Bacillus subtilis and application thereof

Abstract

The invention discloses bacillus subtilis G190 and application of the bacillus subtilis G190 in culture of eriocheir sinensis. The bacillus subtilis is used for fermenting soybean meal to prepare an aquatic product feed, the feed is used for breeding the eriocheir sinensis, and the survival rate and flavor substances of the eriocheir sinensis can be remarkably increased. The effective implementation of the research can provide a scientific theoretical basis for the action effect, regulation and control mechanism, popularization and application of the probiotic bacillus feed, and effectively serves the further development of the sustainable ecological breeding technology of the eriocheir sinensis.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, in particular to Bacillus subtilis and application thereof. Background Art

[0002] Chinese mitten crab (Eriocheir sinensis) has become one of the most popular freshwater economic aquatic products for Chinese consumers due to its plump meat and rich yellow roe. The most critical stage in Chinese mitten crab farming is the "fattening period" of the last 45 days. In traditional farming models, fresh-frozen fish is often used as the main feed during this stage. With the rapid growth of the Chinese mitten crab farming industry and the increasing pressure on environmental protection, this type of fresh-frozen fish feeding model can no longer fully meet people's urgent needs for ecological and green farming technology. Although fresh-frozen fish as a source of animal protein feed can improve the nutritional value of Chinese mitten crab and increase its growth rate, fresh-frozen fish has problems such as uneven quality, high feed coefficient, damage to the farming environment, impact on food safety and increase in farming costs, which affect the sustainable development of Chinese mitten crab ecological farming. Soybean meal is the most common protein source in Chinese mitten crab formula feed. It has the advantages of rich high-quality protein, wide sources and low prices. However, soybean meal has problems such as poor palatability and anti-nutritional factors, which limit the effective utilization of protein in soybean meal. Bacillus is a probiotic widely used in aquaculture. It has the characteristics of high temperature resistance, dryness resistance, acid resistance, etc., and its ability to resist adverse environments is stronger than other probiotics. In addition, Bacillus also has the characteristics of producing antibacterial small molecule metabolites, competing for intestinal adhesion sites, regulating the immunity of Chinese mitten crabs, and producing a variety of anti-nutritional factor digestive enzymes. The rational use of Bacillus to ferment soybean meal in Chinese mitten crab feed can decompose the difficult-to-digest components, improve the palatability and flavor substances of soybean meal, and increase the content of soluble protein and small molecule peptides. At present, research on the use of probiotics in Chinese mitten crab farming has been carried out one after another, but most probiotics are derived from terrestrial environments. There is little research on probiotics isolated from water sources, especially the lack of development and comprehensive utilization of probiotic Bacillus from the intestinal tract of Chinese mitten crabs. Summary of the invention

[0003] The object of the present invention is to provide a Bacillus subtilis and application thereof to solve the problems raised in the above background technology.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] A Bacillus subtilis, characterized in that the Bacillus subtilis is named Bacillus subtilis G190;

[0006] The Bacillus subtilis is deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2024, with a deposit number of CGMCC NO.32678 and a strain number of BS2208G190.

[0007] The application of the Bacillus subtilis is characterized in that the Bacillus subtilis G190 is used to ferment soybean meal to prepare fermented soybean meal.

[0008] An aquatic product feed, characterized in that the formula of the feed calculated by weight is:

[0009]

[0010] On the basis of the above scheme, the formula of the feed calculated by weight is:

[0011]

[0012] On the basis of the above scheme, the formula of the feed calculated by weight is:

[0013]

[0014]

[0015] The application of the above-mentioned aquatic product feed is characterized in that the aquatic product feed is used for breeding Chinese mitten crab.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0017] By screening the probiotics in the intestine of Chinese mitten crab, a high temperature and acid resistant Bacillus subtilis G190 was obtained. The bacteria has good stress resistance, can better attach to the host intestine and play a probiotic role, and has no toxic effects on cells, Chinese mitten crabs, and mice. The bacteria were further used to ferment soybean meal in Chinese mitten crab feed, and the optimal fermentation conditions were obtained: 93.1h, feed-water ratio: 1:0.498, inoculation amount: 15.76%, and fermentation temperature: 36.6℃. Finally, through the comparison of breeding experiments of different types of feeds, it was proved that the aquatic product feed prepared with soybean meal fermented by probiotic Bacillus G190 can significantly improve the survival rate and flavor substances of Chinese mitten crab. The effective implementation of the present invention can provide a scientific theoretical basis for the effect, regulation mechanism and promotion and application of probiotic Bacillus feed, and effectively serve the further development of sustainable ecological breeding technology of Chinese mitten crab. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Effects of different feeds on growth indicators of Chinese mitten crab: (A) average body weight (B) hepatopancreas index (C) gonad index (D) weight gain rate (E) survival rate, D group: unfermented soybean meal group; F group: fermented soybean meal group; M group: semi-fermented soybean meal group; S group: chilled mackerel group;

