Bacillus subtilis and application thereof

Abstract

The application discloses bacillus subtilis G190 and application thereof in Eriocheir sinensis breeding. After soybean meal is fermented by the bacillus subtilis G190, aquatic product feed is prepared, and the Eriocheir sinensis is bred by using the feed, so that the survival rate and flavor substances of the Eriocheir sinensis can be remarkably improved. The effective implementation of the research can provide scientific theoretical basis for the effect, regulation mechanism and popularization and application of the probiotic bacillus feed, and effectively serve further development of sustainable ecological breeding technology of the Eriocheir sinensis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquaculture, in particular to a bacillus subtilis and application thereof. BACKGROUND

[0002] Chinese mitten crab (Eriocheir sinensis) is one of the most popular freshwater economic aquatic products in China due to its delicious and fatty meat. The most critical stage in the cultivation of Chinese mitten crab is the last 45 days of the "fattening period". In the traditional cultivation mode, ice-fresh fish is often used as the main feed during this period. With the rapid growth of the Chinese mitten crab cultivation industry and the increasing environmental pressure, this ice-fresh fish feeding mode cannot fully meet the urgent needs of people for ecological and green cultivation technology. Although ice-fresh miscellaneous fish as an animal protein feed source can improve the nutritional value of Chinese mitten crab and increase its growth rate, it has problems such as uneven quality, high feed coefficient, destruction of the ecological environment, impact on food safety, and increased cultivation costs, which affect the sustainable development of Chinese mitten crab ecological cultivation. Soybean meal is the most common protein source in Chinese mitten crab formula feed, which has the advantages of high-quality protein, wide source, and low price. However, soybean meal has poor palatability and contains anti-nutritional factors, which limits the effective utilization of protein in soybean meal. Bacillus is a widely used probiotic in aquaculture, which has the characteristics of high temperature resistance, dry resistance, and acid resistance, and stronger resistance to adverse environments than other probiotics. In addition, bacillus also has the characteristics of producing antibacterial small molecule metabolites, competing for intestinal adhesion sites, regulating Chinese mitten crab immunity, and producing various anti-nutritional factor digestion enzymes. Reasonable utilization of bacillus for soybean meal fermentation in Chinese mitten crab feed can decompose the indigestible components in soybean meal, 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 cultivation has been carried out, but most of the probiotics are derived from terrestrial environments, and research on probiotics isolated from aquatic environments is less, especially the development and comprehensive utilization of probiotic bacillus from the intestinal tract of Chinese mitten crab. SUMMARY

[0003] The present application aims to provide a bacillus subtilis and application thereof to solve the problems in the background art.

[0004] To solve the above technical problems, the present application 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 preserved in China General Microbiological Culture Collection Center (CGMCC) on November 18, 2024, and the preservation number is CGMCC NO.32678, and the strain number is BS2208G190.

[0007] The application of the above-mentioned 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 is characterized by a formula calculated by weight fraction:

[0009]

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

[0011]

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

[0013]

[0014]

[0015] The application of the above-mentioned aquatic product feed is characterized in that the aquatic product feed is applied to breed Eriocheir sinensis.

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

[0017] Through screening of probiotics in the intestinal tract of Eriocheir sinensis, a high-temperature-resistant and acid-resistant bacillus subtilis G190 is obtained, which has good stress resistance, can better adhere to the intestinal tract of the host and play a probiotic function, and has no toxic effect on cells, Eriocheir sinensis and mice. Further application of the bacillus subtilis G190 to ferment soybean meal in the feed of Eriocheir sinensis obtains the best fermentation conditions: 93.1 h, material-water ratio: 1:0.498, inoculation amount: 15.76%, fermentation temperature: 36.6 DEG C. Finally, through the comparison of breeding experiments of different types of feeds, it is proved that the aquatic product feed prepared by using the soybean meal fermented by the probiotic bacillus G190 can significantly improve the survival rate and flavor substances of Eriocheir sinensis. The effective implementation of the present application can provide scientific theoretical basis for the effect, regulation mechanism and popularization and application of probiotic bacillus feed, and effectively serve the further development of sustainable ecological breeding technology of Eriocheir sinensis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1Effects of different feeds on growth indexes of Eriocheir sinensis: (A) average weight (B) hepatosomatic index (C) gonadosomatic 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: fresh mackerel group;

