Bacillus subtilis with high selenite tolerance and application thereof

Abstract

The invention discloses bacillus subtilis NKX with high selenite tolerance and application of the bacillus subtilis NKX, and relates to the technical field of microorganisms. The strain bacillus subtilis (Bacillus sp.) NKX provided by the invention is sent to the China Center for Type Culture Collection on January 2, 2025 for preservation, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2025008. The strain can tolerate selenite with the highest concentration of 25600 mg / L, the concentration of the selenite converted into nano-selenium exceeds 6000 mg / L, and the conversion rate exceeds 96%. The strain provided by the invention provides a strain resource for high tolerance of selenite, and has application potential for high conversion efficiency of nano-selenium in production and preparation of nano-selenium.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Bacillus subtilis with high selenite tolerance and its application. Background Art

[0002] Selenium is an essential trace element for humans, animals and plants to maintain normal physiological functions, and generally exists in the form of inorganic selenium in nature. However, inorganic selenium, especially sodium selenite, has high toxicity and has great defects as a selenium supplement preparation. Therefore, researchers have developed yeast selenium and then nano-selenium. Compared with inorganic selenium and organic selenium, nano-selenium is recognized internationally as the organic selenium form with the best effect and the safest, so it is increasingly favored by the market.

[0003] Microorganisms are used to reduce selenate to produce nano-selenium. Its characteristics are small particle size, uniform and stable structure, high temperature resistance, safety and environmental friendliness, and it has better biocompatibility than nano-selenium prepared by physical synthesis method and chemical reduction method, and has a broader market space.

[0004] Bacillus also has strong stress resistance and is one of the main microorganisms for the production of microbial nano-selenium. Previous studies have shown that the selenite tolerance of Bacillus does not exceed 5000 mg / L, and the concentration of transformed nano-selenium does not exceed 5000 mg / L. If a Bacillus strain with high selenite tolerance can be obtained through strain screening, and then the conversion rate of elemental selenium synthesis can be increased by optimizing the culture conditions and the concentration of transformed nano-selenium can be increased, it can not only accelerate the conversion speed of nano-selenium, but also improve the utilization rate of raw materials and reduce costs, and has considerable prospects in the microbial nano-selenium industry. Summary of the Invention

[0005] The purpose of the present invention is to provide a Bacillus subtilis that can tolerate high concentrations of selenite and its application, so as to solve the problem of low selenite tolerance of existing Bacillus subtilis and improve the conversion efficiency of nano-selenium.

[0006] To achieve the above purpose, the present invention provides a Bacillus subtilis ( Bacillus sp.) NKX that has been deposited at the China Center for Type Culture Collection on January 2, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025008. This Bacillus subtilis NKX can be used to prepare nano-selenium, and can also be used to prepare growth-promoting bacterial agents or feed additives.

[0007] The Bacillus subtilis NKX provided by the present invention can be applied in the breeding field, including promoting the growth of breeding animals and increasing the weight gain rate.

[0008] The present invention also provides a growth-promoting bacterium agent or feed additive comprising the above-mentioned Bacillus subtilis NKX.

[0009] Preferably, the growth-promoting bacterium agent or feed additive is a bacterial suspension of Bacillus subtilis NKX, and its preferred concentration is 1×10 9 cfu / g, and the addition amount in the growth-promoting bacterium agent or feed additive is 5 mL / kg.

[0010] The growth-promoting bacterium agent or feed additive provided by the present invention can be applied to the aquaculture field, including promoting the growth of aquaculture animals, increasing the weight gain rate, regulating the intestine, increasing the number of beneficial intestinal bacteria, and reducing the number of harmful bacteria, and has significant application potential in the aquaculture field.

[0011] The present invention has the following advantages: The present invention isolates and transforms a strain of Bacillus subtilis from the intestine of Pelteobagrus fulvidraco. This strain can tolerate the highest concentration of selenite of 25,600 mg / L, and the concentration of selenite converted into nano-selenium exceeds 6,000 mg / L, and the conversion rate reaches more than 96%. It provides strain resources for the high tolerance of selenite, and at the same time, the high conversion efficiency of nano-selenium has a significant effect in the preparation of nano-selenium. Description of the Drawings

[0012] Figure 1 It is the strain morphology of Bacillus subtilis NKX in the present invention, namely the Gram staining result.

