Application of composite single-cell protein as fish meal substitute source in largemouth bass culture

Abstract

The invention provides application of composite single-cell protein as a fish meal substitute source in largemouth bass culture, and belongs to the technical field of aquatic feed application. The composite single-cell protein obtained by combining chlorella and clostridium ethanol protein according to the mass ratio of 1: (3.5-4.5) can partially or completely replace fish meal in largemouth bass feed. The invention further provides a largemouth bass feed which is low in fish meal content or completely free of fish meal, and the obtained largemouth bass feed can effectively improve the growth performance and muscle quality of largemouth bass and improve the intestinal flora structure while remarkably reducing the cost.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic feed application, and particularly relates to application of composite single-cell protein as a fish meal substitute in largemouth bass breeding. Background Art

[0002] Largemouth bass (Micropterus salmoides) is a freshwater farmed fish native to North America. It is favored by aquaculture farmers and consumers for its exquisite meat and significant economic value. This typical carnivorous fish feed uses fish meal as the main protein source. Usually, the content of fish meal in aquatic feed exceeds 30%, accounting for most of the feed cost. In addition, the price of fish meal is gradually rising, which has further increased the cost of breeding. Therefore, it is particularly important to develop a green, healthy, sustainable and nutritionally complete fish meal alternative for the development of largemouth bass breeding.

[0003] Fish meal contains rich protein, essential amino acids and vitamins, and has low anti-nutritional factors, is easy to digest and absorb by aquatic animals, and is a traditional high-quality protein source in aquaculture. Other protein sources (such as plant protein sources, animal protein sources and single-cell protein sources) usually have problems such as low digestibility, poor palatability and amino acid deficiency when replacing fish meal, and it is difficult to achieve the breeding effect when adding fish meal. At present, although plant protein sources, animal protein sources and single-cell protein sources are reported to be added in largemouth bass feed, due to the significant differences in sources, body absorption rates, nutritional values, etc. of different protein sources, and the ratio of different protein sources also has unpredictable effects on the growth performance, muscle quality, intestinal flora changes of largemouth bass, etc., therefore, providing an effective replacement for fish meal and not reducing the growth performance and muscle quality of largemouth bass, and a protein source that can also improve the intestinal flora structure is still one of the key issues to be solved in this field. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a composite single-cell protein obtained by combining Chlorella and Clostridium ethanolum protein in a mass ratio of 1:3.5-4.5 as a fish meal substitute in largemouth bass farming.

[0005] The present invention also aims to provide a largemouth bass feed, which has a low fish meal content or contains no fish meal at all, has low cost, and can effectively improve the growth performance and muscle quality of largemouth bass and improve the intestinal flora structure.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of a composite single-cell protein as a fish meal substitute in largemouth bass farming. The composite single-cell protein is obtained by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:3.5-4.5; the composite single-cell protein replaces 45-100% of the fish meal.

[0008] Preferably, the composite single-cell protein increases the terminal body weight, weight gain rate and specific growth rate of largemouth bass; the composite single-cell protein reduces the feed coefficient of largemouth bass.

[0009] Preferably, the composite single-cell protein improves the protein efficiency ratio of largemouth bass and increases the muscle protein content of largemouth bass.

[0010] Preferably, the composite single-cell protein increases the water-holding capacity of the muscles of the largemouth bass and reduces the loss rate of the juice and muscle and the fat loss rate of the largemouth bass.

[0011] Preferably, the composite single-cell protein increases the muscle hardness and muscle elasticity of largemouth bass.

[0012] Preferably, the composite single-cell protein reduces the relative abundance of Mycoplasma and Aeromonas in the intestine of largemouth bass, and increases the relative abundance of Cetacea and Clostridium in the intestine of largemouth bass.

[0013] The present invention also provides a largemouth bass feed, which comprises, by mass percentage, 26% fish meal, 4.8% chlorella, 19.2% ethanol clostridium protein, 20% wheat flour, 15% corn protein, 5.1% soybean oil, 5.1% soybean lecithin, 1.5% multivitamin premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.08% microcrystalline cellulose.

