CN115895930A - 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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- AIHDCSAXVMAMJH-GFBKWZILSA-N levan Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 title claims abstract description 18
- 241000193830 Bacillus <bacterium> Species 0.000 title abstract description 27
- 229920002670 Fructan Polymers 0.000 claims abstract description 31
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- 238000004321 preservation Methods 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 19
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Abstract
The invention discloses a salt-tolerant bacillus for producing levan and application of the 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 fructan under the conditions mentioned in the invention, the yield is 50-60 g/L, and the salt-tolerant bacillus SCU-E has wide application prospects in the aspects of food medicine, biological materials, industrial emulsifiers and the like.
Description
Technical Field
The invention relates to a microbial strain technology, in particular to fructan-producing halophilic bacillus and application of fructan produced by the halophilic bacillus.
Background
Fructan is an important linear homopolysaccharide, and is polymerized from beta-D-fructofuranose. Fructans mainly include inulin-type fructans and Levan-type fructans. Inulin-type fructans are mainly composed of beta- (2, 1) fructoside linkages. Inulin type fructosan can enhance immunity and increase bone density, and can be used as substitute for fat, sugar and functional food. Levan-type fructans consist of a large number of beta- (2, 6) fructosyl linkages and a small number of beta- (2, 1) fructosyl linkage branches. The Levan type fructan has wide application, can be used as a food additive in food, and is also an ideal soluble dietary fiber prebiotic. Medically, the antioxidation of Levan fructan has the function of preventing atherosclerosis linked to oxidative stress; it also has fibrinolytic activity and is used to make lytic agents. In addition, levan fructan has important application value in the fields of anti-obesity, anti-inflammation, immunoregulation and anti-tumor.
The application of the bacillus in fermentation products is mature, and the bacillus is an internationally recognized food safety level safe 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 which is commonly used at present for producing various industrial enzymes.
Disclosure of Invention
Aiming at the prior art, the invention provides a strain of Bacillus halodurans sp.SCU-E115 capable of producing levan, a method for producing levan by using the strain and application of the levan.
Different salt-tolerant strains are obtained from the saline-alkali soil of Gansu province of China by a standard dilution coating flat plate method. The strain is morphologically identified, the strain is preliminarily determined to be Bacillus (Bacillus sp.), and is named as salt-tolerant Bacillus SCU-E115 (Bacillus sp. SCU-E115), the salt-tolerant Bacillus is preserved in China general microbiological culture Collection center (No. 3 of Xilu No. 1 of Xingyang district, beijing City) in 7-9 th of 2021, and the strain preservation number is: CGMCC No.22860.
The invention extracts DNA, after 16S rDNA gene amplification and sequencing, uploads the sequence to EzTaxon of an EzBioCloud server for online BLAST comparison analysis, and a phylogenetic tree is constructed by using an adjacency method (Neighbor-Joining) and a Maximum Likelihood method (Maximum-Likeliod) in a MEGA7.0 program.
The invention also provides a culture method of the bacillus, which comprises the following steps:
inoculating the bacillus SCU-E115 into a fermentation culture medium, culturing at 35-37 ℃ and pH7.0 +/-0.3, and performing shaking culture on a shaking table.
Another purpose of the embodiments of the present invention is to provide a method for producing levan by fermentation of the above-mentioned Bacillus, comprising the following steps:
inoculating the bacillus SCU-E115 into a fermentation culture medium, and carrying out shake culture for 2 days at the temperature of 35-37 ℃ and under the environment of pH7.0 +/-0.3. After the fermentation is finished, adding physiological saline to dilute the fermentation liquid, centrifuging to remove thalli, and removing protein in the centrifugate through a series of operations. Then adding ethanol into the levan solution for precipitation, centrifuging to collect white precipitate, dissolving the precipitate with water, and freeze-drying with a freeze dryer to obtain levan.
The fermentation medium comprises the following components: 3-5 g/L of peptone, 2-3 g/L of beef extract, 30-100 g/L of 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。
A toxicological research experiment is carried out on the levan generated by the bacillus SCU-E115, the levan is harmless to mice, reaches the edible safety level, and is expected to be developed into health care medicines and food additives.
