Application of Halobacillus LMVS and feed

Abstract

The present invention discloses uses of Haloclonius LMVS and a feed. Adding Haloclonius LMVS, native to Lateolabranch seabass, to a feed containing 15% defatted mealworm powder can increase Lateolabranch seabass yield, improve feed utilization, and enhance the seabass's digestive tract's anti-inflammatory capacity, making its digestive tract more intact and healthier. Haloclonius LMVS, native to Lateolabranch seabass, can effectively improve Lateolabranch seabass's utilization of defatted mealworm feed.

Description

Technical Field

[0001] The present invention relates to the field of microbial feed, in particular to application of Halobacillus halogenides LMVS and a feed. Background Art

[0002] The striped seabass (Lateolabrax maculatus), also known as seabass, belongs to the order Perciformes, family Perciformes, genus Lateolabrax. It is a eurythermal and euhaline fish found in the shallow coastal waters of China, South Korea, and Japan, and is also found in coastal areas of Shandong, Zhejiang, Fujian, Guangdong, and other provinces in my country. According to the 2022 China Fisheries Statistical Yearbook, the total production of striped seabass in my country reached 199,000 tons in 2021. The demand for its feed is large, and fish meal is an important source of protein in aquatic feed. With the development of the aquaculture industry, the demand for fish meal has increased, and the supply has been insufficient, and the price has been rising year by year. In order to reduce feed costs, many studies on fish meal replacements have been launched and have made some progress. Mealworms, as a source of insect protein in feed, have attracted much attention. The specific allergenic proteins present in mealworms cause abnormalities in the intestinal tract of the striped seabass and reduce growth performance. Although the feed is heated during production, there is much evidence that new antigenic sites may also appear in the protein after heating.

[0003] Adding intestinal probiotics to feed can effectively increase animals' feed utilization and improve their growth performance. Native probiotics refer to bacteria that have a very close and stable relationship with the host, are isolated from the animal body, and are beneficial to maintaining the growth and health of the host. On the one hand, native probiotics can colonize in the intestines and form a biological barrier, spatially preventing the colonization of pathogenic microorganisms and playing a protective role; on the other hand, they prevent the invasion of pathogens by competing for nutrients or producing antagonistic effects. Compared with other types of microecological preparations, after the native bacteria of animal origin are isolated, screened and made into probiotic additives, they are easier to colonize, reproduce and exert specific probiotic effects in the corresponding animals. They are not only highly homologous and highly targeted, but also ensure safety, making them ideal probiotic preparations. Summary of the Invention

[0004] The purpose of the present invention is to provide a microorganism that can increase the yield of sea bass fed with defatted mealworm powder feed; improve feed utilization rate; and enhance the anti-inflammatory ability of the digestive tract, thereby making the digestive tract tissue structure more complete and healthier.

[0005] To achieve the above object, the present invention provides a use of Virgibacillus salarius LMVS for preparing feed.

[0006] Furthermore, Virgibacillus salarius LMVS is used to prepare defatted mealworm meal feed for Lateolabrax japonicus.

[0007] Furthermore, the feed refers to fish feed.

[0008] Furthermore, the feed has the effects of increasing fish production and improving the intestinal health of fish.

[0009] Furthermore, the feed has the effect of improving fish's utilization of defatted mealworm feed.

[0010] The present invention also provides a feed, characterized in that it contains Virgibacillus salarius LMVS.

[0011] Furthermore, the defatted mealworm powder in the feed replaces 50 wt% of the fish meal.

[0012] Strain name: Virgibacillus salarius LMVS;

[0013] Deposit date: April 3, 2023;

[0014] Depository: General Microbiology Center of China Culture Collection of Microorganisms (CGMCC), No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0015] Deposit number: CGMCC No.27011.