[0013] Figure 2 It is the phylogenetic tree constructed by Bacillus subtilis NKX in the present invention based on 16S rDNA.

[0014] Figure 3 It is the comparison of the conversion of sodium selenite in the fermentation broth of different strains in the present invention.

[0015] Figure 4 It is the growth situation of Bacillus subtilis NKX under different concentrations of selenite in the present invention. Detailed Embodiments

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] Note: The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0018] Isolation and Identification of the Strain in Experimental Example 1 I. Isolation of the Strain Samples were collected from the aquaculture base of Sichuan Mianxing Agricultural Development Co., Ltd., which is located in Yufeng Village, Jianhua Township, Youxian District, Mianyang City. The intestinal contents of 30 healthy Pelteobagrus fulvidraco in the culture pond were mixed. 5 g of the intestinal contents were taken into a sterile Erlenmeyer flask, and 50 mL of sterile normal saline was added. The mixture was cultured on a shaker at 150 rpm at 37 °C for 30 min to obtain a bacterial suspension. Under sterile conditions, 1 mL of the bacterial suspension was taken and added to a centrifuge tube containing 9 mL of sterile water, and shaken well to obtain a bacterial suspension diluted to 10 -1 , and the bacterial suspension was continuously diluted to 10 -2 ~10 -7 according to the concentration gradient dilution method. 100 μL was taken from each of the bacterial suspensions diluted to 10 -4 , 10 -5 , 10 -6 , 10 -7 and evenly spread on the LB plate medium. The plates were cultured at 37 °C for 1 - 2 d. The dominant colonies with better growth and a larger proportion of colonies were selected, and the screened strains were isolated by streaking on the plate. The plates were cultured at 37 °C for 1 - 2 d to obtain a strain, denoted as NKX.

[0019] II. Identification of the Strain 1. Morphological Identification of the Strain The colony morphology of the NKX strain isolated and grown on the plate was observed. As shown in A of Figure 1 , it can be seen that the NKX strain colonies are milky white, forming irregular dirty white colonies, with a rough surface, folds, opaque, flat, and a lighter water-soaked band at the edge. The strain was subjected to Gram staining, and the result is shown in B of Figure 1 . It can be seen that after Gram staining of NKX, the bacterial cells were stained purple, showing Gram-positive. Under an optical microscope, the spores of the strain are oval or cylindrical, with slightly swollen or non-swollen ends.

[0020] 2. Molecular Biology Identification Using conventional methods, the obtained NKX strain was sent to the company for 16S rDNA sequencing, and the 16S rDNA gene sequence of the strain is shown in SEQ ID NO.1 as follows.

[0021] NKX 16S rDNA gene sequence (SEQ ID NO.1):

[0022] The 16s rDNA sequence was compared on NCBI BLAST, and it was found that the sequence similarity between the 16s rDNA of the strain and that of Bacillus subtilis was as high as 99.79%. In the phylogenetic tree, it was clustered into a branch with Bacillus subtilis, see Figure 2 As shown. It can be seen that the strain is Bacillus subtilis ( Bacillus sp.) NKX, and the strain was sent to the China Center for Type Culture Collection for preservation on January 2, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025008.

[0023] Experimental Example 2 Screening of selenite-transforming strains (10) Six experimental groups and one control group were designed. No strains were added to the control group. The strains used in the five experimental groups were NKX, YZ, JS, SICC 1.1136 (Bacillus licheniformis), SICC 1.947 (Bacillus amyloliquefaciens), and SICC1.950 (Bacillus subtilis). Strains numbered with SICC were purchased from the Sichuan Microbial Resources Platform Strain Collection Management Center. Strain YZ was a Bacillus strain introduced from Sichuan Agricultural University. Strain JS was a Bacillus strain isolated from a water purification and conditioning composite bacterial agent.

