention.

Claims

1. The low-starch expanded floating feed for carnivorous fish is characterized in that raw materials prepared from the feed contain clostridium autoethanolate protein for partially replacing fish meal, and the weight percentage of the clostridium autoethanolate protein accounts for 9-34.5% of the raw materials for preparing the feed.

2. The low starch expanded feed according to claim 1, wherein the ratio of the fish meal to the clostridium ethanolate protein in the raw materials is, in parts by weight: FM 0-30; ECM 9-34.5; wherein FM is low temperature dried fish meal, ECM is Clostridium ethanolicum protein.

3. The low starch expanded feed according to claim 2, wherein the ratio of the fish meal to the clostridium ethanolate protein in the raw materials is, in parts by weight: FM 10-20; ECM 18-27.

4. The low starch expanded feed according to claim 2, wherein the ratio of the fish meal to the clostridium ethanolate protein in the raw materials is, in parts by weight: FM 10; the ECM 27.

5. The low-starch expanded feed according to any one of claims 1 to 3, wherein the starch source used in the raw material is both tapioca flour and wheat flour.

6. The low starch expanded feed of claim 5, wherein the tapioca flour and the wheat flour are used in the following proportions: 5 parts of cassava flour and 6 parts of wheat flour, wherein the parts are parts by weight.

7. The low-starch expanded floating feed as claimed in claim 1 to 3, wherein the other nutrient components in the raw materials are: 14.8 parts of concentrated cottonseed protein, 16.3 parts of concentrated soybean protein, 6 parts of wheat flour, 5 parts of cassava flour, 6 parts of wheat gluten and 8.9-11.1 parts of 6 soybean oil, wherein the parts are parts by weight.

8. The low starch expanded feed according to claim 7, wherein the raw materials further comprise 1-3.1 parts by weight of cellulose, 1-1.8 parts by weight of monocalcium phosphate, and 1.4 parts by weight of premix of vitamins and minerals.

9. The low-starch expanded feed according to any one of claims 1 to 3, which is prepared by the following steps: crushing solid raw materials, mixing the raw materials for feed, and then carrying out tempering, extrusion and expansion, granulation and air drying to obtain feed particles, wherein the feed particles are characterized in that the moisture content of the tempered material is 27-30%.

10. The low-starch expanded feed according to claim 9, wherein the oil-based material is added after the pellets are formed.