[0014] The present invention also provides a largemouth bass feed, which comprises, by mass percentage, 9.6% chlorella, 38.4% ethanol clostridium protein, 20% wheat flour, 15% corn protein, 5.9% soybean oil, 5.9% soybean lecithin, 1.5% multivitamin premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.48% microcrystalline cellulose.

[0015] The present invention also provides application of the largemouth bass feed in improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass.

[0016] The present invention also provides application of the largemouth bass feed in preparing a product for regulating intestinal flora disorder of largemouth bass.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention is based on the research of chlorella and ethanolic clostridium protein, and it is found that the composite single-cell protein obtained by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:3.5-4.5 can be used as a fish meal (partial or full) substitute source for largemouth bass breeding, and based on this, a largemouth bass feed with low fish meal content or no fish meal is further provided, which can significantly improve the terminal body weight, weight gain rate and specific growth rate of largemouth bass while effectively reducing feed cost; reduce the feed coefficient of largemouth bass; improve the protein efficiency ratio of largemouth bass; improve the muscle protein content of largemouth bass; improve the muscle water holding capacity of largemouth bass; reduce the loss rate and fat loss rate of largemouth bass juice muscle; improve the muscle hardness and muscle elasticity of largemouth bass; reduce the relative abundance of harmful bacteria such as mycoplasma and Aeromonas in the intestine of largemouth bass, and increase the relative abundance of beneficial bacteria such as Cetacea and Clostridium in the intestine of largemouth bass, so as to achieve the effect of significantly improving the growth performance and muscle quality of largemouth bass and improving the intestinal flora structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of each diet group on the growth performance of largemouth bass;

[0020] Figure 2 The effect of each group of feed on the water holding capacity of largemouth bass muscle;

[0021] Figure 3 The effects of each diet group on the muscle texture characteristics of largemouth bass;

[0022] Figure 4 PCoA analysis of the effects of each diet group on the intestinal flora of largemouth bass;

[0023] Figure 5 The effects of each diet group on the intestinal flora of largemouth bass at the phylum level;

[0024] Figure 6 This is the effect of each group of feed on the intestinal flora of largemouth bass at the genus level. DETAILED DESCRIPTION

[0025] The present invention provides the use of composite single-cell protein as a fish meal substitute in largemouth bass farming, wherein the composite single-cell protein is obtained by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:3.5-4.5, preferably by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:4; the composite single-cell protein partially or completely replaces the use of fish meal, and the composite single-cell protein replaces 45-100% of the fish meal, preferably replaces 50% of the fish meal. The present invention adopts the rich nutritional components of chlorella and the high protein content of ethanolic clostridium protein. After the two are compounded in a specific ratio, the dependence of largemouth bass feed on fish meal can be reduced, and comprehensive nutritional support can be provided for largemouth bass. On the basis of reducing the amount of fish meal and reducing the feed cost, the growth performance of largemouth bass can be promoted, the muscle quality of largemouth bass can be improved, and the structure of intestinal flora can be improved.

[0026] After the composite single-cell protein is used to replace fish meal in largemouth bass feed, the present invention can significantly improve the terminal body weight, weight gain rate and specific growth rate of largemouth bass; reduce the feed coefficient of largemouth bass; improve the protein efficiency ratio of largemouth bass; improve the muscle protein content of largemouth bass; improve the muscle water holding capacity of largemouth bass; reduce the loss rate of juice muscle and fat loss rate of largemouth bass; improve the muscle hardness and muscle elasticity of largemouth bass; reduce the relative abundance of harmful bacteria such as mycoplasma and Aeromonas in the intestine of largemouth bass, increase the relative abundance of beneficial bacteria such as Cetacea and Clostridium in the intestine of largemouth bass, and improve the abundance of intestinal flora of largemouth bass.

