Application of ethanolic clostridial protein in promoting growth and improving intestinal flora of Litopenaeus vannamei

Abstract

The invention belongs to the technical field of aquatic feed application, and discloses the application of ethanol clostridial protein in promoting the growth of Penaeus vannamei and improving intestinal flora. The invention takes Penaeus vannamei as the research object, studies the influence of ethanol clostridial protein as a partial replacement source of fish meal on Penaeus vannamei, and finds that it has a positive influence on the growth performance, immune performance, muscle fatty acid synthesis, intestinal flora and other aspects of Penaeus vannamei, thereby providing a theoretical basis for realizing ethanol clostridial protein as a partial replacement source of fish meal.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic feed application, and more specifically, relates to the application of ethanol clostridium protein in promoting the growth of Litopenaeus vannamei and improving intestinal flora. Background Art

[0002] Vannamei shrimp, commonly known as white shrimp, is a tropical shrimp with wide temperature and salt tolerance. The shrimp has delicious meat and high unit yield, and is currently one of the most important breeding objects in my country. Studies have shown that Vannamei shrimp has a high demand for protein, and the optimal protein level of its shrimp feed is 35% to 40%. Fish meal, as a marine animal protein raw material, is irreplaceable or special in the diet of aquatic animals. The use of high-quality fish meal is the main method to meet the high protein demand of Vannamei shrimp. Fish meal is a representative raw material from marine fish, and has advantages in nutrient composition patterns such as amino acid composition patterns and digestibility. This is the main advantage of fish meal over other feed raw materials. It is reported that 35% of the global catch is used to produce fish meal, but in recent years, due to the rise in fish meal prices, the cost of feed has greatly increased, so reducing the price of fish meal is an important key to saving breeding costs. Fish meal has always been in an irreplaceable position in the feed used in global aquaculture. At the current rate of consumption, fish meal resources will no longer be able to meet the growing demand for aquaculture. Shrimp feed is also heavily dependent on fish meal. About 50% of the cost of shrimp farming comes from feed input, of which fish meal costs account for a large proportion. At present, the proportion of fish meal in the current commercial shrimp feed formula is maintained at more than 25%. It is urgent to find other available protein sources to effectively replace fish meal in shrimp feed, so as to reduce the proportion of fish meal in shrimp feed, reduce feed costs, and reduce the shrimp farming industry’s strong dependence on fish meal. Therefore, reducing the fish meal content in Vannamei shrimp feed and developing new protein sources can not only reduce feed costs, but also reduce the environmental burden, which plays an important role in promoting the sustainable development of the Vannamei shrimp feed industry.

[0003] Animal protein, plant protein, and microbial protein are important protein sources. Animal protein sources such as blood meal, chicken meal, and meat and bone meal have high protein content and can replace some fish meal, but their essential amino acids such as isoleucine and lysine are insufficient, and the fat saturation in animal protein sources is high, which affects the palatability of fish; plant protein sources such as soybeans, rapeseed meal, and plant protein isolates have high protein content and are rich in mineral elements and vitamins, which can partially replace fish meal, but the anti-nutritional factors in plant protein sources such as hemagglutinin and trypsin inhibitors will affect the growth of aquatic animals and the flavor of feed; microbial protein often comes from bacteria, fungi, yeast, unicellular algae, etc. Single-cell protein is a type of microbial protein, which has a high protein content and a balanced amino acid ratio, but a low digestibility, and safety also needs further confirmation. Single-cell protein, also known as bacterial protein or microbial protein, refers to the bacterial protein obtained by microalgae, bacteria, and true algae using various substrates during their growth to cultivate individual cells or filamentous microorganisms under suitable culture conditions. Single-cell protein feed has the advantages of high production efficiency, abundant raw materials, and small production area. The development of new protein sources that can replace fish meal is the current and future development direction of shrimp farming and even the entire aquatic industry, and is of great significance to the healthy development of the Vannamei shrimp industry. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the above problems existing in the prior art. Firstly, application of ethanolic clostridium protein as a partial substitute source for fish meal is provided.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The invention firstly provides the use of ethanol clostridial protein as a partial substitute source of fish meal in promoting the growth performance of Penaeus vannamei. The growth performance includes average final weight, weight gain rate and specific growth rate.