[0019] Figure 2 Effects of different feeds on sensory evaluation indexes of Chinese mitten crab: (A) male crabs (B) female crabs; Group D: unfermented soybean meal group; Group F: fermented soybean meal group; Group M: semi-fermented soybean meal group; Group S: chilled mackerel group;

[0020] Figure 3 The contents of umami amino acids, sweet amino acids, and bitter amino acids in four groups: (A)-(C) muscle; (D)-(F) hepatopancreas; (G)-(I) gonads; Group D: unfermented soybean meal group; Group F: fermented soybean meal group; Group M: semi-fermented soybean meal group; Group S: chilled mackerel group;

[0021] Figure 4 GMP, IMP, AMP and EUC values ​​in four groups: (A) male (B) female; Group D: unfermented soybean meal group; Group F: fermented soybean meal group; Group M: semi-fermented soybean meal group; Group S: chilled mackerel group;

[0022] Figure 5 The heat map of the correlation of volatile substances among four groups: (A) male (B) female; Group D: unfermented soybean meal group; Group F: fermented soybean meal group; Group M: semi-fermented soybean meal group; Group S: chilled mackerel group;

[0023] Figure 6 The petal maps and OTUs of the intestinal flora of Chinese mitten crab in different groups: (A) is the petal map; (B) is the OTUs;

[0024] Figure 7 Diversity analysis of intestinal flora in different groups of Chinese mitten crab: (A) Observed OTUs (B) Chao index (C) Shannon index (D) Simpson index (E) Goods coverage index. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0026] Example 1 Screening of Bacillus subtilis G190

[0027] Sample pretreatment: Clean and disinfect the body surface of Chinese mitten crab; remove the intestine with tweezers in a clean bench, add sterile physiological saline in a sterile homogenizer, and homogenize thoroughly.

[0028] Place the homogenate in a 90°C water bath for 10 minutes, dilute it in a 50-fold gradient, take 100 μL of the diluted solution, spread it evenly, and invert it in a constant temperature incubator at 28°C for 24h-36h. Select a suitable plate (50-300 CFU / plate) and purify it by streaking multiple times; extract DNA from the purified strain, amplify it by PCR, and detect it by agarose gel electrophoresis. If it is qualified, it will be sent to Shanghai Paisonno Biotechnology Co., Ltd., China for 16S rRNA sequencing.

[0029] The isolated bacteria were inoculated into broth medium and activated at 37°C and 150 r / min for 24 h. Screening of acid-resistant bacteria: 5% bacterial suspension (v / v) was added to 100 mL broth medium and the bacterial concentration was adjusted to about 10 after 24 h of shaking culture. 8 CFU / mL, then 5mL was added to 100mL broth medium with initial pH of 4.0 and 5.0, respectively, and after shaking and culturing for 4h, plate coating was performed to count, and strains with a survival rate of more than 50% were selected. Among them, there was only one Bacillus subtilis, which was named G190.

[0030] The Bacillus subtilis G190 has good stress resistance, can secrete amylase, protease and cellulase, has good hydrophobicity and self-aggregation, can better adhere to the host intestine and exert probiotic properties. In addition, safety evaluation tests on Vero cells, Chinese mitten crabs and mice showed that G190 did not cause any damage or death to these three models. Therefore, Bacillus subtilis G190 has the probiotic potential to be used in sustainable and healthy aquaculture.

[0031] Example 2: Fermentation of soybean meal by Bacillus subtilis G190

[0032] The soybean meal used in feed was fermented with Bacillus subtilis G190 under the following fermentation conditions: fermentation time: 93.1h, feed-water ratio: 1:0.498, inoculation amount: 15.76%, fermentation temperature: 36.6℃. After fermentation, the content of H in soybean meal reached 1.47×10 10 cfu / g, and the fermented soybean meal is used for subsequent preparation of aquatic product feed.

[0033] Example 3 Preparation and application of aquatic product feed

[0034] According to the following formula, the fermented soybean meal obtained in Example 2 was used to prepare aquatic product feed.

