CN116064279B - Siamese bacillus and application thereof in low-salt fermentation of food - Google Patents

Siamese bacillus and application thereof in low-salt fermentation of food Download PDF

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CN116064279B
CN116064279B CN202210966683.0A CN202210966683A CN116064279B CN 116064279 B CN116064279 B CN 116064279B CN 202210966683 A CN202210966683 A CN 202210966683A CN 116064279 B CN116064279 B CN 116064279B
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rxl01
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siamese bacillus
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CN116064279A (en
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张素芳
鲍捷
梁悦琪
林心萍
董亮
陈映羲
梁会朋
纪超凡
代艺伟
李冬梅
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Dalian Polytechnic University
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Abstract

The invention discloses a Siamese bacillus strain and application thereof in low-salt fermentation of foods, and belongs to the technical field of foods. The invention provides a Siamese bacillus DL-RXL01, the preservation number is as follows: CGMCC No.23532. The Siamese bacillus DL-RXL01 has high growth speed, can inhibit pathogenic bacteria from growing, has excellent fermentation performance, is resistant to gastrointestinal tract environment, has excellent probiotics performance, and has the enzyme activity of 820U/mL when Leu-pNA (L-leucine-4-nitroaniline) is taken as a substrate; the enzyme activity was 230U/mL using Cbz-Glu-Tyr (N-benzyloxycarbonyl-L-glutamyl-L-tyrosine) as substrate. The strain can be used as a starter to ensure the edible safety and flavor quality of low-salt fermented soybean paste, bean paste and other foods, remarkably improve the nutritive value of the fermented foods, and can also be used for preparing feeds to improve the nutritive substances.

Description

Siamese bacillus and application thereof in low-salt fermentation of food
Technical Field
The invention relates to a Siamese bacillus strain and application thereof in low-salt fermentation of foods, and belongs to the technical field of foods biology.
Background
Fermented foods such as thick broad-bean sauce, fermented soybean sauce and the like have unique color, fragrance, taste and shape and are deeply favored by consumers. Fermented foods such as soybean paste and soybean paste are mostly naturally fermented, and sun-cured night dew is obtained, and the sodium chloride addition amount in the production of the fermented foods is usually up to 18-20% in order to ensure edible safety. Higher sodium chloride content is likely to cause hypertension, cardiovascular diseases and the like of consumers, and is unfavorable for health. With increasing attention to health along with the promotion of sugar and salt reduction and food safety, low-salt fermented foods are favored by more and more consumers.
The bacillus has important influence on the safety, flavor and nutritional quality of the low-salt fermented bean product. Researches show that the bacillus can inhibit the growth of harmful bacteria in the low-salt fermented bean products through lipopeptid, antibacterial peptide and bacteriocin, so that the microbial safety of the low-salt fermented bean products is ensured; the bacillus can metabolize carbohydrate, protein and fat to produce flavor substances such as acids, esters, peptides and the like, so that the sensory and flavor quality of the low-salt fermented bean product is improved. In recent years, research and use of spore bacteria useful in low-salt fermented bean products have been attracting attention.
Chinese patent CN111248409A discloses a low-salt thick broad-bean sauce and a preparation method thereof, which are characterized in that staphylococcus botulinum, bacillus subtilis, and mixed starter of Weissella and Lussella combined yeast are added in the fermentation stage of thick broad-bean sauce mash to prepare the low-salt thick broad-bean sauce; chinese patent CN108967904a uses aspergillus oryzae, pediococcus halophilus, mucor elegans, parsley, dill, oregano beans for fermentation; fermenting the capsicum by utilizing Pediococcus halophilus and Pediococcus halophilus; mixing fermented soybean paste and fermented soy sauce with fermented chilli sauce, and performing post fermentation on pichia pastoris and leuconostoc mesenteroides to prepare the low-salt soybean paste. The current low-salt fermentation is a fermentation strategy of a compound microbial inoculum for preparing low-salt thick broad-bean sauce by multi-strain cooperation and functional substance mixed fermentation, and the population advantage is preferentially achieved by multi-strain large-dose inoculation, so that excessive propagation of harmful bacteria is prevented, but the threat of pollution of the harmful bacteria is not completely eliminated.
The use of Siamese bacillus to prepare fermented food in various documents and patents is one of future probiotics with development potential, such as strengthening Daqu by adding Siamese bacillus and candida utilis in patent CN112725114A, and preparing the strong aromatic white spirit rich in ethyl acetate; in patent 107734972a, fermented milk is prepared by inoculating bacillus siamensis and lactic acid bacteria; fermenting with Siamese bacillus in patent CN112795519A to prepare acetoin-rich vinegar; it is indicated in the literature that bacillus siamensis is one of the main bacterial groups in the fermentation process of korean soybean paste (Jeong D W, kim H R, jung G, et al Bacterial Community Migration in the Ripening of Doenjang, a Traditional Korean Fermented Soybean Food [ J ]. Journal of Microbiology & Biotechnology, 2014, 24 (5)).