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

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

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

[0022] Figure 5 Heat map of volatile substances related to four groups: (A) male (B) female; D group: unfermented soybean meal group; F group: fermented soybean meal group; M group: semi-fermented soybean meal group; S group: fresh mackerel group;

[0023] Figure 6 Petals and OTUs of intestinal flora of Eriocheir sinensis in different groups: (A) petals (B) OTUs;

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

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Example 1 Screening of Bacillus subtilis G190

[0027] Sample pretreatment: The surface of Eriocheir sinensis was cleaned and disinfected; the intestinal tract was taken out with forceps in a clean bench, and sterile saline was added in a sterilized homogenizer for homogenization.

[0028] The homogenate was placed in a 90°C water bath for 10 min, diluted by 50 times gradient, and 100 μL of the diluted solution was taken and uniformly coated, then cultured in a constant temperature incubator at 28°C for 24-36 h. Suitable plates (50-300 CFU / plate) were selected and purified by multiple streaks; the purified strain was extracted for DNA, and after PCR amplification, agarose gel electrophoresis was used for detection, and after passing the test, it was sent to Shanghai Pisen Life Sciences and Technology Co., Ltd. for 16S rRNA sequencing.

[0029] The isolated bacteria were inoculated in broth medium and activated at 37°C with 150 r / min shaking for 24 h. Acid-resistant bacteria screening: 5% bacterial suspension (v / v) was added to 100 mL broth medium, and after shaking culture for 24 h, the bacterial concentration was adjusted to about 10 8 CFU / mL, and then 5 mL was added to 100 mL broth medium with initial pH of 4.0 and 5.0, respectively, and after shaking culture for 4 h, plate coating counting was performed, and strains with survival rate above 50% were selected. Only one strain of Bacillus subtilis was selected, 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 intestinal tract of the host and exert probiotic performance. In addition, safety evaluation tests on Vero cells, Eriocheir sinensis and mice showed that G190 did not cause any damage and death to the three models. Therefore, Bacillus subtilis G190 has the potential to be applied to sustainable healthy aquaculture.

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

[0032] According to the following fermentation conditions, Bacillus subtilis G190 was used to ferment soybean meal used in feed: fermentation time: 93.1 h, solid-liquid ratio: 1:0.498, inoculation amount: 15.76%, fermentation temperature: 36.6°C. After fermentation, the number of bacteria in the soybean meal reached 1.47 x 10 10 cfu / g, which was used for subsequent preparation of aquatic feed.

[0033] Example 3 Preparation and application of aquatic feed

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

[0035]

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

[0037] The soybean meal and shrimp shell powder are crushed by a Bohai HBM-103B hammer mill and then sieved through an 80-mesh screen; the fish meal and meat and bone meal are directly sieved through an 80-mesh screen; the black soldier fly dry larvae are crushed by a Midea MJ-PB13S69 cell wall breaking machine and then sieved through a 40-mesh screen; the fish oil, soybean phospholipid and squid paste are weighed according to the proportion and then mixed uniformly; the Haematococcus pluvialis powder, Eriocheir sinensis premix, calcium dihydrogen phosphate and yeast hydrolysate are mixed manually according to the proportion from low to high, and then put into a Zhigao ZG-LZ710 stirrer, and the black soldier fly powder, shrimp shell powder, flour and soybean meal sieved in advance are added according to the proportion while stirring, and the mixture of fish oil, soybean phospholipid and squid paste is gradually added during the mixing process, and after all the feed raw materials are mixed uniformly, a spray is used to add water uniformly at a weight of 15%-20%, so that the feed can be granulated after being clumped without falling apart. The feed is granulated by a Xino SXC-12 meat grinder with a particle size of 3 mm, and the feed is stored in an aluminum foil bag after being dried indoors.

[0038] The above aquatic product feed is used for breeding Eriocheir sinensis (as shown in F group in the following table), and the control groups include D group (using unfermented soybean meal), M group (using semi-fermented soybean meal, i.e. the ratio of fermented soybean meal to unfermented soybean meal is 1:1) and S group (fresh mackerel).

