CN115895930B - Salt-tolerant bacillus and application of levan produced by same - Google Patents
Salt-tolerant bacillus and application of levan produced by same Download PDFInfo
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- 241000193830 Bacillus <bacterium> Species 0.000 title abstract description 29
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 claims abstract description 8
- 239000003995 emulsifying agent Substances 0.000 abstract description 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a salt-tolerant bacillus for producing levan and application of levan produced by the salt-tolerant bacillus. The preservation number of the salt-tolerant Bacillus sp.SCU-E115 is CGMCC No.22860. The salt-tolerant bacillus SCU-E115 can ferment and produce extracellular levan under the conditions mentioned in the invention, the yield is 50-60 g/L, and the salt-tolerant bacillus SCU-E has wide application prospect in the aspects of food medicine, biological materials, industrial emulsifying agents and the like.
Description
Technical Field
The invention relates to a microbial strain technology, in particular to salt-tolerant bacillus for producing levan and application of levan produced by the salt-tolerant bacillus.
Background
Levan is an important linear homopolysaccharide and is polymerized by beta-D-fructofuranose. Levan mainly comprises inulin type Levan and Levan type Levan. Inulin-type levan is mainly composed of beta- (2, 1) fructosyl bonds. Inulin-type levan can enhance immunity and bone density, and can be used as substitute for fat, sugar and functional food. Levan consists of a large number of beta- (2, 6) fructosyl bonds and a small number of beta- (2, 1) fructosyl bonds branches. Levan type Levan has wide application, can be used as a food additive in food, and is also an ideal soluble dietary fiber prebiotic. Medically, levan has antioxidant properties against oxidative stress-linked atherosclerosis; it also has fibrinolytic activity and is used to make a lytic agent. In addition, levan has important application value in the fields of anti-obesity, anti-inflammation, immunoregulation and anti-tumor.
Bacillus is already very mature in its application in fermentation products and is an internationally accepted food-safe safety strain. The bacillus has a series of characteristics of strong stress resistance, high growth speed, simple culture, strong protein secretion capacity and the like, and is a microorganism for producing various industrial enzymes commonly used at present.
Disclosure of Invention
Aiming at the prior art, the invention provides a salt-tolerant Bacillus sp.SCU-E115 capable of producing levan, a method for producing levan and application of levan.
Different salt-tolerant strains are obtained from saline-alkali soil in Gansu province of China by a standard dilution coating plate method. The strain was morphologically identified, and the strain was preliminarily determined to be Bacillus sp, and designated as salt-tolerant Bacillus SCU-E115 (Bacillus sp. SCU-E115), and the salt-tolerant Bacillus was deposited at the general microbiological center of the chinese microbiological bacterial culture management committee at 7 months 9 of 2021 (No. 3 of the north chen west way 1, the region of facing yang, beijing) with a bacterial deposit number of: cgmccno.22860.
The invention uses Neighbor-Joining and Maximum Likelihood (Maximum-Likelihood) methods in MEGA7.0 program to construct phylogenetic tree by extracting DNA, amplifying and sequencing 16S rDNA gene, uploading the sequence to EzTaxon of EzBioCloud server for online BLAST comparison analysis.
The invention also provides a method for culturing the bacillus, which comprises the following specific steps:
inoculating the bacillus SCU-E115 into a fermentation culture medium, and performing shake culture by a shaking table, wherein the culture temperature is 35-37 ℃ and the pH value is 7.0+/-0.3.
Another object of the embodiments of the present invention is to provide a method for producing levan by fermenting the bacillus, which comprises the following specific steps:
the bacillus SCU-E115 was inoculated into a fermentation medium and cultured with shaking at 35 to 37℃and pH 7.0.+ -. 0.3 for 2 days. After the fermentation, the fermentation broth was diluted with physiological saline, and after the cells were removed by centrifugation, proteins in the supernatant were removed by a series of operations. And then adding ethanol for precipitation into the levan solution, centrifuging to collect white precipitate, dissolving the precipitate with water, and freeze-drying by a freeze dryer to obtain levan.
The fermentation medium disclosed by the invention comprises the following components: 3-5 g/L peptone, 2-3 g/L beef extract, 30-100 g/L sucrose and NaNO 3 0.4～0.6g/L，MgSO 4 0.3～0.4g/L，K 2 HPO 4 0.2～0.3g/L，pH7.0±0.3。
The levan produced by bacillus SCU-E115 is subjected to toxicology exploration experiments, is harmless to mice, achieves the edible safety level, and is expected to be developed into health-care medicines and food additives.
The study of the emulsifying activity of levan produced by bacillus SCU-E115 shows that levan can be used as an emulsifier in different industrial fields in the future.