Disclosure of Invention
Aiming at the problems, the invention provides a probiotic Siamese bacillus capable of producing antibacterial substances and application thereof in low-salt fermentation of foods.
The invention is realized by the following technical scheme:
the invention provides a Siamese bacillus strain DL-RXL01 separated from self-fermented foods, and the preservation number is CGMCC No.23532. The Siamese bacillus DL-RXL01 is collected from farm soybean paste in Liaoning province and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in 10 month 08 of 2021. The preservation address is the microbiological institute of China academy of sciences of national institute of sciences No. 3, north Chen West Lu No. 1, chaoyang district of Beijing, china.
In one embodiment, the nucleotide sequence of 16s rRNA of the Siamese bacillus strain DL-RXL01 is shown in SEQ ID No. 1.
The present invention provides a composition comprising a strain of bacillus siamensis DL-RXL01, a fermentation broth and/or a fermentation supernatant.
In one embodiment, the strain DL-RXL01 of the Siamese bacillus is inoculated into a culture medium for fermentation culture to obtain a bacterial liquid of the fermentation liquid.
In one embodiment, the fermentation supernatant refers to a supernatant obtained by centrifuging and collecting a fermentation liquid.
The invention provides a microbial inoculum, which contains a Siamese bacillus strain DL-RXL01.
The invention also provides a starter which contains the bacillus siamensis strain DL-RXL01.
In one embodiment, the strain of Siamese bacillus DL-RXL01 in the microbial inoculum and the fermentation agent is in a live bacterial form.
In one embodiment, the microbial inoculum and the fermentation agent may further contain a lyoprotectant.
In one embodiment, the microbial agent or the fermenting agent is a liquid microbial agent, a fermenting agent or a solid microbial agent, a fermenting agent.
The invention provides a preparation method of the microbial inoculum or the fermentation agent, which comprises the steps of culturing a Siamese bacillus strain DL-RXL01 to obtain a microbial inoculum: selecting a single colony of Siamese bacillus DL-RXL01, inoculating the single colony to 10 mL of LB culture medium, and carrying out aerobic culture at 30-37 ℃ and 200-250 rpm for 24-h to obtain bacterial liquid A; then taking 500 mu L of the bacterial liquid A, inoculating the bacterial liquid A into 50mL of LB culture medium, and carrying out aerobic culture for 24 h at the temperature of 30-37 ℃ and the rpm of 200-250 rpm to obtain bacterial liquid B; then 10 mL of the bacterial liquid B is inoculated into 1000mL of LB culture medium, and is subjected to aerobic culture for 24 h at 30-37 ℃ and 200-250 rpm to obtain bacterial liquid C; placing the bacterial liquid C in 8000 rpm for centrifugation for 10min, collecting bacterial cells, diluting the bacillus siamensis DL-RXL01 bacterial cells with 0.9% sodium chloride aqueous solution by mass fraction to prepare 10 10 -10 12 CFU/mL of bacterial suspension.
In one embodiment, the culturing is performed at 37 ℃. The culture medium used for the culture includes, but is not limited to, a medium commonly used for spore bacteria such as LB culture and nutrient broth culture.
The bacterial liquid obtained can be directly prepared into liquid bacterial agent or fermenting agent by adding auxiliary materials allowed in the field of microbial preparations, or can be prepared into dry bacterial agent or fermenting agent by vacuum freeze drying after the bacterial cells in the bacterial liquid are mixed with auxiliary materials allowed in the field of microbial preparations such as freeze-drying protective agent and the like.
Based on the functions and characteristics of the Siamese bacillus strain DL-RXL01, the invention provides application of the Siamese bacillus strain DL-RXL01 or the microbial inoculum or the fermentation agent in preparing foods.
In one embodiment, the food product comprises a fermented food product.
In one embodiment, the fermented food product is preferably a fermented bean product; more preferably a low salt fermented soy product; most preferred are low salt fermented soybean paste, soybean paste and soy sauce.
The invention provides an application of a Siamese bacillus strain DL-RXL01 or a microbial inoculum or a fermentation agent in improving amino acid nitrogen content in food.
In one embodiment, the food product comprises a fermented food product.
In one embodiment, the fermented food product is preferably a fermented bean product; more preferably a low salt fermented soy product; most preferred are low salt fermented soybean paste, soybean paste and soy sauce.