[0027] The present invention also provides a largemouth bass feed, which includes fish meal, composite single-cell protein, wheat flour, corn protein, soybean oil, soybean lecithin, multivitamin and multi-mineral premix, choline chloride, calcium dihydrogen phosphate, antioxidants and microcrystalline cellulose; the feed is obtained by partially or completely replacing fish meal with composite single-cell protein on the basis of traditional feed, including a ratio in which the fish meal content is 0; the composite single-cell protein is obtained by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:3.5 to 4.5, and is preferably obtained by combining chlorella and ethanolic clostridium protein in a mass ratio of 1:4. Preferably, when the composite single cell protein replaces 50% of the fish meal, the feed is 26% fish meal, 4.8% chlorella, 19.2% ethanolic clostridial protein, 20% wheat flour, 15% corn protein, 5.1% soybean oil, 5.1% soybean lecithin, 1.5% multivitamin premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.08% microcrystalline cellulose by mass percentage. When the composite single cell protein replaces 100% of the fish meal, the feed is 9.6% chlorella, 38.4% ethanolic clostridial protein, 20% wheat flour, 15% corn protein, 5.9% soybean oil, 5.9% soybean lecithin, 1.5% multivitamin premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.48% microcrystalline cellulose by mass percentage.

[0028] The antioxidant in the feed of the present invention is preferably ethoxyquinoline; the multivitamin and multimineral premix in the feed of the present invention is a vitamin and mineral premix: vitamin A, 500000 IU / kg; vitamin D3, 100000 IU / kg; vitamin E, 4000 mg / kg; vitamin K3, 1000 mg / kg; vitamin B1, 500 mg / kg; vitamin B2, 1000 mg / kg; vitamin B6, 1000 mg / kg; Vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0029] The preparation method of the largemouth bass feed of the present invention is preferably as follows: all feed raw materials are crushed by a grinder and all are sieved through a 60-80 mesh sieve. Accurately weigh each feed raw material according to the feed formula, and use a step-by-step mixing method to mix all powders evenly (choline chloride is added in a water-soluble form). Add a weighed fat source (soybean oil and soybean lecithin) to the mixed powder, and add 30% of the feed weight (300 mL of water is added to every 1 kg of feed) of purified water. The powder and oil are manually mixed, and then all the raw materials are sieved through a 40-60 mesh sieve to prevent fat from clumping and to mix it evenly with the material. Finally, the mixed material is extruded into a 2.0-2.5 mm granular feed using a twin-screw extruder.

[0030] The present invention also provides the use of the largemouth bass feed in improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass, and in preparing a product for regulating intestinal flora disorder of largemouth bass.

[0031] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In a specific embodiment of the present invention, fish meal was purchased from Guangdong Dongteng Feed Co., Ltd., the chlorella species was Chlorella vulgaris, provided by the Institute of Hydrobiology, Chinese Academy of Sciences, and ethanolic Clostridium protein was purchased from Beijing Shoulang Biotechnology Co., Ltd.

[0033] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] A largemouth bass feed: 26wt% fish meal, 4.8wt% chlorella, 19.2wt% ethanol clostridium protein, 20wt% wheat flour, 15wt% corn protein, 5.1wt% soybean oil, 5.1wt% soybean lecithin, 1.5wt% multivitamin premix, 0.2wt% choline chloride, 1wt% monocalcium phosphate, 0.02wt% antioxidant and 2.08wt% microcrystalline cellulose. The antioxidant is ethoxyquinoline; the multivitamin mineral premix is ​​a vitamin and mineral premix: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0037] Preparation method of largemouth bass feed: All feed raw materials are crushed by a grinder and all are sieved through a 70-mesh sieve. Accurately weigh each feed raw material according to the feed formula, and use a step-by-step mixing method to mix all the powders evenly (choline chloride is added in a water-soluble form). Add the weighed fat source (soybean oil and soybean lecithin) to the mixed powder, and add 30% of the feed weight (300mL of water per 1kg of feed) of purified water. Mix the powder and oil manually, and then sieve all the raw materials through a 50-mesh sieve. Use a twin-screw extruder to extrude the mixed material into 2.0-2.5mm granular feed.