[0016] In previous studies, the inventors of the present invention successfully replaced 50% of the fish meal in the japonica seabass feed with defatted mealworm powder. However, the inventors found that in these results, no protein source could completely replace the fish meal in the feed. Plant protein contains anti-nutritional factors such as phytic acid and lectins, which can easily affect the growth and intestinal health of animals. Although mealworms are animal proteins, they still cannot completely replace fish meal. The inventors analyzed that, on the one hand, the proportion of soybean meal in the replaced feed formula is still 30%-35%. It is speculated that after the proportion of fish meal is reduced, this part of soybean meal may aggravate the damage to the intestines, resulting in the proportion of defatted mealworms replacing fish meal being maintained at only 50%. On the other hand, it is speculated that the specific allergenic proteins present in mealworms may cause abnormalities in the japonica seabass intestine and reduce growth performance. Although the feed is heated during production, there is a lot of evidence that new antigenic sites may appear in proteins after heating. The invention separates a strain of Virgibacillus salarius LMVS from the intestine of a sea bass fed with defatted mealworms instead of 50% of fish meal. Experiments show that the addition of the Virgibacillus salarius LMVS to defatted mealworms instead of half of the fish meal and then feeding the sea bass can increase the yield of the sea bass fed with defatted mealworm powder, improve feed utilization, and enhance the anti-inflammatory ability of the digestive tract. At the same time, the digestive tract tissue structure of the sea bass is more complete and healthier. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the effect of adding potential probiotics to defatted mealworm powder feed on the intestinal tissue morphology of sea bass.

[0018] Figure 2 This is a graph showing the effect of adding potential probiotics to defatted mealworm powder feed on the expression of genes related to tight junction proteins in the intestine of sea bass.

[0019] Figure 3 This is a graph showing the effect of adding potential probiotics to defatted mealworm powder feed on the expression of genes related to intestinal inflammatory factors in sea bass. DETAILED DESCRIPTION

[0020] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0021] Example 1:

[0022] 1. Separation technology:

[0023] Defatted mealworms were used to replace 50% of fish meal to feed sea bass. After the breeding, 5 larger individuals were selected. The body surface was disinfected with 75% alcohol. The intestines were removed under sterile conditions and placed in a centrifuge tube. They were mixed with 0.8% sterile saline in a ratio of 1:9 and homogenized at low temperature. Part of it was used to screen Bacillus. The homogenate was inactivated at 55℃ for 15 minutes and diluted 10 2 , 10 3 , 10 4 100 μl of each of the 3 gradient dilutions was applied. The other part was used to screen lactic acid bacteria. 2 , 10 3 , 10 4 Spread 100 μl of each of three dilutions onto MRS agar. Incubate at 33°C for 24 hours. Pick a single strain for streak purification. Mix the purified strain with 50% glycerol at a 1:1 volume ratio in a cryovial and store at -80°C until ready for use.

[0024] 2.16S rDNA Sequencing and Analysis

[0025] The isolated strains were centrifuged at 20000 r / min for 30 s to collect the bacteria. TM Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit according to the manufacturer's instructions. The reaction system consisted of 1 μL of DNA template (~50 ng), 1 μL of the kit's included forward primer (10 μM), 1 μL of the kit's included reverse primer (10 μM), 25 μL of premix, and 22 μL of deionized water. The extracted DNA served as a template for amplification of the 16S rDNA gene fragment using the bacterial 16S rDNA amplification primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO: 1) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′, SEQ ID NO: 2). PCR products were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing. After removal of low-quality bases and assembly, the sequences were blasted against the NCBI database and the Korean ezbiocloud database (https: / / www.ezbiocloud.net / ). The results are shown in Table 1. The identification result is Virgibacillus salarius, and its identification sequence is shown in SEQ ID NO: 3.

[0026] Table 1 LMVS sequence comparison results

[0027]

[0028] Sequence of LMVS:

[0029]

[0030] SEQ ID NO:3.

[0031] 3. Physiological and biochemical indicators

[0032] The physiological and biochemical tests were used to identify the indicators (see Table 2). The LMVS bacteria were positive in the citrate utilization test, propionate utilization test, gelatin liquefaction test, and nitrate reduction test, and negative in the VP test, D-xylitol, 7% NaCl growth, pH 5.7 growth, and starch test.

[0033] Table 2 LMVS physiological and biochemical identification results

[0034]

[0035]

[0036] Note: “+” indicates positive identification; “-” indicates negative identification.

[0037] 4. Hemolytic activity and pathogen antagonism test

[0038] Hemolytic test: This test adopts the spot method. 8 The test bacterial solution (cfu / mL) was spotted onto blood agar and incubated at 33°C for 24 hours, observing the clear zone in the blood agar. α-hemolysis, when a 1-2 mm grass-green ring appears around the test colony, indicates mild hemolysis and mild virulence. β-hemolysis, when a 2-4 mm, well-defined, completely transparent hemolytic ring forms around the test colony, indicates hemolysis and strong virulence. γ-hemolysis, when no hemolytic ring appears around the colony, indicates no hemolysis and no virulence. The results are shown in Table 3.