Drawings
FIG. 1 shows the strain morphology of bacillus SCU-E115 under a scanning electron microscope;
FIG. 2 is a phylogenetic tree of the strain constructed by the Bacillus SCU-E115 based on the NJ method;
FIG. 3 is an infrared spectrum of levan produced by Bacillus SCU-E115;
FIG. 4 shows the morphology of levan produced by Bacillus SCU-E115 under a scanning electron microscope.
Detailed Description
The invention is further described in detail below with reference to examples and figures.
Embodiment one: screening and isolation of Bacillus salis sp.SCU-E115
1. Screening strains: taking 1g of saline-alkali soil in Gansu province, adding sterilized normal saline for dissolving, and diluting to 10 5 Multiple times. The strains were screened step by step using 1%, 4%, 7%, 10% NaCl using screening medium. Spreading 0.1mL of soil dilution on a screening culture medium plate, and standing at 3Culturing for 2-3 days at 0 ℃, selecting a colony with good growth at 7% salt concentration, picking a single colony, diluting again, coating and culturing, and carrying out passage purification to obtain a pure culture.
Screening culture medium, namely weighing 7.5g of casein peptone, 10g of yeast powder, 100g of NaCl and 1.5mLMS mixed solution [ 0.05g/L compatible substance (betaine, proline, glycine, D-sorbitol, glutamate) mixed solution ], adding 1000mL of distilled water, regulating pH to 8.0, adding 20g of agar powder, stirring, heating, dissolving, packaging, and sterilizing at 121 ℃ for 20min by high-pressure steam.
2. Culturing and identifying strains: selecting single colony of the pure culture in the step 1, inoculating to a seed culture medium, and placing in a shaking table at 30 ℃ for shaking culture for 2 days. The morphology of the cells was observed directly with an electron microscope (FIG. 1). And then taking bacterial liquid on a glass slide to carry out gram staining experiment operation, wherein gram staining is positive, and the bacterial body is rod-shaped.
3. Pure bacterial DNA was extracted, the 16S rDNA gene sequence was amplified by PCR, sent to sequencing company for sequencing, and the sequence was uploaded to EzTaxon of EzBioCloud' S server for online BLAST alignment analysis, which showed the highest sequence similarity (98.4%) with Paenibacillus licheniformis (B.lichenifermis). Phylogenetic tree was constructed using the Neighbor-Joining method (Neighbor-Joining) and the Maximum Likelihood method (Maximum-Likelihood) in the MEGA7.0 program (fig. 2).
Embodiment two: bacillus salioleus sp.SCU-E115 growth characteristics
1. LB liquid media (pH=7.0) with NaCl concentrations of 0%, 4%, 6%, 7%, 10%, 12%, 15% and 17 were prepared, respectively, and the tolerance range of NaCl concentration and the optimal growth concentration were determined.
2. Buffer systems (pH 4.0-5.0:0.1M citric acid/0.1M sodium citrate;pH 6.0-8.0:0.1M KH) 2 PO 4 /0.1M NaOH；pH 9.0–10.0:0.1M NaHCO 3 /0.1M Na 2 CO 3 ；pH 11.0:0.05M Na 2 HPO 4 0.1M NaOH) to prepare liquid culture medium of LB+7% (w/v) NaCl with pH value of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and analyzing pH tolerance range and optimal growth after inoculating culturepH。
3. Preparing LB+7% (w/v) NaCl liquid culture medium and solid culture medium (pH=7.0), quantitatively inoculating fresh seed liquid into each test tube, respectively placing in 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, 45 deg.C, 52 deg.C and 60 deg.C, shake culturing at 180rpm for 48 hr, taking out and measuring OD 600 A value; simultaneously, inoculating the solid flat plate by streaking, culturing at 4 ℃ and 10 ℃ for two weeks, and observing the growth condition; and finally determining the growth temperature range and the optimal growth temperature of the experimental strain.
4. The results show that the NaCl tolerance range of the salt tolerant Bacillus sp.SCU-E115 is 0-17% (the optimal growth salt concentration is 6-8%), the growth temperature range is 10-52 ℃ (the optimal growth temperature is 30 ℃), and the pH growth range is 6.0-10.0 (the optimal growth pH is 8.0).
Embodiment III: production of extracellular levan by salt-tolerant bacillus SCU-E115
A method for producing extracellular levan by using salt-tolerant bacillus SCU-E115:
1. seed liquid culture: the purified strain was inoculated in a seed medium and cultured on a shaker at 37℃for 1 day with shaking.
2. Fermentation culture: the seed solution was inoculated into a fermentation medium and cultured with shaking at 37℃for 2 days.