The invention provides application of a Siamese bacillus strain DL-RXL01 or a microbial agent or a fermentation agent in preparation of a product for inhibiting pathogenic microorganisms.
In one embodiment, the pathogenic microorganism comprises staphylococcus aureus @Staphylococcus aureus) Pathogenic coliform bacteriaEscherichia coli) Listeria monocytogenesListeria monocytogenes) Enterococcus faecalisEnterococcus faecalis) Salmonella (Salmonella)Salmonella entericasubsp enterica serovar Typhi) shigellaShigella flexneri) Pseudomonas aeruginosaPseudomonas aeruginosa）。
In one embodiment, the product includes, but is not limited to, a feed additive, a biological bacteriostat, a probiotic formulation, or a medicament.
In one embodiment, the product has an addition of 10% of the strain of Bacillus siamensis DL-RXL01 9 -10 12 CFU/kg or 10 9 -10 12 CFU/L。
In a real worldIn an embodiment, the addition amount of the Siamese bacillus strain DL-RXL01 in the product is 10 12 CFU/kg。
The invention also provides a method for preparing the low-salt fermented food, which is to add the Siamese bacillus strain DL-RXL01 or the microbial inoculum or the starter in the food fermentation process.
In one embodiment, the addition amount of the strain of Siamese bacillus DL-RXL01 is 10 9 -10 12 CFU/kg or 10 9 -10 12 CFU/L。
The beneficial effects are that:
the invention provides a strain DL-RXL01 of bacillus siamensis, which is separated from traditional natural fermentation northeast soybean paste, and has high growth speed, and the culture OD of 12 h can reach 6.4. The strain has obvious inhibition effect on pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like, has excellent aminopeptidase and carboxypeptidase activities, and can better ensure the flavor quality of fermentation products. Moreover, the Siamese bacillus strain DL-RXL01 can resist the gastrointestinal tract environment and has excellent probiotics. The strain can be used as a starter to ensure the edible safety and flavor quality of low-salt fermented foods, shorten the fermentation period and remarkably improve the nutritional value of low-salt fermented bean products.
Preservation of biological materials
The invention provides the Siamese bacillusBacillus siamensis) DL-RXL01, taxonomically named Siamese bacillusBacillus siamensisThe microbial strain is preserved in China general microbiological culture collection center (CGMCC) at 10 and 08 in 2021, with the preservation number of CGMCC No.23532 and the preservation address of North Chenxi Lu No. 1 and 3 in the Chaoyang area of Beijing city, china academy of sciences of microorganisms.
Drawings
FIG. 1 is a photograph of LB culture of Siamese bacillus DL-RXL 01;
FIG. 2 shows detection of antibacterial effect of Siamese bacillus DL-RXL 01; (A) The inhibition effect of Siamese bacillus DL-RXL01 on staphylococcus aureus and the inhibition effect of Siamese bacillus DL-RXL01 on 7 pathogenic microorganisms.
FIG. 3 is a photograph showing the protease activity screening of Siamese bacillus DL-RXL01.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art; reagents and materials used in the following examples are commercially available unless otherwise specified.
The various culture mediums used in the invention are prepared by adopting a conventional method, and the molecular biological operation such as the specific test conditions and methods are not noted in the examples, and refer to SambrookJ et al, scientific Press, 2002 and molecular cloning experiment guidelines (third edition); or with reference to the product specifications.
The preparation of the culture medium used in the invention is as follows:
(1) Common nutrient broth solid medium: 5g of peptone, 3g of beef extract, 5g of sodium chloride, 15g of agar, 1000mL of distilled water, adjusting the pH to 7.0, autoclaving for 20min and pouring into a flat plate.
(2) LB liquid medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 1L of distilled water, adjusting the pH to 7.0 and sterilizing for 20min.
(3) LB solid medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar, distilled water to 1L, pH to 7.0, autoclaving for 20min and pouring into a plate.
(4) Protease activity screening medium: and preparing a casein culture medium into a solution A and a solution B respectively.
And (3) solution A: weighing Na 2 HPO 4 •7 H 2 O1.07 g and casein 5g, adding a proper amount of distilled water, and heating to dissolve. And (2) liquid B: weighing KH 2 PO 4 0.36 g, adding water for dissolution. A. After mixing the solution B, agar 20g is added, and finally distilled water is used for fixing the volume to 1L. Sterilizing at 121deg.C for 15 min, and pouring into plate or making oxford cup plate.
(5) Amylase activity screening medium: 2.5g of soluble starch, 5g of peptone, 2.5g of ammonium sulfate, 3g of monopotassium phosphate, 0.25g of calcium chloride hexahydrate, 20g of agar and 1000ml of water. Sterilizing at 121deg.C for 15 min, and pouring into plate or making oxford cup plate.