[0007] The invention also provides the use of ethanol clostridium protein as a partial substitute source for fish meal in promoting the ability of Penaeus vannamei to resist Vibrio parahaemolyticus infection.

[0008] The invention also provides the use of ethanol clostridium protein as a partial substitute source for fish meal in promoting serum immunity of Penaeus vannamei.

[0009] Preferably, the alcoholic Clostridium protein significantly increases the activities of superoxide dismutase, lysozyme, aspartate aminotransferase, alanine aminotransferase and phenoloxidase.

[0010] The present invention also provides the use of ethanol clostridium protein as a partial substitute source for fish meal in regulating the intestinal flora of Penaeus vannamei.

[0011] Preferably, the alcoholic Clostridium protein can increase the number of probiotics in the shrimp intestine and improve the diversity of intestinal flora.

[0012] Preferably, the alcoholic Clostridium protein can increase the abundance of Planctomycetes and Ruegeria in the intestinal tract of shrimp.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention takes Litopenaeus vannamei as the research object and studies the effect of ethanol clostridial protein as a partial substitute for fish meal on Litopenaeus vannamei. The results show that it has a positive effect on the growth performance, immune performance, muscle fatty acid synthesis, intestinal flora and other aspects of Litopenaeus vannamei, thereby providing a theoretical basis for realizing ethanol clostridial protein as a partial substitute for fish meal. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of ethanol clostridial protein replacing fish meal on the growth performance of Litopenaeus vannamei;

[0016] Figure 2 The effect of ethanol clostridial protein replacing fish meal on the disease resistance of Penaeus vannamei;

[0017] Figure 3 The effect of ethanol clostridial protein replacing fish meal on the immune parameters of Penaeus vannamei;

[0018] Figure 4 Venn diagram comparison of intestinal flora OUT of shrimps with different levels of fishmeal replaced by ethanolic clostridial protein;

[0019] Figure 5 Effects of replacing different levels of fish meal with ethanolic clostridial protein on the intestinal flora of shrimp at the phylum (A) and genus (B) levels. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Unless otherwise specified, the test methods used in the following embodiments and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0022] Example 1

[0023] Ethanol Clostridium protein is a high-quality single-cell protein with a high protein content. The crude protein content can reach 80%. It is rich in various trace elements, does not contain anti-nutritional factors, and is rich in 18 kinds of amino acids, among which the content of thiocyanate and lysine is relatively high, and arginine is relatively lacking. Compared with traditional plant proteins, it does not contain anti-nutritional factors. Compared with animal proteins, it has lower biogenic amines, salmonella, etc. The production of ethanol Clostridium protein is green, energy-saving and environmentally friendly.

[0024] The shrimp Penaeus vannamei was used as the research object to study the effects of different levels of ethanolic clostridial protein added in feed on the growth performance, feed utilization, immunity and intestinal flora of Penaeus vannamei, providing technical support for the development of new feed protein sources. With the addition of fish meal as the control, 15%, 30% and 45% of fish meal protein were replaced by ethanolic clostridial protein in equal amounts, and lysine, methionine and arginine were balanced to prepare 4 kinds of feeds for feeding Penaeus vannamei for 8 weeks to study the effects of ethanolic clostridial protein replacing fish meal protein in feed on the growth performance, feed utilization and immunity of Penaeus vannamei, providing technical support for the development of new feed protein sources. The breeding experiment lasted for 8 weeks, with 4 treatments (respectively recorded as FM, CAP15, CAP30 and CAP45 groups), 3 replicates for each treatment, and 40 shrimps for each replicate. Quantitative feeding was carried out at 7:00, 11:00, 17:00 and 21:00 every day, and the feeding amount was calculated according to 8-10% of body weight. The feeding degree was observed after 1 hour of feeding. After the breeding experiment, the shrimps were fasted for 24 hours, weighed, and the number of survivors was recorded. The survival rate, weight gain rate, and specific growth rate were calculated. At the same time, samples of hemolymph, intestine, hepatopancreas, and other tissues of Penaeus vannamei were collected, and digestive enzymes, antioxidant capacity, and immunity were analyzed. The intestinal flora of Penaeus vannamei was determined and analyzed using the HiSeq2500 PE250 instrument (Illumina, USA). A challenge experiment was conducted, and 30 shrimps were selected from each treatment group for challenge, injected with Vibrio parahaemolyticus, and observed continuously for 72 hours. The number of dead individuals was counted every 4 hours to calculate the survival rate.