3. Extraction of extracellular polysaccharide: diluting the obtained fermentation broth with normal saline, centrifuging at 6000rpm for 20min to remove thalli, and adding into the centrifugate according to the ratio of 1:1 in the fermentation broth to remove residual protein, precipitating polysaccharide solution with ethanol, centrifuging at 6000rpm for 10min, and collecting precipitate. And precipitating the obtained polysaccharide, and freeze-drying the dissolved solution to obtain extracellular polysaccharide produced by bacillus SCU-E115, wherein the yield is 50-60 g/L.
Seed liquid culture medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH7.0.
Fermentation medium: 3-5 g/L peptone, 2-3 g/L beef extract, 30-100 g/L sucrose and NaNO 3 0.4～ 0.6g/L，MgSO 4 0.3～0.4g/L，K 2 HPO 4 0.2～0.3g/L，pH7.0±0.3。
Embodiment four: identification of polysaccharide Structure
1. The functional groups of the polysaccharide were analyzed by potassium bromide tabletting and the polysaccharide infrared spectrum was measured by placing on a fourier infrared spectrometer (fig. 3). Extracellular polysaccharide produced by bacillus SCU-E115 is Levan type Levan through atlas analysis.
2. And then a small amount of extracellular polysaccharide is placed on the treated carrying net, an observation loading sheet is manufactured, and the morphology of the polysaccharide is directly observed under an electron microscope (figure 4).
Fifth embodiment: toxicology experiments on levan
1. Healthy male and female ICR mice were weighed, labeled, and then fasted for 4 hours, and were free to drink water. Each mouse was fed at a dose of 10000mg levan/Kg body weight, and any toxic symptoms and behavioral changes were closely monitored during 6 hours of feeding and continued for 14 days. Individual body weights were recorded on days 1, 7 and 14 thereafter. Finally, the mice inhale CO 2 Anesthesia, and general pathological examination of the major viscera.
2. For male and female mice, no clinical symptoms associated with poisoning were found during the 14-day experiment following oral levan. At the end of the experiment, the body weight of the mice did not change significantly. Necropsy showed no significant changes were found in the heart, kidneys and spleen, among other important organs. The single oral administration of 10000mg/kg body weight has no lethal or toxic effect on mice, and achieves the safe eating grade. Therefore, levan produced by the salt-tolerant Bacillus sp.SCU-E115 is expected to be applied to the aspects of food industry, biological materials and the like.
Example six: emulsion Activity experiment of levan
1. 3mL of sunflower seed oil or 3mL of mineral oil and 2mL of a solution of levan having a concentration of 2mg/mL were added to a glass tube, and maintained at 25℃for 1, 24, 48 and 72 hours, respectively, to form an upper emulsion layer and a lower aqueous layer, and the emulsion index was examined at regular intervals. The sunflower seed oil emulsion prepared from levan has good stability after 24 hours of storage, and the mineral oil emulsion has low stability. An effective emulsifier should be able to retain at least 50% of the original emulsion volume within 24 hours after formation. It is speculated that the levan may be used in the future as an emulsifier in different industrial fields.
The above embodiments are only for illustrating the technical solution of the present invention, and it is obvious to those skilled in the art that modifications can be made to the technical solution of the above embodiments, and these modifications are also included in the scope of the claims of the present invention.
Sequence listing
<110> university of Sichuan
<120> application of salt-tolerant bacillus and levan produced by same
<141> 2021-08-26
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1460
<212> DNA
<213> salt-tolerant Bacillus SCU-E115 (Bacillus sp. SCU-E115)
<400> 1
taccctggaa ggcgcgtgct atacatgcag tcgagcggac cgacgggagc ttgctccctt 60
aggtcagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact 120
ccgggaaacc ggggctaata ccggatgctt gattgaaccg catggttcaa tcataaaagg 180
tggcttttag ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240
ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300
agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360
tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaaac tctgttgtta 420
gggaagaaca agtaccgttc gaatagggcg gtaccttgac ggtacctaac cagaaagcca 480
cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540
ttgggcgtaa agcgcgcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600
cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660
tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720
ctgtaactga cgctgaggcg cgaaagcgtg gggagcgaac aggattagat accctggtag 780
tccacgccgt aaacgatgag tgctaagtgt tagagggttt ccgcccttta gtgctgcagc 840
aaacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg 900
acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960
accaggtctt gacatcctct gacaacccta gagatagggc ttccccttcg ggggcagagt 1020
gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080
cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg 1140
gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200
tacacacgtg ctacaatggg cagaacaaag ggcagcgaag ccgcgaggct aagccaatcc 1260
cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg 1320
ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380
cgtcacacca cgagagtttg taacacccga agtcggtgag gtaacctttg gagccagccg 1440
ccgaagtgac agatgtagta 1460
Claims (1)
1. Bacillus sp.SCU-E115 strain for producing levan with the accession number: CGMCC No.22860.
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