[0038] Example 2

[0039] A largemouth bass feed: 9.6wt% chlorella, 38.4wt% ethanol clostridium protein, 20wt% wheat flour, 15wt% corn protein, 5.9wt% soybean oil, 5.9wt% soybean lecithin, 1.5wt% multivitamin premix, 0.2wt% choline chloride, 1wt% monocalcium phosphate, 0.02wt% antioxidant and 2.48wt% microcrystalline cellulose. The antioxidant is ethoxyquinoline; the multivitamin mineral premix is ​​a vitamin and mineral premix: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; niacin, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg.

[0040] Preparation method of largemouth bass feed: All feed raw materials are crushed by a grinder and all are sieved through a 60-mesh sieve. Accurately weigh each feed raw material according to the feed formula, and use a step-by-step mixing method to mix all the powders evenly (choline chloride is added in a water-soluble form). Add the weighed fat source (soybean oil and soybean lecithin) to the mixed powder, and add 30% of the feed weight (300mL of water per 1kg of feed) of purified water. Mix the powder and oil manually, and then sieve all the raw materials through a 60-mesh sieve. Use a twin-screw extruder to extrude the mixed material into 2.0-2.5mm granular feed.

[0041] Test Example 1 Determination of changes in functional component content

[0042] The largemouth bass was used as the research object to study the effects of composite single-cell protein in feed on the growth performance, muscle quality and intestinal flora of largemouth bass: the breeding experiment set up three treatment groups: fish meal group (FM100), 50% replacement group (FM50) and 100% replacement group (FM0), and prepared three kinds of isonitrogen and isolipid feeds (as shown in Table 1), the protein addition level was 50%, and each treatment had 3 replicates, with 40 fish in each replicate. The breeding experiment lasted for 8 weeks, and the experimental fish were fed twice a day (07:00 and 17:00), and the experimental fish were fed each time until they were apparently full (most of the experimental fish stopped fighting for food) and the feed feeding amount was recorded.

[0043] Table 1 Feed formula for each group

[0044]

[0045]

[0046] Note: a The multivitamin and multimineral premix is ​​a vitamin and mineral premix: vitamin A, 500000IU / kg; vitamin D3, 100000IU / kg; vitamin E, 4000mg / kg; vitamin K3, 1000mg / kg; vitamin B1, 500mg / kg; vitamin B2, 1000mg / kg; vitamin B6, 1000mg / kg; vitamin B12, 2.0mg / kg; nicotinic acid, 4000mg / kg; calcium pantothenate, 2000mg / kg; biotin, 10.0mg / kg; folic acid, 100mg / kg; vitamin C, 15000mg / kg; iron, 10000mg / kg; copper, 300mg / kg; zinc, 5000mg / kg; manganese, 1200mg / kg; iodine, 80mg / kg; cobalt, 20mg / kg; selenium, 30mg / kg. The preparation method of the feed is the same as that of Example 1. The test feed was air-dried at room temperature and stored in a ziplock bag at -20°C for later use. In the FM50 and FM100 groups, due to the reduction of fish meal, the fat content of the added Chlorella and Clostridium ethanolicum protein was different from that of the fish meal, and the final feed had to be prepared with equal nitrogen and fat, so the fat content was adjusted. After the adjustment, microcrystalline cellulose was used to balance the final weight to 100%.

[0047] According to the laboratory analysis standards established by AOAC (2005), the crude protein, crude fat, ash and moisture content of each group of feed were tested. The crude protein content of the sample was determined by Kjeldahl nitrogen determination (method 960.52), the crude fat content was determined by Soxhlet extraction (method 948.22), the ash content was determined by carbonizing the sample and then burning it in a muffle furnace at 550°C for 16 hours (method 942.05), and the moisture content was determined by drying in an oven at 105°C to constant weight (method 934.01). The protein and fat content of each group of feed was not much different, and the results are shown in Table 2.