[0035]

[0036] In the above formula, the processing requirements of some ingredients are as follows:

[0037] Soybean meal and shrimp shell powder are crushed by Bohan HBM-103B hammer mill and then passed through 80 mesh screen for use; fish meal, flour, and meat and bone meal are directly passed through 80 mesh screen for use; dried black soldier fly worms are crushed by Midea MJ-PB13S69 wall breaker and then passed through 40 mesh screen for use; fish oil, soybean lecithin, and cuttlefish paste are weighed in proportion and mixed for use. Haematococcus pluvialis powder, Chinese mitten crab premix, calcium dihydrogen phosphate, and yeast hydrolyzate are gradually mixed manually according to the proportion of the formula from low to high, and then put into Zhigao ZG-LZ710 mixer, and the pre-screened black soldier fly worm powder, shrimp shell powder, flour, and soybean meal are added in turn according to the formula proportion while stirring. During the mixing process, fish oil, soybean lecithin, and cuttlefish paste mixture are gradually added. After all the feed raw materials are mixed evenly, water is evenly added by weight at 15%-20% using a sprayer, so that the feed can be granulated without falling apart after being clenched. The feed was pelletized using a Sino SXC-12 meat grinder with a particle size of 3 mm. The feed was dried indoors and stored in aluminum foil bags for later use.

[0038] The above-mentioned aquatic feed was used to carry out a breeding experiment on Chinese mitten crab (Group F as shown in the table below), and the control groups included: Group D (using unfermented soybean meal), Group M (using semi-fermented soybean meal, i.e., the ratio of fermented soybean meal to unfermented soybean meal was 1:1) and Group S (chilled mackerel).

[0039]

[0040] The experimental results show that:

[0041] (1) The fermented formula feed prepared with soybean meal fermented by Bacillus subtilis G190 significantly increased the average body weight and weight gain rate of male crabs ( Figure 1 The results showed that fermented feed could meet the growth and development needs of Chinese mitten crabs. The survival rate of male crabs in group F (fermented soybean meal) was 27.5% higher than that in group S (frozen mackerel). Figure 1 (E) Probiotic fermented formula feed can improve the survival rate of Chinese mitten crab.

[0042] (2) In terms of flavor changes, the grassy, ​​fat, and meaty flavor scores of male crabs in group F (fermented soybean meal) were higher than those in the other groups ( Figure 2 ), probiotic fermented formula feed can improve the flavor of all edible parts of Chinese mitten crab. The umami amino acids in the muscles and sweet amino acids in the gonads of female crabs in group F (fermented soybean meal) were significantly higher than those in the other three groups, and the bitter amino acids in the hepatopancreas were the lowest ( Figure 3); The contents of GMP, IMP and AMP in the hepatopancreas of male crabs in group F (fermented soybean meal) were significantly higher than those in the other three groups; the contents of GMP and IMP in the hepatopancreas of female crabs in group F (fermented soybean meal) were significantly higher than those in the other three groups; the EUC value of the hepatopancreas in group F (fermented soybean meal) was the highest ( Figure 4 ); Group F (fermented soybean meal) had the highest content of aldehydes, with hexanal, tetradecanal, heptaldehyde and phenylacetaldehyde significantly higher than those of the other three groups, and the lowest content of trimethylamine ( Figure 5 ).

[0043] (3) At the level of intestinal flora, probiotic fermented formula feed can increase the species richness and diversity of the intestinal flora of Chinese mitten crab.

[0044] OTU (operational taxonomic unit) analysis showed that the total number of OTUs in the four groups (male and female) was 2938, and the four groups had 19 OTUs in common. The number of OTUs in male crabs was higher than that in female crabs. The number of OTUs in male crabs in groups D (unfermented soybean meal) and F (fermented soybean meal) was the highest, 689 and 538 respectively. The highest average number of OTUs in the samples was in male crabs in group F (fermented soybean meal) ( Figure 6 ).

[0045] Analysis of intestinal flora α diversity showed that in male crabs, Chao and Shannon indexes of group F (fermented soybean meal) were higher than those of the other three groups, but there was no significant difference (P>0.05); in female crabs, Shannon index of group F (fermented soybean meal) was significantly higher than that of the other three groups (P<0.05), while Simpson index of group D (unfermented soybean meal) was significantly lower than that of the other three groups (P<0.05). In terms of β diversity, PCoA analysis results showed that the four groups of samples were roughly clustered, and the clustering of group S (frozen mackerel) samples was slightly better than that of the other three groups ( Figure 7 ).

[0046] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0047] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis G190, characterized in that The Bacillus subtilis is named as Bacillus subtilis G190, and is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCCNO.32678.

2. The use of Bacillus subtilis according to claim 1, characterized in that: The Bacillus subtilis G190 is used to ferment soybean meal to prepare fermented soybean meal.

3. A fishery feed, characterized in that: The formula of the feed calculated by weight is:

4. A fishery feed, characterized in that: The formula of the feed calculated by weight is:

5. A fishery feed, characterized in that: The formula of the feed calculated by weight is:

6. An application of aquatic product feed according to any one of claims 3 to 5, characterized in that: The aquatic product feed is used for breeding Chinese mitten crab.

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