CN112574900B - Paenibacillus and method for producing levan by using same - Google Patents

Paenibacillus and method for producing levan by using same Download PDF

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CN112574900B
CN112574900B CN201910922636.4A CN201910922636A CN112574900B CN 112574900 B CN112574900 B CN 112574900B CN 201910922636 A CN201910922636 A CN 201910922636A CN 112574900 B CN112574900 B CN 112574900B
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levan
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张建法
程龙
程瑞
王世明
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Hebei Feng Chuan Biotechnology Co ltd
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Nanjing University of Science and Technology
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Abstract

The invention discloses a paenibacillus strain and a method for producing levan by the paenibacillus strain. The preservation number of the paenibacillus FP01 is CCTCC NO: m2019349. The paenibacillus FP01 can ferment and produce extracellular fructan, the yield can reach 92g/L, the sucrose conversion rate can reach 50 percent, and the conversion rate is close to the highest theoretical conversion value. The weight average molecular weight of the fructan produced is about 1X 10 6 Da, the molecular weight is relatively uniform. The fermentation medium adopted by the invention has definite components, simple production process and high fermentation yield, and is suitable for industrial large-scale production.

Description

Paenibacillus and method for producing levan by using same
Technical Field
The invention belongs to the technical field of levan zymophyte, and relates to a paenibacillus strain and a method for producing levan by using the same.
Background
The levan is a linear isopolysaccharide formed by connecting beta-D-fructofuranose. Fructans in nature are mainly divided into two groups according to the type of linkage: inulin-type fructan (inulin-type) is connected by beta- (2,1) bonds to form a main chain, and is most abundant in medicinal plants, particularly in compositae and platycodon grandiflorum; secondly, levan-type fructan (levan-type), also called levan, is connected by beta- (2,6) bonds to form a main chain, some contain a small amount of beta- (2,1) branched chains, mainly come from microorganisms, and is synthesized by the catalytic action of fructan synthetase produced by the microorganisms. Generally, plant-derived fructans have a low Degree of Polymerization (DP) (2-60) and bacteria-derived fructans have a high molecular weight (DP)>10 3 )(M.Jimenez-Sanchez,R.Perez-Morales,F.M.Goycoolea,et al,Self-assembled high molecular weight inulin nanoparticles:Enzymatic synthesis,physicochemical and biological properties,Carbohydrate polymers 215(2019)160-169.)。
The fructan belongs to non-starch polysaccharide, can not be digested and absorbed by human bodies or animals, can be decomposed and utilized by beneficial flora in colon such as lactobacillus and bifidobacterium after entering the colon, generates substances such as short chain fatty acid beneficial to organisms, and the like, promotes intestinal health, and is a well-known prebiotic. In the food industry, fructans have achieved GRAS certification by the FDA in the united states. In addition, the high molecular fructan has wide application in the fields of medicines, cosmetics, nano materials and the like due to the special rheological property and excellent moisturizing performance. Compared with plant levan, the microbial levan has more abundant and various structures, controllable microbial fermentation production, small environmental factor, high yield and is more suitable for industrial preparation. The levan-producing microorganisms identified to date are a wide variety of microorganisms including bacteria, yeasts and fungi. Chinese patent 200510106534.3 discloses a strain of Cellulomonasp. nov.GJT07 for producing levan, which has a yield of 15g/L of levan after fermentation optimization. Chinese patent application 201710981158.5 discloses a strain of Bacillus amyloliquefaciens B.amyloliquefaciens 66 with high levan yield, the yield of levan produced by fermentation can reach 100g/L, 400g/L of sucrose is required as a carbon source, and the conversion rate is only about 25%. As a substrate for producing levan by microbial fermentation, a sucrose molecule is composed of 1 molecule of glucose and 1 molecule of fructose. Theoretically, if all of the fructose in the sucrose molecule is used to synthesize levan, the conversion rate of levan synthesis can reach 50%. Therefore, further exploring a fructan-producing strain with high sucrose conversion rate has practical significance for the industrial production of fructan.
Disclosure of Invention
The invention aims to provide a paenibacillus strain with high sucrose conversion rate and high levan yield and a method for producing levan by using the paenibacillus strain.