[0024] (11) The above five strains were inoculated into LB plate culture medium and cultured at 37°C for 24 h. A single colony was picked and inoculated into a flask containing 100 mL of LB sterile liquid culture medium. The culture was shaken at 37°C, 150 rpm for 24-48 h and the bacterial solution was diluted to a concentration of 1 × 10 9 cfu / g as seed solution.

[0025] (3) Prepare nano-selenium fermentation medium with the following formula: 10 g / L molasses, 5 g / L brown sugar, 5 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 0.3 g / L potassium dihydrogen phosphate, and 0.3 g / L magnesium sulfate. Dispense the medium into 250 mL culture bottles, 99 mL per bottle, sterilize and cool, add 1 mL of bacterial solution, and then add sodium selenite to make it Se. +4 The concentration was 1600 mg / L, and the culture was carried out at 37°C and 150 rpm for 5 days. The conversion of sodium selenite in the fermentation broth of different strains was observed. The results are shown in Figure 3 As shown. It can be seen that except for the fermentation liquid of NKX, YZ, and JS strains, which obviously turned red, the fermentation liquid of other strains did not obviously turn red compared with CK. Compared with other strains, these three strains can effectively convert sodium selenite. Among them, the selenite in the present invention can also be selected from other selenites besides sodium selenite.

[0026] Experimental Example 3: Study on the Conversion Rate of Strains to Sodium Selenite at Different Concentrations (1)Design three experimental groups and one control group, with three replicates in each group. The control group CK does not add any strains, and the experimental groups are inoculated with strains NKX, YZ, and JS respectively.

[0027] Prepare a nano-selenium fermentation medium with the following formula: 10 g / L molasses, 5 g / L brown sugar, 5 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 0.3 g / L potassium dihydrogen phosphate, and 0.3 g / L magnesium sulfate. Dispense the medium into 1000 mL culture flasks, 495 mL per flask. After sterilization and cooling, add 5 mL of bacterial liquid, and then add sodium selenite to make the Se +4 concentrations 1600 mg / L, 3200 mg / L, 4800 mg / L, 6400 mg / L, and 8000 mg / L respectively. Incubate at 37 °C and 150 rpm for 9 days, and detect the conversion of sodium selenite in the fermentation broth of different strains. The results are shown in Table 1. Strains NKX, YZ, and JS can all partially convert selenite in the fermentation broth into nano-selenium. Among them, strain NKX has a higher tolerance concentration to Se +4 and the conversion rate is as high as 83.65% at a Se +4 concentration of 6000 mg / L, and it can still convert 62.94% at 8000 mg / L. However, the tolerance concentration of strain JS to Se +4 does not exceed 6400 mg / L, and the conversion rate of strain YZ is only 9.01% at 6400 mg / L. Strain NKX can be used for the production of high-concentration nano-selenium.

[0028] Table 1: Conversion Efficiency of Strains to Sodium Selenite at Different Concentrations

[0029] Experimental Example 4: Determination of the Tolerance Degree of Strain NKX to Sodium Selenite (1)Inoculate Bacillus subtilis NKX into an LB plate medium and culture at 37 °C for 24 h. Pick a single colony and inoculate it into a triangular flask containing 100 mL of sterile LB liquid medium. Incubate at 37 °C and 150 rpm for 24 - 48 h, and dilute the bacterial liquid concentration to 1×10 9 cfu / g as the seed liquid.

[0030] (2) Prepare LB liquid medium and dispense it into 250 mL Erlenmeyer flasks, 99 mL per flask. After sterilization and cooling, add 1 mL of the seed solution to each flask, and then add sodium selenite to make its concentrations 0, 1600, 3200, 4800, 6400, 8000, 9600, 11200, 12800, 14400, 16000, 17600, 19200, 20800, 22400, 24000, 25600, 27200 mg / L respectively. Incubate at 37 °C and 150 rpm for 48 h. After fermentation, take 25 mL from each concentration and place it in a colorimetric tube to observe the growth of the strain and the conversion of sodium selenite in the fermentation broth under different concentrations, as shown in Figure 4 shown.