The research on the emulsifying activity of the fructan produced by the bacillus SCU-E115 shows that the fructan 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 a strain of Bacillus SCU-E115 constructed based on the NJ method;
FIG. 3 is an infrared spectrum of fructan produced by Bacillus SCU-E115;
FIG. 4 shows the morphology of fructan produced by Bacillus SCU-E115 under scanning electron microscope.
Detailed Description
The present invention will be described in more detail with reference to the following examples and the accompanying drawings.
The first embodiment is as follows: screening and separation of salt-tolerant Bacillus sp.SCU-E115
1. And (3) screening strains: taking 1g saline-alkali soil of Gansu province, adding sterilized normal saline to dissolve, and diluting to 10% 5 And (4) doubling. The screening culture medium is adopted, and the strains are screened step by using 1%, 4%, 7% and 10% NaCl. Spreading 0.1mL of soil diluent on screening culture medium plate, and culturing at 30 deg.CCulturing for 2-3 days, selecting bacterial colony with good growth at 7% salt concentration, selecting single bacterial colony, diluting, coating and culturing again, and carrying out passage purification to obtain pure culture.
Screening culture medium, weighing 7.5g casein peptone, 10g yeast powder, 100g NaCl and 1.5mL LMS mixed solution [ compatible substance (betaine, proline, glycine, D-sorbitol, glutamate) mixed solution with concentration of 0.05g/L ], adding 1000mL distilled water, adjusting pH to 8.0, adding 20g agar powder, stirring, heating for dissolving, packaging, and sterilizing with high pressure steam at 121 deg.C for 20min.
2. Culturing and identifying strains: and (3) selecting a single colony of the pure culture obtained in the step (1) to be inoculated into a seed culture medium, and placing the seed culture medium in a shaking table at the temperature of 30 ℃ for 2 days. The form of the cells was directly observed by an electron microscope (FIG. 1). And then taking the bacterial liquid on a glass slide for a gram staining experiment operation, wherein gram staining is positive, and the bacteria are rod-shaped.
3. Pure thallus DNA is extracted, a 16S rDNA gene sequence is amplified by utilizing PCR, the DNA sequence is sent to a sequencing company for sequencing, the sequence is uploaded to EzTaxon of an EzBioCloud server for online BLAST comparison analysis, and the sequence similarity of the thallus DNA and the sequence of the Bacillus licheniformis (B.licheniformis) is the highest (98.4%). The phylogenetic tree was constructed using the Neighbor-Joining method (Neighbor-Joining) and the Maximum Likelihood method (Maximum-likehood) in the MEGA7.0 program (FIG. 2).
The second embodiment: growth characteristics of Bacillus halodurans Bacillus sp.SCU-E115
1. LB liquid media (pH = 7.0) were prepared with NaCl concentrations of 0%, 4%, 6%, 7%, 10%, 12%, 15%, and 17, respectively, and their NaCl concentration tolerance ranges and optimal growth concentrations were determined.
2. The buffer system (pH 4.0-5.0 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) was prepared as a liquid medium of LB +7% (w/v) NaCl having pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and the pH tolerance range and optimum growth pH were analyzed after the inoculation culture.
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 at 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 48h, taking out, and determining OD 600 A value; simultaneously, after streaking and inoculating the solid plate, culturing the solid plate at 4 ℃ and 10 ℃ for two weeks, and observing the growth condition of the solid plate; 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).
Example three: production of extracellular fructan by bacillus halodurans SCU-E115
The method for producing the extracellular fructan by the salt-tolerant bacillus SCU-E115 comprises the following steps:
1. seed liquid culture: the purified strain was inoculated in a seed medium and cultured with shaking on a shaker at 37 ℃ for 1 day.
2. Fermentation culture: inoculating the seed solution into a fermentation culture medium, and performing shake culture at 37 deg.C for 2 days.