[0039] Pathogen antagonism test: This test adopts the spot inoculation method. Aeromonas hydrophila is used as the pathogen indicator bacteria, and 100ul is taken at a concentration of 10 6 cfu / mL of pathogen indicator bacteria solution was spread on nutrient agar medium, and 10ul of the solution was taken to a concentration of 10 8 cfu / mL of the test bacteria solution was added to the culture medium and incubated at 33°C for 24 hours to observe whether there was an inhibition zone. The results are shown in Table 3.

[0040] Table 3. Test results of hemolytic and antibacterial activity of strains

[0041] strain Hemolytic Antibacterial properties LMVS γ -

[0042] Note: “α” indicates slightly hemolytic; “β” indicates hemolytic; “γ” indicates non-hemolytic; “-” indicates non-bacteriostatic; “+” indicates bacteriostatic.

[0043] Example 2:

[0044] 1. Materials:

[0045] Lateolabrax samples: After the defatted mealworm powder replaced about 50% of fish meal in the aquaculture experiment, the intestines of 5 large lateolabrax were selected as samples.

[0046] Main culture medium and materials:

[0047] Nutrient broth (NB): peptone 10.0 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, pH 7.2 ± 0.2, 25°C.

[0048] Nutrient agar medium (NA): peptone 10.0 g / L, beef extract powder 3.0 g / L, NaCl 5.0 g / L, agar powder 15.0 g / L, pH 7.3±0.1, 25°C.

[0049] MRS medium: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2, 25°C.

[0050] Preparation of bacterial suspension: Single colonies of each LMVS were picked and inoculated into NB liquid medium at 30°C until the mid-to-late logarithmic period. The cells were centrifuged at 12,000 rpm for 10 min, and the cell pellets were collected. The cells were then rinsed with sterile saline and centrifuged twice to prepare a bacterial suspension. The bacterial suspension concentration was adjusted to 1 × 10 based on the plate count results corresponding to the absorbance value (A600 nm) in the preliminary test. 8 cfu / mL for future use.

[0051] Preparation of experimental feed: According to the nutritional requirements of Lateolabranch Seabass, the experimental feed was prepared with fish meal, defatted mealworm powder and soybean meal as the main protein sources, and fish oil and soybean oil as the main fat sources. There were two experimental feeds: control group TM and bacteria-added group H2. In the bacteria-added group H2, 1 ml of Bacillus halogenisis LMVS bacterial solution was added to the surface of the feed by spraying on the control group TM, and the bacterial count in the feed finally reached 1×10 8 The experimental feed used yttrium trioxide as an exogenous indicator, and the experimental feed formula is shown in Table 4 (control group feed formula).

[0052] Table 4 Feed formula and nutritional level (dry matter basis %)

[0053]

[0054]

[0055] 2. Lateolabrax japonicus aquaculture experiment

[0056] A culture experiment with L. japonicum was conducted at the Dragon Boat Pond in Jimei, Xiamen. Fry were purchased from the Huifeng Farm in Zhangzhou. The fry were placed in 1000-liter aquariums and fed an unbacterial diet for two weeks to acclimate to the environment. Then, 120 robust, uniformly sized L. japonicum (3.7 ± 0.3 g) were randomly assigned to six 500-liter freshwater aquariums. The fry were fed two experimental diets, with three replicates of each diet. For eight weeks, the fry were fed twice daily (at 8:00 AM and 5:00 PM) to a full meal. After each feeding, the tank contents were siphoned out. Fecal samples were collected from the L. japonicum starting in the sixth week of the experiment. Feces were collected 30 minutes after each feeding and filtered through gauze. The feces were then scraped with a metal spoon and placed in labeled ziplock bags. The bags were then stored at -20°C for determination of apparent nutrient digestibility. During the breeding period, the water temperature was 27±2℃, dissolved oxygen ≥6.0mg / L, ammonia nitrogen <0.1mg / L, and pH 7.0-7.5.