[0048] Table 2 Nutritional composition of feed in each group

[0049]

[0050]

[0051] Note: Crude protein, crude lipids, ash and moisture contents are all measured values.

[0052] After the breeding experiment, the animals were fasted for 24 hours, weighed, and the number of survivors was recorded. The survival rate, final average weight, weight gain rate, specific growth rate, feed coefficient, and protein efficiency ratio were calculated.

[0053] Survival rate (SR, %) = 100 × (final number of fish / initial number of fish);

[0054] Final average body weight (FBW) = (final body weight / final number of fish);

[0055] Weight gain rate (WGR, %) = 100 × (final body weight - initial body weight) / initial body weight;

[0056] Specific growth rate (SGR, %) = 100 × [ln (final body weight - ln (initial body weight)] / day;

[0057] Feed conversion ratio (FCR) = feed intake / (final body weight-initial body weight);

[0058] Protein efficiency ratio (PER, %) = 100 × (final body weight - initial body weight) / protein intake.

[0059] The results are as follows Figure 1 The results showed that after 8 weeks of feeding, there was no significant difference in the survival rate of largemouth bass among the groups (P>0.05). The final average weight, weight gain rate and specific growth rate of the FM50 group were significantly higher than those of the FM100 and FM0 groups. At the same time, the feed coefficient of the FM50 group was significantly lower than that of the FM100 group, and the protein efficiency ratio was significantly higher than that of the FM100 group (P<0.05).

[0060] After the breeding experiment, the fish were fasted for 24 hours, and the muscles and intestines of largemouth bass were collected for muscle quality and intestinal flora analysis. The muscle quality of largemouth bass was analyzed using a TA.XT.PLUS texture analyzer, and the intestinal flora of largemouth bass was determined using a Hiseq2500 PE250 instrument (Illumina, USA). All values ​​are expressed as mean ± standard deviation (Mean ± SD), and one-way analysis of variance (ANOVA) was used. When there were significant differences between treatment groups (P < 0.05), multiple comparisons were analyzed using Tukey's test. The changes in the growth performance, muscle texture and intestinal flora composition of largemouth bass with different replacement levels of composite single-cell protein were compared to explore the regulatory effects of composite single-cell protein on the growth, muscle quality and intestinal flora of largemouth bass. The results are shown in Tables 3 to 4 and Figure 2 to Figure 6 shown.

[0061] Table 3 Nutritional components of largemouth bass muscle in each group

[0062] Group Crude protein (%) Crude fat (%) Ash content (%) Moisture (%) FM100 <![CDATA[19.69±0.17 b ]]> <![CDATA[1.45±0.07 a ]]> 1.45±0.03 <![CDATA[77.34±0.69 ab ]]> FM50 <![CDATA[20.18±0.06 a ]]> <![CDATA[1.48±0.07 a ]]> 1.40±0.03 <![CDATA[76.49±0.29 b ]]> FM0 <![CDATA[19.19±0.17 c ]]> <![CDATA[1.12±0.02 b ]]> 1.40±0.08 <![CDATA[78.29±0.18 a ]]>

[0063] Note: Muscle composition is based on wet matter (%). Data are expressed as Mean±SD, n=3.

[0064] As shown in Table 3, there were significant differences in crude protein content among different groups of largemouth bass muscles. The crude protein content of FM50 group was significantly higher than that of FM100 group and FM0 group, among which the crude protein content of FM0 group was the lowest (P<0.05), which indicated that FM50 group had a certain improvement on muscle protein content; there was no significant difference in crude fat content between FM50 group and FM100 group, but both were significantly higher than FM0 group (P<0.05); there was no significant difference in ash content among the three groups; in terms of moisture content, FM0 group was significantly higher than FM50 group (P<0.05), but the difference was not significant compared with FM100 group. In short, the different fish meal content significantly affected the crude protein, crude fat and moisture content, but had no significant effect on the ash content.