[0025] Table 1 Conventional nutritional composition of ethanolic Clostridium protein

[0026]

[0027] Table 2 Experimental feed composition and nutritional level (dry matter basis) %

[0028]

[0029]

[0030] Note: 1Each kilogram of vitamin premix contains: Vitamin A 450000IU, Vitamin D3 150000IU, Vitamin E 5000mg, Vitamin K 3500mg, Vitamin B1 500mg, Vitamin B2 700mg, Vitamin B6 600mg, Vitamin B 12 2mg, calcium pantothenate 2000mg, niacin 3500mg, biotin 6mg, inositol 8000mg, folic acid 150mg. Each kilogram of mineral premix contains: magnesium 20000mg, iron 2000mg, zinc 8000mg, manganese 2000mg, copper 1500mg, cobalt 80mg, selenium 10mg, iodine 100mg.

[0031] 1. Growth performance of Penaeus vannamei after 8 weeks of rearing Figure 1 As shown in the data, when the ratio of ethanol clostridial protein to fish meal was 15% and 30%, the final average weight, weight gain rate and specific growth rate of Penaeus vannamei were higher than those of the fish meal group, among which the CAP15 group had a significant difference compared with the FM group, but there was no significant difference in the feed coefficient of Penaeus vannamei in each replacement group and the fish meal group. The results show that an appropriate amount of ethanol clostridial protein replacing fish meal can effectively improve the growth performance of Penaeus vannamei.

[0032] 2. After 8 weeks of feeding, 30 shrimps were selected from each treatment group for challenge with Vibrio parahaemolyticus. The challenge results were as follows: Figure 2 As shown in the results, in the groups where the proportion of ethanol clostridial protein replacing fish meal was 15% and 30%, the ability of Penaeus vannamei to resist Vibrio parahaemolyticus infection was significantly improved compared with the fish meal group. This result shows that the appropriate amount of ethanol clostridial protein replacing fish meal can effectively improve the disease resistance of Penaeus vannamei.

[0033] 3. Figure 3 As shown in the results, feeding diets with different proportions of fish meal replaced by ethanol clostridium protein can regulate the serum immune and antioxidant enzyme activities of Penaeus vannamei to varying degrees. Compared with the control group, the acid phosphatase and alkaline phosphatase activities and malondialdehyde content in the hepatopancreas of Penaeus vannamei in all replacement groups decreased significantly, the superoxide dismutase and lysozyme activities increased significantly, and aspartate aminotransferase, alanine aminotransferase and phenoloxidase showed a trend of first increasing and then decreasing, reaching the highest level in the 15% replacement group. The results show that replacing fish meal with ethanol clostridium protein can effectively improve the immune enzyme activities in Penaeus vannamei and help improve the immunity of shrimp.

[0034] 4. Figure 4 As shown in the figure, different numbers and types of microbial populations appeared in the intestines of Penaeus vannamei fed with ethanol clostridial protein instead of different levels of fish meal. It can be seen that replacing different levels of fish meal with Chlorella in the feed can regulate the microbial flora in the intestines of Penaeus vannamei.