Example one strain isolation and identification:
1. isolation and purification of Strain DL-RXL01
(1) Sample: the soybean paste is collected from peasant family of Liaoning province.
(2) The separation and screening method adopts an enrichment-screening culture method, and is concretely as follows:
taking finished raw soybean paste 1 g, adding into 10 mL LB liquid medium, transferring into a homogenizing bag, beating for 30 min, centrifuging for 5 min to remove sauce residue, transferring the turbid liquid part containing bacteria into a 50mL centrifuge tube under aseptic condition, supplementing LB medium to 10 mL, and culturing at 37deg.C and 200 rpm for 3 h. The culture solution is properly diluted and then coated on protease activity screening culture medium, and after the liquid is absorbed completely, the culture solution is inversely cultured at 37 ℃ for 24-48 h. The strain with obvious transparent circle is selected, streaked to LB solid medium, and single colony is separated. And (3) continuously purifying for three generations, picking single bacterial colony, performing microscopic examination, and performing oxford cup protease activity analysis after confirming that the bacterial colony is not polluted, wherein the bacterial strain is found to have better protease activity, and is stored in a frozen state and named as DL-RXL01.DL-RXL01 strain was first liquid-cultured with 10 mL of LB for 24 h, then inoculated in an inoculum size of 1% (v/v) into a 250mL Erlenmeyer flask containing 50mL of LB medium, and further shake-cultured at 200-250 rpm and 37℃for 48 h. The culture supernatant was then collected after centrifugation at 8000 rpm for 10min and subjected to the following analysis (FIG. 3).
(3) Analysis of amylase activity. The culture supernatant of the strain DL-RXL01 as initially screened in the above (2) was subjected to amylase activity analysis by using a starch oxford cup plate. Incubation at 37℃for 24 h revealed that the transparent circles appeared around the oxford cup of the DL-RXL01 supernatant, indicating that the DL-RXL01 strain had amylase activity.
(4) And (5) antibacterial activity analysis. Is prepared from staphylococcus aureusStaphylococcus aureusATCC 25923), pathogenic E.coliEscherichia coliATCC 25922), listeria monocytogenesListeria monocytogenesATCC 19115 enterococcus faecalisEnterococcus faecalisATCC 29212, salmonellaSalmonella entericaSubsp enterica serovar Typhi CMCC (B) 50071) Shigella ]Shigella flexneriCMCC (B) 51572) Pseudomonas aeruginosaPseudomonas aeruginosa ATCC 27853) and the like as indicator bacteria, and analyzing the bacteriostatic activity of the culture supernatant of the DL-RXL01 strain by the oxford cup method (oxford cup method reference: khan M N, lin H, li M, et al Identification and growth optimization of a Marine Bacillus DK1-SA11 having potential of producing broad spectrum antimicrobial compositions Pakistan Journal of Pharmaceutical Sciences, 2017, 30 (3): 839-853.). Incubation at 37℃for 24 h showed transparent circles around oxford cups, and the diameter of the inhibition circles was measured and analyzed in summary. The 7-strain indicator bacteria of the antibacterial substance produced by the DL-GBS01 strain have good antibacterial activity, especially staphylococcus aureus and salmonella, and the diameter of a inhibition zone can reach 21.3 mm and 22.4 mm. The antibacterial substance produced by DL-RXL01 strain has better antibacterial activity on harmful bacteria (figure 2).
2. Identification of Strain DL-RXL 01:
(1) The strain DL-RXL01 grows well on LB solid plates, and is cultured at 37 ℃ for 24-48 h, as shown in figure 1, to form round, slightly flat, neat-edged and milky colonies; microscopic examination shows that the bacterial cells are rod-shaped, have a length greater than a width and a size of 0.5-1 multiplied by 1.5-4 mu m.
(2) The thallus sample is directly processed by boiling method to obtain PCR template, which is specifically as follows: inoculating purified strain DL-RXL01 on LB solid medium, culturing at 28deg.C for 24H, picking single colony with sterilized toothpick, and placing in 16S-free H containing 10 μl 2 In Microtube of O, after heat denaturation at 99℃for 10 minutes, centrifugation was performed, and 5. Mu.L of the supernatant was used as a template for PCR reaction.
PCR reaction 50. Mu.L: 2 XPCR Mix 25. Mu.L, primer Forward Primer 1. Mu.L, primer Reverse Primer 1. Mu.L, template supernatant 5. Mu.L, ddH 2 O was filled to 50. Mu.L. Primer sequence Forward Primer: ggttacttgttacgactt; reverse Primer: agagttgatcctggcttag. PCR procedure: 94 ℃ for 5 min;94 ℃ for 1 min,55 ℃ for 1 min,72 ℃ for 1.5 min,30 cycles; 10 at 72 DEG CAnd (5) min. After the PCR is finished, the PCR product is directly sent to Shanghai to be sequenced, and the sequencing result shows that the PCR product has the nucleotide sequence of SEQ ID No. 1 in a sequence table.