[0065] Figure 2 The effect of each group of feed on the water holding capacity of largemouth bass muscle. The stronger the muscle water holding capacity, the slower the degradation rate of muscle protein and the slower the reduction rate of flavor substances, thus keeping the various nutrients in the muscle from being lost and ensuring the quality of the fish meat. Figure 2 The test results showed that compared with the FM100 and FM0 groups, the FM50 group significantly enhanced the water holding capacity of the largemouth black bass muscles and reduced the juice loss rate and fat loss rate (P<0.05).

[0066] Figure 3 The effects of different feed groups on the muscle texture characteristics of largemouth bass. The muscle hardness and elasticity of fish are the main indicators for evaluating muscle quality. Figure 3 The test results showed that the muscle hardness and elasticity of the FM50 group were significantly higher than those of the FM100 group and the FM0 group (P<0.05). In addition, the muscle chewiness of the FM50 group was significantly higher than that of the FM0 group, while the muscle chewiness and adhesiveness of the FM100 group were significantly higher than those of the FM0 group (P<0.05). However, there were no significant differences in cohesion and recovery among the three groups (P>0.05).

[0067] Figure 4 PCoA analysis of the effects of each feed group on the intestinal flora of largemouth bass. Figure 4 The results showed that there were different clusters among the groups, indicating that there were certain differences in the intestinal flora among the groups. The α diversity index of the intestinal flora of largemouth bass was analyzed, and the results are shown in Table 4.

[0068] Table 4 Effects of each feed group on the α diversity index of intestinal flora of largemouth bass

[0069] Group Sobs Shannon Simpson Chao1 ACE FM100 <![CDATA[301.67±53.50 b ]]> 1.86±0.45 0.52±0.18 <![CDATA[367.86±36.79 b ]]> <![CDATA[364.29±31.95 b ]]> FM50 <![CDATA[526.33±95.05 a ]]> 3.17±1.06 0.54±0.15 <![CDATA[566.57±90.73 a ]]> <![CDATA[564.35±96.91 a ]]> FM0 <![CDATA[363.33±38.89 ab ]]> 2.68±1.34 0.58±0.27 <![CDATA[428.09±50.23 ab ]]> <![CDATA[442.56±47.33 ab ]]>

[0070] The Sobs index in Table 4 showed that the FM50 group had a significantly higher species richness, which was significantly higher than the FM100 group, while the FM0 group had no significant difference compared with these two groups (P<0.05). The Shannon index showed that the FM50 group had the highest diversity, but the differences between the groups did not reach a significant level. The Simpson index showed that the uniformity of the FM0 group was slightly higher, but the differences between the groups were not significant. The results of the Chao1 and ACE indices were similar. The species richness of the FM50 group was significantly higher than that of the FM100 group, while the FM0 group was between the FM50 and FM100 groups in both indicators, and the differences between them were not significant (P<0.05). Overall, the change in fish meal content significantly affected the species richness of the microbial community, while the effects on diversity and uniformity were relatively limited.