[0057] Sample collection: After the breeding experiment, the animals were fasted for 24 h, anesthetized with eugenol (1:10000), and weighed and counted. Three fish were randomly selected from each barrel, placed in ziplock bags, and stored at -20°C for whole-body composition analysis. Feed and dried fecal samples (0.1-0.2 g) were weighed from each experimental group and digested with nitric acid. Yttrium concentrations were determined using an inductively coupled plasma atomic emission spectrometer (ICP-OES, Prodigy 7, Leeman Labs, USA). Yttrium content in the samples was calculated using the formula: Apparent Digestibility Coefficient (ADC) = (1 - Yttrium content in feed / Yttrium content in feces) × 100. Ten fish were randomly selected from each barrel and weighed. Blood was collected from the tail vein. The blood was allowed to rest in a 1.5 mL centrifuge tube for 12 hours and then centrifuged at 2500 rpm for 10 minutes. Serum was collected and stored at -80°C for determination of serum lysozyme (LZM) and diamine oxidase (DAO) activities. Liver and abdominal fat were dissected and weighed to calculate liver index and abdominal fat percentage. The intestines of two fish were randomly sampled from each bucket for measurement of intestinal digestive enzyme activity. The intestines of two fish were also randomly sampled from each bucket for measurement of intestinal inflammatory factors and tight junction protein-related gene expression. The intestines of two fish were randomly sampled from each bucket, the surface fat removed, and 2 cm of midgut was placed in a 5 mL cryovial containing 4% formaldehyde fixative and stored at 4°C. Intestinal tissue sections were prepared using H&E staining for intestinal histomorphological analysis.

[0058] Data statistics and analysis: The experimental data were subjected to independent sample T test (Student's t test) using SPSS26.0 statistical software. The significant difference level was P<0.05. All experimental data were expressed as mean ± standard error (Mean ± SE).

[0059] 3. Results:

[0060] 1) Effects of adding potential probiotics to defatted mealworm meal feed on the growth performance of Lateolabrax japonicus:

[0061] The effects of adding LMVS strain to the feed on the growth performance of Lateolabrax are shown in Table 5.

[0062] Table 5 Effects of adding potential probiotics to defatted mealworm meal feed on the growth performance of Lateolabrax japonicus

[0063] Group Control groupTM Bacteria-added group H2 Feeding rate 3.11±0.12 3.04±0.03 Weight gain rate% 987.83±16.23 1082.91±16.68* Survival rate (%) 98.33±1.67 97.50±1.44 Feed efficiency 0.85±0.03 0.88±0.01 Protein deposition rate 33.83±1.21 37.03±0.78 Specific growth rate 4.26±0.03 4.41±0.02*

[0064] Note: The data in the same column marked with "*" indicate that the treatment group is significantly different from the control group (P<0.05).

[0065] As shown in Table 5, the addition of the LMVS strain to the feed significantly increased the weight gain and specific growth rate of L. japonicus compared to the control group TM (P < 0.05). There were no significant differences in feed efficiency, survival rate, or protein deposition between the H2 group and the control group (P > 0.05).

[0066] This shows that after adding the LMVS strain, the growth rate of the sea bass increases, while it does not affect the feed efficiency and survival rate during the breeding process.

[0067] 2) Effects of adding potential probiotics to defatted mealworm meal feed on the body parameters of Lateolabrax japonicus

[0068] The results are shown in Table 6.

[0069] Table 6 Effects of adding potential probiotics to defatted mealworm feed on the body parameters of Lateolabrax japonicus

[0070]

[0071]

[0072] As shown in Table 6, the addition of the LMVS strain to the experimental diet had no significant effect on the liver-to-body ratio, organ-to-body ratio, or abdominal fat percentage of L. japonicus (P>0.05), but significantly reduced the fatness of L. japonicus (P<0.05). This suggests that the LMVS strain increases yield by improving overall growth rather than improving the quality of L. japonicus' internal organs.

[0073] 3) Effects of adding potential probiotics to defatted mealworm meal feed on the nonspecific immunity of Lateolabrax japonicus

[0074] The results are shown in Table 7.

[0075] Table 7 Effects of adding potential probiotics to defatted mealworm meal feed on nonspecific immunity of Lateolabrax japonicus

[0076]

[0077] As shown in Table 7, there was no significant effect on the nonspecific immune capacity of L. japonicus in each group (P>0.05).

[0078] 4) Effects of adding potential probiotics to defatted mealworm meal feed on digestive enzyme activities in the intestine of Lateolabrax japonicus

[0079] The results are shown in Table 8.

[0080] Table 8 Effects of adding potential probiotics to defatted mealworm meal feed on digestive enzyme activities in the intestine of Lateolabrax japonicus

[0081] Group Protease (U / mg prot) Lipase (U / g prot) Amylase (U / g prot) Control groupTM 296.65±61.86 1.96±0.09 49.42±0.65 Bacteria-added group H2 300.47±33.74 2.11±0.05 43.51±5.39

[0082] As shown in Table 8, compared with the control group TM, there was no significant change in the intestinal digestive enzyme activity of the H2 seabass in the bacteria-added group (P>0.05).