The sequence of the strain DL-RXL01 16s rRNA is as follows:
CCTCTGTCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACACAGAGCTTTACGATCCGAAAACTTCATCACTCACGCGCGTGCTCCGTCAGACTTTCGTCATTGCGAGATTCCTACTGCTGCTTCCGTAGAGTCTGGCCGTGTTCTCAGTCCAGTGTGACGGATCCACCCCCTCTCTCAAGT
sequencing results show that the nucleotide sequence with SEQ ID No. 1 in the sequence table is subjected to Blastn analysis, and the strain is found to be compared with Siamese bacillusBacillus siamensis) The highest homology of (3) was 99.6%.
Morphological and 16s rRNA identification shows that the strain DL-RXL01 isBacillus siamensis. And deposited in China general microbiological culture Collection center, deposit number: CGMCC No.23532. Preservation address: the institute of microbiology, national institute of sciences, 1, 3, north chen west way, north, south, face, chinese, postal code: 100101.
example Siamese bacillus DL-RXL01 biological Activity assay and evaluation of Probiotics
1. Aminopeptidase and carboxypeptidase activity of Siamese bacillus DL-RXL01 were determined:
taking out a storage glycerol pipe of a Siamese bacillus DL-RXL01 strain from a refrigerator at the temperature of minus 80 ℃, taking a loop of fungus-containing ice residues into an LB culture medium containing 10 mL, and carrying out shaking culture at the temperature of 37 ℃ at 200-250 rpm for 24 h; then, the cells were transferred to a 250mL flask containing 50mL of LB medium at an inoculum size of 1% (V/V), and the culture was continued at 200-250 rpm and 37℃with shaking for 48 h. Then, centrifugation was carried out at 8000 rpm for 10min, and the culture supernatant was collected for aminopeptidase and carboxypeptidase activity analysis.
Reference (Chinese brewing, 2017,36 (2): 99-101; chinese brewing, 2010, 216 (3): 30-33) the aminopeptidase and carboxypeptidase activities of bacillus siamensis DL-RXL01 were measured, and the results show that the enzyme activity of the supernatant of a culture solution of bacillus siamensis DL-RXL01 cultured at 48 h under the conditions of 37 ℃ and 200 rpm at the shake flask level was 820U/mL by taking Leu-pNA (L-leucine-4-nitroaniline) as a substrate; the enzyme activity was 230U/mL using Cbz-Glu-Tyr (N-benzyloxycarbonyl-L-glutamyl-L-tyrosine) as substrate. The Siamese bacillus DL-RXL01 has aminopeptidase and carboxypeptidase activities.
2. Acid resistance detection of strain of Siamese bacillus DL-RXL 01:
the number of viable bacteria is 3×10 8 CFU/mL of Siamese bacillus DL-RXL01 bacterial suspension is inoculated into LB culture medium with pH of 3.0 according to the proportion of 5% (V/V), incubated at 37 ℃, and 0 h and 2 h samples are taken for plate counting to calculate the survival rate of the strain. The result shows that after 2 h is cultivated in LB culture medium with pH of 3.0, the number of viable bacteria of the strain of the Siamese bacillus DL-RXL01 is changed from 10 8 CFU/mL was reduced to 10 7 CFU/mL. The Siamese bacillus DL-RXL01 has good tolerance to acidic environment.
3. Bile salt tolerance test of Siamese bacillus DL-RXL 01:
in the acid resistance test (pH 3.0), the Siamese bacillus DL-RXL01 passes through the stomach to reach the intestinal tract after 2 h, and the viable count is reduced to 3×10 7 CFU/mL. Therefore, the initial viable count of the bile salt tolerance test was adjusted to3×10 7 CFU/mL. Centrifugally collecting the activated strain DL-RXL01 of Siamese bacillus, washing the LB culture medium for 2 times, re-suspending in LB culture medium containing 0.3% (W/V) sodium taurocholate, and regulating the concentration of the active bacteria to 3×10 7 CFU/mL, incubation at 37℃was performed, and 0 h and 3 h samples were taken for colony counting to calculate strain survival. The result shows that the viable count of the Siamese bacillus strain DL-RXL01 is reduced in the 0.3% bile salt environment, but the viable count is still 10 after 3 h 6 CFU/mL or more, is higher than the critical value of the bacterial number of the functional characteristic of the living bacteria. The Siamese bacillus DL-RXL01 has good tolerance to small intestine environment.