[0031] Among them, from left to right are ck (without sodium selenite), sodium selenite concentrations from 27200 to 1600 mg / L. The results show that the bacteria grow to some extent when the sodium selenite concentration is 25600 mg / L and below, and the fermentation broth turns red. The strains grow best at concentrations of 1600, 3200, and 4800 mg / L, followed by 6400 and 8000 mg / L. The growth of the strains gradually weakens at concentrations of 9600 - 27200 mg / L. When the sodium selenite concentration is above 27200 mg / L, the growth of Bacillus subtilis strain (NKX) is significantly inhibited, and the fermentation broth does not turn red significantly. The highest tolerance concentration of this bacterium to sodium selenite is 25600 g / mL, and the suitable concentration is 1600 - 8000 mg / L.

[0032] Experimental Example 5 Optimization of Fermentation Conditions for Microbial Nano Selenium (1) Prepare nano selenium fermentation medium, and the formula is: 10 g / L molasses, 5 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 0.3 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate.

[0033] (2) Experimental group: The experimental group is repeated three times. Dispense the nano selenium fermentation medium into 1000 mL fermentation flasks, 468 mL per flask. Sterilize at 121 °C under high pressure for 30 min. After cooling, add 5 mL of the corresponding NKX seed solution and 7.01 g of sterilized sodium selenite to make the total volume of the fermentation broth 500 mL and the concentration of Se +4 be 6400 mg / L. Add 5 mL of molasses to the fermentation broth of the experimental group on the 3rd, 5th, and 7th days of fermentation. Add 5 mL of the NKX seed solution to the fermentation broth 6 days after fermentation. Incubate with shaking at 37 °C and 150 rpm for 9 days.

[0034] (3) Control group: The control group had three replicates. The nano-selenium culture medium was aliquoted into 1000 mL fermentation flasks, with 488 mL of the fermentation medium. After cooling, 5 mL of the corresponding strain seed solution was added, and 7.01 g of sterilized sodium selenite was added. Incubate with shaking at 37 °C and 150 rpm for 9 days.

[0035] (4) Detection of nano-selenium fermentation results: The nano-selenium content was detected by the method for detecting amorphous red elemental selenium in NYSL-1010-2024, and the sodium selenite content was detected by the method for determining sodium selenite content in HG-2937-1999. The results of the determination of the sodium selenite content in the fermentation broth of each group are shown in Table 2. It can be seen that after 9 days of cultivation, the content of Se +4 in the experimental group was significantly lower than that in the control group. The conversion rate of Se +4 in the experimental group reached over 96%, and the content of the converted amorphous elemental selenium reached over 6023 mg / L. This method can be used to produce a nano-selenium fermentation broth with a concentration of 6000 mg / L.

[0036] Table 2 Nano-selenium fermentation results

[0037] Experimental Example 6 Determination of the growth-promoting performance of strains (1) The yellow catfish used in the experiment were purchased from the aquaculture base of Sichuan Mianxing Agricultural Development Co., Ltd. Before the experiment, the yellow catfish were temporarily raised with the yellow catfish feed produced by Zhejiang Haida Feed Co., Ltd. for 2 weeks to adapt to the environment. Before the experiment, they were fasted for 24 h. Select yellow catfish with uniform size and good condition, and place them in 4 culture barrels respectively, divided into four groups (CK, Group 1, Group 2, Group 3). CK (fed with Haida yellow catfish feed), Group 1 (fed with Haida yellow catfish feed + 5 mL / kg SICC 1.950 seed solution 1×10 9 cfu / g), Group 2 (fed with Haida yellow catfish feed + 5 mL / kg SICC 1.1136 seed solution 1×10 9 cfu / g), Group 3 (fed with Haida yellow catfish feed + 5 mL / kg NKX seed solution 1×10 9 cfu / g). There are 30 fish in each barrel, and the barrel capacity is 300 L of water. Feed twice a day (8:00, 17:30), and keep the feeding amount of each barrel consistent, based on the satiety level of one of the groups. If fish die, the feed needs to be converted according to the remaining number of fish tails for feeding. The culture water is well water with sufficient aeration. The experiment was carried out for 8 weeks, with continuous oxygenation for 24 hours. The water temperature was 25 ± 1 °C, the nitrite concentration was lower than 0.05 mg / L, the ammonia nitrogen concentration was lower than 0.5 mg / L, and pH = 7.0 - 8.0.