[0071] Figure 5 The effects of each diet group on the intestinal flora of largemouth bass at the phylum level. Figure 6 The effects of each group of feed on the intestinal flora of largemouth bass at the genus level. At the phylum level, the top five phyla in terms of intestinal microbial community richness of largemouth bass are Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteriota, and Fusobacteriota. Among them, Firmicutes and Proteobacteria are the dominant phyla, and compared with the FM100 and FM0 groups, the relative classification of Firmicutes in the FM50 group increased, while Proteobacteria decreased. At the genus level, the top six genera with the highest intestinal microbial community richness of largemouth bass are Mycoplasma, Plesiomonas, Clostridium sensu stricto_1, Cetobacterium, Vogesella, and Aeromonas. Figure 6 The results showed that feeding with different fish meal contents (FM100, FM50, FM0) significantly affected the relative abundance of the four bacteria ( Figure 6B). Mycoplasma is a harmful bacterium in the intestinal microbiota and a potential pathogen that causes intestinal inflammation. At the same time, Aeromonas is the most common pathogen in the intestine of carp. After infection, fish will suffer from diseases such as sepsis, enteritis and skin ulcers, which seriously threaten the health of fish. The results showed that the relative abundance of Mycoplasma decreased significantly with the decrease of fish meal content, especially between FM100 and FM50 and between FM100 and FM0 (P<0.05). In Aeromonas, FM50 was significantly lower than FM100 and FM0 groups (P<0.05). Cetobacterium can ferment peptides and carbohydrates, and plays an important role in the digestion and nutrition of fish. The vitamin B12 it produces is an intestinal microecological regulator that helps stabilize the intestinal flora, and can inhibit the reproduction of pathogens to a certain extent and enhance the body's immune ability, indicating that the presence of this bacterium is extremely important for the intestinal health and flora stability of fish. Clostridium can promote the production of butyrate in the intestine. After feeding composite single-cell protein to replace 50% of fish meal, the relative abundance of Cetobacterium and Clostridium_sensu_stricto_1 in the intestine of largemouth bass was significantly increased compared with the FM100 and FM0 groups. The above results show that the addition of composite single-cell protein to replace part of fish meal can regulate the composition of intestinal flora by increasing the content of potential probiotics, thereby having a beneficial effect on largemouth bass.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of composite single cell protein as a fish meal substitute in largemouth bass farming, characterized in that: The composite single-cell protein is obtained by combining chlorella and ethanol clostridium protein in a mass ratio of 1:3.5-4.5; the composite single-cell protein replaces 45-100% of fish meal.

2. The use according to claim 1, characterized in that: The composite single-cell protein increases the terminal body weight, weight gain rate and specific growth rate of largemouth bass; and the composite single-cell protein reduces the feed coefficient of largemouth bass.

3. The use according to claim 1, characterized in that: The composite single-cell protein improves the protein efficiency ratio of the largemouth bass and increases the muscle protein content of the largemouth bass.

4. The use according to claim 1, characterized in that: The composite single-cell protein improves the water-holding capacity of the muscles of the largemouth bass and reduces the loss rate of the muscles and fat loss rate of the largemouth bass.

5. The use according to claim 1, characterized in that: The composite single-cell protein improves the muscle hardness and muscle elasticity of largemouth bass.

6. The use according to claim 1, characterized in that: The composite single-cell protein reduces the relative abundance of Mycoplasma and Aeromonas in the intestine of largemouth bass, and increases the relative abundance of Cetiformis and Clostridium in the intestine of largemouth bass.

7. A largemouth bass feed, characterized in that: The feed comprises, by mass percentage, 26% fish meal, 4.8% chlorella, 19.2% ethanolic clostridial protein, 20% wheat flour, 15% corn protein, 5.1% soybean oil, 5.1% soybean lecithin, 1.5% multivitamin premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.08% microcrystalline cellulose.

8. A largemouth bass feed, characterized in that: The feed comprises, by mass percentage, 9.6% chlorella, 38.4% ethanolic clostridium protein, 20% wheat flour, 15% corn protein, 5.9% soybean oil, 5.9% soybean lecithin, 1.5% multivitamin and mineral premix, 0.2% choline chloride, 1% monocalcium phosphate, 0.02% antioxidant and 2.48% microcrystalline cellulose.

9. Use of the largemouth bass feed according to any one of claims 7 to 8 in improving the growth performance of largemouth bass and / or improving the meat quality of largemouth bass.

10. Use of the largemouth bass feed according to any one of claims 7 to 8 in the preparation of a product for regulating intestinal flora disorder of largemouth bass.

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