4. Antioxidant experiment of Siamese bacillus DL-RXL01
(1) DPPH radical scavenging test: 0.2 mmol/L DPPH:0.0078 The g DPPH is dissolved by absolute ethyl alcohol, the volume is fixed to 100 mL, and the mixture is placed in a dark place and is prepared for use. Will 10 8 The Siamese bacillus DL-RXL01 bacterial solution of CFU/mL is mixed with the DPPH solution of 0.2 mM according to the volume ratio of 1:1, and is cultured for 30 min in the dark at 25 ℃. Singly use 10 8 CFU/mL of Siamese bacillus DL-RXL01 stock solution and ethanol served as blanks, and PBS and 0.2 mmol/L DPPH served as controls. The supernatant was collected after centrifugation at 2330 Xg (4120 rpm) for 10 minutes. Absorbance was measured in triplicate at 517 nm.
Calculation of DPPH radical clearance:
ai is the absorbance value of 1 mL DPPH ethanol solution added to the bacterial liquid to be detected of 1 mL;
aj is the absorbance value of the bacterial liquid to be detected of 1 mL and 1 mL absolute ethyl alcohol;
ac was absorbance of 1 mL PBS plus 1 mL DPPH ethanol solution.
(2) ABTS radical scavenging test: ABTS (14 mM) and potassium persulfate (5 mM) were dissolved in 0.1: 0.1M potassium phosphate buffer (pH 7.4), mixed in a ratio of 1:1, and reacted at 25 ℃ for 12-16 h to obtain ABTS working solution. mu.L of Siamese bacillus DL-RXL01 (10) 8 CFU/mL) to 900mu.L of ABTS working fluid and incubated in the dark at 25℃for 15 min. After centrifugation (14000 g,1 min), the absorbance of the supernatant was measured at 734 nm.
Calculation of ABTS radical clearance:
as is the absorbance value of 100 mu L of bacteria liquid to be detected and 900 mu L of ABTS working solution;
ac is 1 mL of ABTS working fluid;
the results show that the DPPH clearance activity of the Siamese bacillus DL-RXL01 is 90.12 percent, and the ABTS clearance activity is 92.89 percent, which shows that the strain DL-GBS01 has good antioxidant activity.
Example three Siamese bacillus DL-RXL01 safety verification
1. Antibiotic susceptibility test of Siamese bacillus DL-RXL 01:
the antibiotic susceptibility spectrum of the Siamese bacillus DL-RXL01 was characterized using a paper sheet diffusion method. Preparing activated bacteria into 1×10 8 The CFU/mL bacterial suspension is coated on an LB plate by 200 mu L of bacterial liquid, and 11 antibiotic drug sensitive paper sheets of gentamicin (10 mu g), streptomycin (10 mu g), erythromycin (15 mu g), tetracycline (30 mu g), cefalexin (30 mu g), vancomycin (30 mu g), cefazolin (30 mu g), ampicillin (10 mu g), penicillin (10 mu g), minocycline (30 mu g) and amikacin (30 mu g) are carefully placed, wherein the interval of each paper sheet is not less than 24 mm. After 24 h culture at 37 ℃, the diameter of the inhibition zone is measured and counted, and the drug resistance R (less than or equal to 14 mm), the medium-term I (14-20 mm) or the sensitive S (more than or equal to 20 mm) is analyzed and three parallels are made. The result shows that the Siamese bacillus DL-RXL01 is sensitive to 11 antibiotics, the antibiotics resistance does not exist, and the strain safety is good.
2. Hemolysis experiment of Siamese bacillus DL-RXL01
Medium configuration: columbia agar, 5% defibrinated sheep blood.
Siamese bacillus DL-RXL0 is taken1 bacterium (about 1X 10) 8 cfu/mL) was streaked onto a columbia plate containing 5% defibrinated sheep blood, incubated at 37 ℃ for 24 h, after which the presence of a transparent collar was observed. Staphylococcus aureus ATCC 25923 was used as a positive control.
As a result, it was found that a wide (6-8 mm), well-defined, completely transparent hemolytic ring appeared around the strain of Staphylococcus aureus ATCC 25923 positive control group, which was typical of beta hemolysis. The surrounding of the Siamese bacillus DL-RXL01 colony provided by the invention has no hemolysis, and the strain safety is good.
Example preparation of a Bacillus Siamese DL-RXL01 bacterial solution:
1) Sub-packaging of LB culture medium: sterilized LB medium was aseptically dispensed into 50mL centrifuge tubes (10 mL liquid loading), 250mL Erlenmeyer flasks (50 mL liquid loading) and 5L Erlenmeyer flasks (1000 mL liquid loading).