The inventor selects a strain for producing extracellular fructan by fermentation from fermentation wastewater of a food factory, and the strain is identified as Paenibacillus sp by molecular biology and named as Paenibacillus FP 01. The strain is preserved in the China center for type culture collection in 2019, 5 months and 10 days, the preservation address is eight-path Loojia mountain in Wuchang district, Wuhan City, Hubei province, and the preservation number is CCTCC NO: m2019349.
The invention also provides a culture method of the paenibacillus FP01, which comprises the following steps: inoculating paenibacillus FP01 into a fermentation culture medium, culturing at the temperature of 30 +/-2 ℃ and the pH value of 6.8-7.2, and performing shake culture.
The invention further provides a method for producing extracellular fructan by fermenting the paenibacillus FP01, which comprises the following steps:
inoculating paenibacillus FP01 into a fermentation medium, carrying out shake culture at the fermentation temperature of 30 +/-2 ℃ and the pH value of 6.8-7.2, adding physiological saline to dilute the fermentation liquid after the fermentation is finished, centrifuging to remove thalli, removing protein in the supernatant by a Sevage reagent, evaporating and concentrating to obtain a fructan solution, adding ethanol into the fructan solution for precipitation, centrifuging to collect the precipitate, dissolving the precipitate with water, and freeze-drying to obtain Levan-type fructan.
Preferably, the culture time is 48-72 h.
Preferably, the process conditions for removing the bacteria by centrifugation are as follows: the rotating speed is 8000-10000 rpm, and the centrifugation time is 15-20 min.
Preferably, the process conditions for centrifugally collecting the precipitate are as follows: the rotating speed is 3000-6000 rpm, and the centrifugation time is 5-10 min.
The fermentation medium comprises the following components: 20-180 g/L of sucrose and NaH 2 PO 4 1 g/L，CaCl 2 0.07 g/L，MgCl 2 0.2 g/L，FeSO 4 0.0125 g/L，K 2 NO 3 3 g/L，MnSO 4 0.003 g/L，ZnCl 2 0.0075 g/L，pH 6.8～7.2。
Compared with the prior art, the invention has the advantages that:
the paenibacillus FP01 can ferment and produce extracellular fructan, the yield can reach 92g/L, the sucrose conversion rate can reach 50 percent, and the conversion rate is close to the highest theoretical conversion value. The weight average molecular weight of the fructan produced is about 1X 10 6 Da, the molecular weight is relatively uniform. The fermentation medium adopted by the invention has definite components, simple production process and high fermentation yield, and is suitable for industrial large-scale production.
Drawings
Table 1 shows the synthetic conversion of fructan at different substrate concentrations;
FIG. 1 is a gram-stained micrograph of Paenibacillus FP 01;
FIG. 2 shows fructan 1 H nuclear magnetic resonance spectroscopy;
FIG. 3 shows fructan 13 C nuclear magnetic resonance spectrum;
FIG. 4 is an infrared spectrum of levan;
FIG. 5 shows the GPC chromatogram of fructan.
Detailed Description
The present invention will be described in more detail with reference to the following examples and the accompanying drawings.
Example 1
1. Separating and screening strains: adding fermented wastewater of food factory in Nanjing into sterilized normal saline, and mixing by vortex oscillation. And (3) standing for 1h, taking the supernatant, performing gradient dilution, coating the supernatant on a screening plate, and culturing in an incubator at 30 ℃ for 3-7 days. And selecting single colonies, diluting, coating and culturing again, and carrying out subculture purification to obtain a pure culture.
Screening a culture medium: sucrose 20g, peptone 2g, NaNO 3 3g，KH 2 PO 4 1g，CaCl 2 0.07g，MgCl 2 0.2g，FeSO 4 0.01g, agar 15g, H 2 O 1000ml，pH7.0。
2. Culturing and identifying strains: inoculating the obtained single colony of the pure culture into a seed culture medium, placing the seed culture medium in a shaking table at 30 ℃, and performing shaking culture for 3 days. A small amount of the bacterial liquid is taken to be arranged on a glass slide for gram staining, and is observed under a microscope (figure 1), and the bacterial body is in a long rod shape, has the length of 2-8 mu m and is gram positive bacteria. The thalli is collected by centrifugation, DNA is extracted, a 16S rDNA gene sequence (SEQ ID NO.1) is amplified by utilizing PCR and sent to a sequencing company for sequencing, and the similarity with the sequence of Paenibacillus polymyxa SC2 is up to more than 99 percent through Blast comparison, the bacillus is bacillus like and is named as bacillus like FP 01.
3. Method for producing extracellular fructan using FP01 strain:
(1) seed culture: inoculating the strain into sterilized seed culture medium, and culturing in 30 deg.C shaking table at 220rpm for 24 hr.
(2) Fermentation culture: inoculating the seed culture medium obtained in the step (1) into a sterilized fermentation culture medium according to the inoculation amount of 1% (v/v), and culturing for 48-96h in a shaking table at the temperature of 30 ℃, wherein the rotation speed of the shaking table is 220 rpm.