[0083] 5) Effects of adding potential probiotics to defatted mealworm meal feed on the intestinal tissue morphology of Lateolabrax japonicus

[0084] The results are as follows Figure 1 and Table 9. From Figure 1 It can be seen that the intestinal villi of each group of sea bass were intact, neatly arranged and extending into the intestinal lumen, and the intestinal muscular layer and submucosa were well developed. As shown in Table 9, the number of villi in the bacteria-added group H2 was significantly increased (P < 0.05), and the number of intestinal goblet cells was also significantly increased (P < 0.05). The increase in the number of villi can increase the intestinal absorption area, thereby allowing for more efficient nutrient absorption. Goblet cells play an important role in maintaining intestinal health. They can secrete large amounts of mucin, and the mucus layer they form enhances the ability to resist pathogens. An increase in the number of goblet cells can strengthen the intestine's ability to resist pathogens.

[0085] Table 9 Effects of adding potential probiotics to defatted mealworm powder feed on the intestinal tissue morphology of Lateolabrax japonicus

[0086]

[0087]

[0088] 6) Effects of adding potential probiotics to defatted mealworm meal feed on the expression of genes related to intestinal tight junction proteins in Lateolabrax japonicus

[0089] The results are as follows Figure 2 , Figure 2 This is a graph showing the effect of adding potential probiotics to defatted mealworm meal feed on the expression of genes related to tight junction proteins in the intestine of Lateolabrax japonicus. Figure 2 The relative expression levels of tight junction protein genes (Ocln, Claudin, and ZO-1) in the bacteria-added group H2 all showed an upward trend, with Claudin being the most significant (P < 0.05). The intestinal mucosal tight junction layer serves as a physical barrier against the invasion of toxins, antigens, and pathogens. Damage to this layer increases its permeability, triggering a series of inflammatory responses. These results suggest that the addition of LMVS bacteria to the feed can promote intestinal health in Lateolabrax japonicus by increasing the relative expression of tight junction protein genes.

[0090] 7) Effects of adding potential probiotics to defatted mealworm meal feed on the expression of genes related to intestinal inflammatory factors in Lateolabrax japonicus

[0091] See the results Figure 3 , Figure 3 This is a graph showing the effect of adding potential probiotics to defatted mealworm meal feed on the expression of genes related to tight junction proteins in the intestine of Lateolabrax japonicus. Figure 3 It can be seen that compared with the control group TM, the relative expression levels of intestinal inflammatory factors IL-4, TNF-β, and IL-10 genes in the bacteria-added group H2 showed an upward trend, among which the relative expression level of IL-4 was significantly higher than that in the control group TM (P<0.05). At the same time, the relative expression level of IL-1β gene in the bacteria-added group H2 was significantly higher than that in the control group TM. In this experiment, the weight gain rate and relative expression levels of inflammatory factors in the bacteria-added group H2 increased significantly, indicating that probiotics may increase the expression of inflammatory factors by enhancing intestinal barrier function and tissue repair ability. Some probiotics have the ability to adhere to intestinal epithelial cells and produce beneficial metabolites, thereby promoting the integrity and repair of the intestinal barrier. These effects may lead to an increase in the relative expression of inflammatory factors.

[0092] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. Bacillus halogenide ( Virgibacillus salarius ) LMVS is used to prepare defatted mealworm powder feed for Lateolabranch; the Bacillus halogenides ( Virgibacillus salarius )LMVS was deposited in the General Microbiology Center of China Culture Collection Administration on April 3, 2023, with the deposit number CGMCC No.27011.

2. The use according to claim 1, characterized in that The feed has the effects of increasing fish production and improving the intestinal health of fish.

3. The use according to claim 1, characterized in that The feed has the effect of improving fish's utilization of defatted mealworm feed.

4. A defatted mealworm powder feed for Lateolabrax japonicus, characterized in that: Contains Bacillus halogenus ( Virgibacillus salarius )LMVS, the Bacillus halogenide ( Virgibacillus salarius )LMVS was deposited in the General Microbiology Center of China Culture Collection Administration on April 3, 2023, with the deposit number CGMCC No.27011.

5. The feed according to claim 4, wherein The defatted mealworm powder in the feed replaced 50 wt % of the fish meal.

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