[0038] (2)After 8 weeks of breeding, the fish were fasted for 24 hours, anesthetized, and then weighed and various growth indexes of the fish were calculated. The results are shown in Table 3. Compared with the control group CK, groups 1, 2, and 3 could effectively improve the weight gain rate, specific growth rate, and feed coefficient of Pelteobagrus fulvidraco, increase the condition factor, and improve the survival rate of Pelteobagrus fulvidraco. Among them, group 3 fed with NKX was significantly better than CK, group 1, and group 2.

[0039] Three Pelteobagrus fulvidraco were selected from each group to detect the colony numbers of Bacillus, Lactobacillus, Escherichia coli, and Bifidobacterium. The statistical results are shown in Table 4. Group 3 was significantly better than the control group, group 1, and group 2 in reducing the colony number of the harmful bacterium Escherichia coli and increasing the colony numbers of the beneficial bacteria Lactobacillus, Bifidobacterium, and Bacillus. The above results indicate that adding the NKX strain of the present invention to the feed can better promote the growth of intestinal beneficial bacteria, inhibit the growth of harmful bacteria, and regulate the balance of the intestinal flora.

[0040] Meanwhile, the NKX strain can be formulated into a growth-promoting bacterium agent or a feed additive by adjusting the bacterial liquid concentration of the NKX to a concentration of 1×10 9 cfu / g. The addition amount in the feed is 5 mL / kg feed, which can significantly promote the growth performance of farmed animals, increase the weight of the animals, contribute to promoting economic benefits, and has potential application value in the preparation of feed and the aquaculture industry.

[0041] Table 3 Effects of NKX on the growth of Pelteobagrus fulvidraco

[0042] Table 4 Effects of NKX on the intestinal flora of Pelteobagrus fulvidraco

[0043] In summary, the present application provides a Bacillus subtilis NKX with high tolerance to selenite. This strain can tolerate selenite at a maximum concentration of 25,600 mg / L and convert selenite into nano-selenium at a concentration exceeding 6,000 mg / L, with a conversion rate of selenite of over 96%. After verification, the NKX strain can regulate the intestine, can be prepared into a growth-promoting bacterium agent or a feed additive, helps to increase the colony number of intestinal beneficial bacteria of Pelteobagrus fulvidraco, reduce the colony number of harmful bacteria, regulate the intestine, is beneficial to the digestion and absorption of feed by Pelteobagrus fulvidraco, improve the feed coefficient of Pelteobagrus fulvidraco, promote the weight gain rate of Pelteobagrus fulvidraco, and increase the condition factor. The strain provided by the present application provides a strain resource for high tolerance to selenite, and at the same time has a significant effect on the high conversion efficiency of nano-selenium in the preparation of nano-selenium.

[0044] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be construed as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and alternatives to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A high selenite-tolerant Bacillus subtilis ( Bacillus sp.) NKX, characterized in that The strain was sent to the China Center for Type Culture Collection for preservation on January 2, 2025. The preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2025008.

2. Use of the Bacillus subtilis NKX as claimed in claim 1 in the preparation of nano-selenium.

3. Use of the Bacillus subtilis NKX as claimed in claim 1 in the preparation of growth-promoting bacteria or feed additives.

4. Use of the Bacillus subtilis NKX as claimed in claim 1 in the field of aquaculture.

5. The use according to claim 4, characterized in that: The application includes promoting the growth of farmed animals and increasing the weight gain rate.

6. A growth-promoting bacterial agent or feed additive comprising the Bacillus subtilis NKX according to claim 1.

7. The growth-promoting bacteria agent or feed additive according to claim 6, characterized in that: The growth-promoting bacteria agent or feed additive is a bacterial suspension of Bacillus subtilis NKX.

8. The growth-promoting bacteria agent or feed additive according to claim 7, characterized in that: The concentration of the Bacillus subtilis NKX suspension is 1×10 9 cfu / g.

9. Use of the growth-promoting bacteria agent or feed additive as described in any one of claims 6 to 8 in the field of breeding.

10. The use according to claim 9, characterized in that: The application includes promoting the growth of farmed animals and increasing the weight gain rate.

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