2) Taking out a storage glycerol pipe of a Siamese bacillus DL-RXL01 strain from a refrigerator at the temperature of minus 80 ℃, taking a loop of fungus-containing ice residues into an LB culture medium containing 10 mL, and carrying out shaking culture at the temperature of 37 ℃ at 200-250 rpm for 24 h; then transferring the strain into a 250mL triangular flask containing 50mL of LB culture medium at an inoculum size of 1% (v/v), and continuously culturing at 200-250 rpm and 37 ℃ in a shaking way for 24 h; finally, the cells were inoculated in an amount of 1% (v/v) into 3L Erlenmeyer flasks containing 750 mL of LB medium, and cultured at 200-250 rpm and 37℃with shaking for 24 h. Placing the bacterial liquid C in 8000 rpm for centrifugation for 10min, collecting bacterial cells, diluting the bacillus siamensis bacterial cells with 0.9% sodium chloride aqueous solution by mass fraction to prepare 10 9 CFU/mL of bacterial suspension. And (5) standby.
Example five Siamese bacillus DL-RXL01 starter powder preparation:
inoculating Siamese bacillus DL-RXL01 into LB culture medium, culturing at 37deg.C for 16-h, centrifuging, mixing thallus with freeze-drying protective agent (2 g glycerol and 3g sucrose are added into 10% skimmed milk per 100-mL mass concentration), pre-freezing at-20deg.C for 2 h, vacuum drying in vacuum freeze-drying machine (cold trap temperature-45deg.C, vacuum degree 10-20 Pa) for 22 h-24 h, and stopping drying when the water content of the bacterial powder is reduced to 2.5% -3%, to obtain bacterial agent in dry powder form, wherein the viable bacteria content of Siamese bacillus in bacterial agent is 10 12 CFU/g。
Example application of bacillus siamensis DL-RXL 01:
(1) Application of DL-RXL01 strain in soy sauce fermentation
According to SB/T10312-1999 high-salt dilute state fermentation soy sauce brewing process, soy sauce fermentation is carried out, raw material soybean meal and bran=7:3 are mixed, water wetting treatment, cooking treatment, cooling treatment and inoculation treatment are carried out, shanghai brewing 3.042 aspergillus oryzae is inoculated, and then yeast making 24 and h are carried out according to 10 9 Inoculating a strain of Siamese bacillus DL-RXL01 into the CFU/mL (g) ratio, preparing starter at a constant temperature of 30 ℃ for 42 h, loosening, weighing, adding salt and water (final 14% salinity), fermenting at a constant temperature of 30 ℃ for 6 months, squeezing, sterilizing, filtering, subpackaging and other procedures to obtain finished soy sauce, and respectively measuring amino acid nitrogen content and detecting escherichia coli in a control group and an experimental group.
According to the ammonia nitrogen content test method, referring to GB 18186-2000, escherichia coli detection is performed by a Real-time PCR method.
(2) Application of DL-RXL01 strain in fermentation of Pi county thick broad-bean sauce
According to the Pi county bean paste standard of GB/T20560-2006 geographical mark product, performing Pi county bean paste fermentation, and controlling salt 3-5% and water 15-25% when bean paste yeast is filled into a fermentation container at the sweet valve stage according to the ratio of 10 8 Inoculating a strain of bacillus siamensis DL-RXL01 in a CFU/mL ratio, and fermenting for 30 days; and after the chilli embryo and the sweet valve are mixed, the salinity is controlled to be 12 percent, and the salinity is controlled to be 10 percent 8 The Siamese bacillus DL-RXL01 strain is inoculated again in the ratio of CFU/mL, and the subsequent fermentation is continued. The fermentation was ended after the experimental and control groups had an amino acid nitrogen (in nitrogen)/(g/100 g) of greater than 0.18. And (5) measuring the amino acid nitrogen content of the finished product and detecting the escherichia coli.
According to the ammonia nitrogen content testing method, according to GB/T20560-2006, escherichia coli detection is performed by a Real-time PCR method.
(3) Application of DL-RXL01 strain in soybean paste fermentation
And (5) fermenting the soybean paste according to GB/T24399-2009 soybean paste. The process flow comprises the following steps: soaking soybean, steaming, cooling, draining, mashing, inoculating Aspergillus oryzae, and Huniang 3.042 →Preparing sauce blocks, preparing yeast, forming yeast, cleaning the sauce blocks, cutting into small blocks, adding salt with the final concentration of 10%, whipping, skimming froth and mixing according to the proportion of 10 10 Inoculating Siamese bacillus DL-RXL01 strain in the ratio of CFU/mL, fermenting, packaging, sterilizing and obtaining the finished product. And after the fermentation is finished, measuring the amino acid nitrogen content of the finished product and detecting the escherichia coli.