[0039]

[0040] The experimental results show that:

[0041] (1) The fermented formula feed prepared by using the soybean meal fermented by Bacillus subtilis G190 significantly increases the average body weight and the weight gain rate of male crabs (A-D in the middle), indicating that the fermented feed can meet the growth and development needs of Eriocheir sinensis, and the survival rate of male crabs in F group (fermented soybean meal) is 27.5% higher than that in S group (fresh mackerel) (E in the middle), and the probiotic fermented formula feed can improve the survival rate of Eriocheir sinensis. Figure 1 Figure 1 (2) In terms of flavor change, the scores of grassy flavor, fat flavor and meaty flavor of male crabs in F group (fermented soybean meal) are higher than those in other groups (F in the middle), and the probiotic fermented formula feed can improve the flavor of each edible part of Eriocheir sinensis, the fresh-tasting amino acids in the muscle of female crabs in F group (fermented soybean meal) and the sweet-tasting amino acids in the gonads are significantly higher than those in the other three groups, and the bitter-tasting amino acid content in the hepatopancreas is the lowest (G in the middle).

[0042] (2) In terms of flavor change, the scores of grassy flavor, fat flavor and meaty flavor of male crabs in F group (fermented soybean meal) are higher than those in other groups (F in the middle), and the probiotic fermented formula feed can improve the flavor of each edible part of Eriocheir sinensis, the fresh-tasting amino acids in the muscle of female crabs in F group (fermented soybean meal) and the sweet-tasting amino acids in the gonads are significantly higher than those in the other three groups, and the bitter-tasting amino acid content in the hepatopancreas is the lowest (G in the middle). Figure 2 Figure 3 ​​The GMP, IMP, and AMP contents of the hepatopancreas of male crabs in group F (fermented soybean meal) were significantly higher than those of the other three groups. The GMP and IMP contents of the hepatopancreas of female crabs in group F (fermented soybean meal) were significantly higher than those of 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 aldehyde content, with significantly higher levels of hexanal, tetradecanal, heptanal, and phenylacetaldehyde compared to the other three groups, and the lowest trimethylamine content. Figure 5 ).

[0043] (3) At the gut microbiota level, probiotic fermented feed can improve the species abundance and diversity of gut microbiota in Chinese mitten crabs.

[0044] OTU (Operating Taxonomic Unit) analysis showed a total of 2938 OTUs across the four groups (male and female), with a total of 19 OTUs shared by all four groups. Male crabs had a higher OTU count than female crabs. The highest OTU counts were found in male crabs in groups D (unfermented soybean meal) and F (fermented soybean meal), with 689 and 538 OTUs respectively. The highest average OTU count among the samples was found in male crabs of group F (fermented soybean meal). Figure 6 ).

[0045] Gut microbiota α-diversity analysis showed that in male crabs, the Chao and Shannon indices of group F (fermented soybean meal) were higher than the other three groups, but the differences were not statistically significant (P>0.05); in female crabs, the Shannon index of female crabs in group F (fermented soybean meal) was significantly higher than the other three groups (P<0.05), while the Simpson index of group D (unfermented soybean meal) was significantly lower than the other three groups (P<0.05). Regarding β-diversity, PCoA analysis showed that the four groups of samples were roughly clustered independently, with group S (fresh mackerel) showing slightly better clustering than the other three groups. Figure 7 ).

[0046] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0047] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent ones. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Bacillus subtilis G190, characterized in that, The Bacillus subtilis strain was named Bacillus subtilis G190 and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC NO.32678.

2. The application of Bacillus subtilis according to claim 1, characterized in that, Fermented soybean meal was prepared by fermenting soybean meal using the Bacillus subtilis G190.

3. An aquatic product feed, characterized in that, The feed formula, calculated by weight parts, is as follows:

4. An aquatic product feed, characterized in that, The feed formula, calculated by weight parts, is as follows:

5. An aquatic product feed, characterized in that, The feed formula, calculated by weight parts, is as follows:

6. The application of the aquatic product feed according to any one of claims 3-5, characterized in that, The aquatic feed is used for the farming of Chinese mitten crabs.

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