[0035] Table 2 lists the species (Sobs), Shannon, Simpson, Chao1 and abundance-based coverage estimation (ACE) indexes in the intestinal flora of Penaeus vannamei in the four groups to evaluate the alpha diversity of the intestinal flora of Penaeus vannamei in the four groups. Compared with the FM group, the Sobs index of the intestinal flora of Penaeus vannamei in the CAP15 group was significantly increased, and the Sobs and Shannon indices of the intestinal flora of Penaeus vannamei in the CAP30 group were significantly increased (P<0.05). There were no significant differences in the Simpson, Chao1 and ACE indices of the intestinal flora of Penaeus vannamei among the four groups. The results show that the appropriate level of ethanol clostridial protein replacing fish meal in the feed can improve the diversity of the intestinal flora of Penaeus vannamei.

[0036] Table 3 Effects of different levels of fish meal replacement by ethanol clostridial protein on the α-diversity index of intestinal flora of Litopenaeus vannamei

[0037]

[0038] like Figure 5 As shown in the figure, at the phylum level, the top five phyla in terms of gut flora richness of Penaeus vannamei are Proteobacteria, Bacteroidetes, Planctomycetes, Verrucomicrobia, and Actinobacteria ( Figure 5 A). Compared with the control group, the abundance of Planctomycetes bacteria in the intestine of Penaeus vannamei increased in all the replacement groups. At the genus level, the top five genera with the highest richness in the intestinal microbial community of Penaeus vannamei were Vibrio, Haloferula, Halocynthiibacter, Ruegeria, and Blastopirellula. Figure 5 B). Compared with the control group, the Vibrio in the CAP30 and CAP45 experimental groups decreased, while the Haloferula and Ruegeria in the experimental groups increased. Many studies have shown that Vibrio, as a common flora in aquatic animals, has been identified as a major member of the shrimp intestine. In aquaculture, Vibrio is one of the main pathogens of some species. Ruegeria, a genus of Gram-negative bacteria, is widely considered to be a probiotic that degrades triglycerides. In this study, after feeding ethanolic Clostridium protein, the relative abundance of Vibrio in the intestine of Penaeus vannamei decreased, while the abundance of Ruegeria increased compared with the control group. The above results show that feeding ethanolic Clostridium protein can simultaneously regulate the composition of the intestinal flora by increasing the content of potential probiotics and reducing the content of pathogenic bacteria in the intestine of Penaeus vannamei, thereby having a beneficial effect on shrimp.

[0039] In summary, the use of ethanol clostridial protein to replace fish meal at a reasonable level can improve the growth performance, disease resistance and immune ability of Penaeus vannamei and the composition of intestinal flora.

[0040] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Application of ethanol clostridial protein in replacing part of fish meal in regulating the intestinal flora of Penaeus vannamei, characterized in that: The ethanol clostridial protein can replace 30% of fish meal to increase the abundance of Planctomycetes and Ruegeria in the shrimp intestine and improve the diversity of intestinal flora; The feed composition is: fish meal 17.5%, soybean meal 25%, peanut meal 10%, flour 24.05%, brewer's yeast 3%, shrimp head powder 5%, ethanol clostridial protein 6.05%, fish oil 2%, soybean oil 2.57%, 50% choline 0.3%, soybean lecithin oil 1%, complex vitamin mineral salt 1%, monocalcium phosphate 1.5%, Vc phosphate 0.1%, microcrystalline cellulose 0.88%, ethoxyquinoline 0.05%; The complex vitamin mineral salt contains 450000IU of vitamin A, 150000IU of vitamin D3, 5000mg of vitamin E, 500mg of vitamin K3, 500mg of vitamin B1, 700mg of vitamin B2, 600mg of vitamin B6, 22mg of vitamin B12, 2000mg of calcium pantothenate, 3500mg of niacin, 6mg of biotin, 8000mg of inositol, 150mg of folic acid, 20000mg of magnesium, 2000mg of iron, 8000mg of zinc, 2000mg of manganese, 1500mg of copper, 80mg of cobalt, 10mg of selenium and 100mg of iodine per kilogram.

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