According to the ammonia nitrogen content testing method, the GB/T24399-2009 is referred, and the escherichia coli detection is performed by a Real-time PCR method.
The results show that the low-salt fermentation sample inoculated with the Siamese bacillus DL-RXL01 and the control sample without the Siamese bacillus DL-RXL01 are free from colibacillus pollution in the fermentation process. Meanwhile, the amino acid nitrogen content of the low-salt fermentation sample inoculated with the Siamese bacillus DL-RXL01 in the fermentation process is far higher than that of the fermentation sample (control group) not inoculated with the Siamese bacillus DL-RXL01 in the fermentation process. The Siamese bacillus DL-RXL01 is more beneficial to the exertion of the enzyme activity in a fermentation system under the low-salt fermentation condition when being used as a starter, and the fermentation is more thorough (Table 1).
TABLE 1 analysis of amino acid nitrogen content in each fermented food
Note that: each fermented food group 1 is an experimental group added with Siamese bacillus DL-RXL01, and group 2 is a control group.
In conclusion, the Siamese bacillus DL-RXL01 has obvious inhibition effect on pathogenic bacteria such as pathogenic escherichia coli and staphylococcus aureus, has excellent amylase, protease, aminopeptidase and carboxypeptidase activities, and can better ensure the flavor quality of low-salt fermentation products. Moreover, the Siamese bacillus strain DL-RXL01 can resist the gastrointestinal tract environment and has excellent probiotics. The strain can be used as a starter to ensure the edible safety and flavor quality of low-salt fermented foods, shorten the fermentation period and remarkably improve the nutritional value of low-salt fermented bean products. Meanwhile, the strain can also be used as a good feed additive or used for preparing feed so as to improve the nutritional value in the feed, promote the growth of livestock and shorten the growth period.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

1. Siamese bacillusBacillus siamensis) DL-RXL01 is preserved in China general microbiological culture Collection center (CGMCC) at 10 and 08 days of 2021, with the preservation number of CGMCC No.23532 and the preservation address of China academy of microorganisms at national academy of sciences No. 3 of North Chenxi Lu 1 in the Chaoyang area of Beijing city.
2. A microbial inoculum comprising the Bacillus siamensis DL-RXL01 according to claim 1.
3. The use of the siamese bacillus DL-RXL01 of claim 1 or the microbial inoculum of claim 2 in the preparation of fermented bean products.
4. Use of the siamese bacillus DL-RXL01 of claim 1 or the microbial inoculum of claim 2 for increasing the amino acid nitrogen content of fermented bean products.
5. The use according to claim 4, wherein the added amount of the Siamese bacillus DL-RXL01 is 10 9 -10 12 CFU/kg or 10 9 -10 12 CFU/L。
6. The use of the siamese bacillus DL-RXL01 as claimed in claim 1 or the microbial inoculum as claimed in claim 2 in the preparation of a medicament for inhibiting pathogenic microorganisms, wherein the pathogenic microorganisms are staphylococcus aureus @Staphylococcus aureus) Coli @Escherichia coli) Listeria monocytogenesListeria monocytogenes) Enterococcus faecalisEnterococcus faecalis) Salmonella enterica subspecies enterica typhoid serotype @Salmonella entericasubsp enterica serovar Typhi), shigella flexneriShigella flexneri ) Pseudomonas aeruginosaPseudomonas aeruginosa）。
7. The use according to claim 6, wherein the added amount of the strain Siamese bacillus DL-RXL01 is 10 9 -10 12 CFU/kg or 10 9 -10 12 CFU/L。
8. A method for preparing a low-salt fermented bean product, characterized in that the bacillus siamensis DL-RXL01 according to claim 1 or the microbial inoculum according to claim 2 is added during fermentation.
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CN108208316A (en) * 2018-01-24 2018-06-29 吉林省农业科学院 Feed containing Siam bacillus
CN111662849A (en) * 2020-07-03 2020-09-15 山东第一医科大学(山东省医学科学院) Bacillus siamensis and application thereof
CN111690578A (en) * 2020-07-31 2020-09-22 山东佐田氏生物科技有限公司 Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof
CN111718881A (en) * 2020-08-13 2020-09-29 石河子大学 Siamese bacillus and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108208316A (en) * 2018-01-24 2018-06-29 吉林省农业科学院 Feed containing Siam bacillus
CN111662849A (en) * 2020-07-03 2020-09-15 山东第一医科大学(山东省医学科学院) Bacillus siamensis and application thereof
CN111690578A (en) * 2020-07-31 2020-09-22 山东佐田氏生物科技有限公司 Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof
CN111718881A (en) * 2020-08-13 2020-09-29 石河子大学 Siamese bacillus and application thereof

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