3. And (3) extraction of extracellular polysaccharide: diluting the obtained fermentation liquor with physiological saline, centrifuging at 6000rpm for 20min to remove bacteria, and adding the fermentation liquor into the centrifugate according to the proportion of 1:1 volume ratio, sevage reagent is added to remove residual protein in the fermentation liquor, then ethanol is used for precipitating polysaccharide liquid, the centrifugation is carried out at 6000rpm for 10min, and the precipitate is collected. Precipitating the obtained polysaccharide, and freeze-drying the solution to obtain the extracellular polysaccharide produced by the bacillus SCU-E115 with the yield of 50-60 g/L.
Seed liquid culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH7.0.
Fermentation medium: 3-5 g/L of peptone, 2-3 g/L of beef extract, 30-100 g/L of 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。
Example four: identification of polysaccharide structures
1. The functional groups of the polysaccharide were analyzed by potassium bromide tabletting method and the polysaccharide infrared spectrum was measured by placing in a Fourier Infrared spectrometer (FIG. 3). Exopolysaccharide produced by bacillus SCU-E115 is Levan type fructan by mapping analysis.
2. Then a small amount of extracellular polysaccharide is placed on the treated carrier net, and an observation mounting is made, and the polysaccharide form is directly observed under an electron microscope (figure 4).
Example five: toxicology of fructan
1. Healthy male and female ICR mice were weighed, labeled, and 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 toxicity symptoms and behavioral changes were closely monitored over 6h of feeding and continued for 14 days. Individual body weights were recorded on days 1, 7 and 14 thereafter. Finally, mice inhaled CO 2 Anaesthetize and perform gross pathological examination of the major organs.
2. No toxicity-related clinical symptoms were found during the 14 day experiment after oral administration of fructan for male and female mice. At the end of the experiment, there was no significant change in the body weight of the mice. Necropsy showed no significant changes in vital organs such as heart, kidney and spleen. 10000mg/kg body weight is taken orally once, and the compound preparation has no lethal or toxic effect on mice and reaches the safe edible level. Therefore, the fructan 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: experiment on emulsifying Activity of Fructosan
1. Adding 3mL sunflower seed oil or 3mL mineral oil and 2mL fructan solution with concentration of 2mg/mL into glass tube, maintaining at 25 deg.C for 1, 24, 48 and 72h respectively to form upper emulsion layer and lower water layer, and checking emulsification index at certain time interval. Therefore, the sunflower seed oil emulsion prepared from the fructan has better stability after being stored for 24 hours, and the mineral oil emulsion has lower stability. An effective emulsifier should be able to maintain at least 50% of the original emulsion volume within 24 hours after formation. It is speculated that the fructan may be used in different industrial fields in the future as an emulsifier.
The above embodiments are only used to illustrate the technical solutions of the present invention, and it is obvious to those skilled in the art that modifications can be made to the technical solutions of the foregoing embodiments, and these modifications are also included in the protection scope of the claims of the present invention.
Sequence listing
<110> Sichuan university
<120> bacillus halodurans and application of fructan produced by bacillus halodurans
<141> 2021-08-26
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1460
<212> DNA
<213> Bacillus halodurans 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 (3)
1. A strain of fructan-producing salt-tolerant Bacillus sp.SCU-E115 has the strain preservation number: CGMCC No.22860, the salt-tolerant Bacillus sp.SCU-E115 is characterized by moderate salt tolerance, positive gram staining and rod shape, and the length is 1.5-4.5 um; the growth range of NaCl concentration is 0-12%, the temperature range capable of growth is 20-50 ℃, and the pH range capable of growth is 4-10.
2. The method for producing levan by fermentation of Bacillus sp.SCU-E115 according to claim 1,inoculating salt-tolerant Bacillus sp.SCU-E115 into a fermentation culture medium, carrying out shake culture in a shaking table with the temperature of 35-37 ℃ and the pH value of 7 +/-0.3 for 2 days, removing thalli and protein in fermentation liquor, concentrating to obtain a fructan solution, adding ethanol to precipitate fructan, and freeze-drying to obtain solid fructan, wherein the molecular weight of the fructan is 10 6 ～10 8 Da。
3. The method of claim 2, wherein the fermentation medium comprises: 3 to 5g/L of peptone, 2 to 3g/L of beef extract, 30 to 100g/L of 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。
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