(3) And (3) extracting extracellular polysaccharide: diluting the fermentation liquor obtained in the step (2) with physiological saline, centrifuging at 6000rpm for 10min to remove thalli, and then performing centrifugation according to the ratio of 1: sevage reagent was added at a volume ratio of 1 to remove residual protein from the fermentation broth. Concentrating the diluted fermentation broth to below 1/2, precipitating with 3 times of 95% ethanol, centrifuging at 6000rpm for 5min, collecting white precipitate, and washing with ethanol. Dissolving the obtained polysaccharide precipitate with distilled water, and lyophilizing to obtain white substance as extracellular polysaccharide.
Seed culture medium: sucrose 20g, NaH 2 PO 4 1g，CaCl 2 0.07g，MgCl 2 0.2g，FeSO 4 0.0125g，K 2 NO 3 3g，MnSO 4 0.003g，ZnCl 2 0.0075g，H 2 O 1000ml，pH7.0。
Fermentation medium: sucrose 100g, NaH 2 PO 4 1g，CaCl 2 0.07g，MgCl 2 0.2g，FeSO 4 0.0125g，K 2 NO 3 3g，MnSO 4 0.003g，ZnCl 2 0.0075g，H 2 O 1000ml，pH7.0。
Fermentation conditions are as follows: the liquid loading was 50ml of medium per 250ml conical flask, the inoculum size was 1% (v/v), the initial pH was 7.0, 30 ℃, 220rpm for 48 h.
4. And (3) identification of polysaccharide structures:
(1) and (3) identification of polysaccharide structures: the lyophilized polysaccharide was weighed 50mg and used 500ul of 99% D 2 Dissolving O, taking trimethylsilane as an internal standard, placing the internal standard in a nuclear magnetic tube, and measuring by an Avance III 500MHz nuclear magnetic resonance spectrometer 1 H-NMR spectra (FIG. 1) and 13 C-NMR spectrum (FIG. 2). An IR spectrum of 50mg of dried polysaccharide was measured by Fourier IR spectroscopy (FIG. 3). The exopolysaccharide obtained by spectrogram analysis is Levan-type fructan.
(2) Identification of polysaccharide molecular weight: by gel chromatography (GP)C) The molecular weight of the polysaccharide was determined. The sample was injected through a 0.22 μ M filter into a gel column at 0.1M NaNO 3 As a mobile phase, polysaccharide GPC spectrum was obtained by detection with a differential refractometer (FIG. 4). The molecular weight of the polysaccharide was determined by GPC, and found to be 1X 10 6 Da。
TABLE 1 fructan production by FP01 fermentation at different sucrose concentrations
Figure BDA0002218009790000041
Table 1 shows the fructan production by fermentation of FP01 strain at different sucrose concentrations. Under the conditions of low-concentration and high-concentration sucrose fermentation, the conversion rate of sucrose can reach 50%.
Sequence listing
<110> Nanjing university of science and technology
<120> Paenibacillus and method for producing levan by using the same
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1429
<212> DNA
<213> Paenibacillus
<400> 1
tatactgcag tcgagcgggg ttaggttaga agcttgcttc taactaacct agcggcggac 60
gggtgagtaa cacgtaggca acctgcccac aagacaggga taactaccgg aaacggtagc 120
taatacccga tacatccttt tcctgcatgg gagaaggagg aaaggcggag caatctgtca 180
cttgtggatg ggcctgcggc gcattagcta gttggtgggg taaaggccta ccaaggcgac 240
gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtag ggaatcttcc gcaatgggcg aaagcctgac ggagcaacgc 360
cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gccagggaag aacgcttggt 420
agagtaactg ctcttgaagt gacggtacct gagaagaaag ccccggctaa ctacgtgcca 480
gcagccgcgg taatacgtag ggggcaagcg ttgtccggaa ttattgggcg taaagcgcgc 540
gcaggcggct ctttaagtct ggtgtttaat cccgaggctc aacttcgggt cgcactggaa 600
actggggagc ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660
agagatgtgg aggaacacca gtggcgaagg cgactctctg ggctgtaact gacgctgagg 720
cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780
aatgctaggt gttaggggtt tcgataccct tggtgccgaa gttaacacat taagcattcc 840
gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacggggac ccgcacaagc 900
agtggagtat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc 960
tctgaccggt ctagagatag acctttcctt cgggacagag gagacaggtg gtgcatggtt 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttatgc 1080
ttagttgcca gcaggtcaag ctgggcactc taagcagact gccggtgaca aaccggagga 1140
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtactacaa 1200
tggccggtac aacgggaagc gaagccgcga ggtggagcca atcctagaaa agccggtctc 1260
agttcggatt gtaggctgca actcgcctac atgaagtcgg aattgctagt aatcgcggat 1320
cagcatgccg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca caccacgaga 1380
gtttacaaca cccgaagtcg gtgaggtaac cgcaaggagc cagccgtcc 1429

Claims (7)

1. Paenibacillus sp. ((II))Paenibacillus sp.) FP01 with the preservation number of CCTCC NO: m2019349.
2. The culture method of paenibacillus FP01 according to claim 1, which is characterized by comprising the following steps: inoculating paenibacillus FP01 into a fermentation culture medium, culturing at the temperature of 30 +/-2 ℃ and the pH value of 6.8-7.2, and performing shake culture.
3. The method for producing extracellular fructan by fermentation of paenibacillus FP01 according to claim 1, which comprises the following steps:
inoculating paenibacillus FP01 into a fermentation medium, carrying out shake culture at the fermentation temperature of 30 +/-2 ℃ and the pH value of 6.8-7.2, adding physiological saline to dilute the fermentation liquid after the fermentation is finished, centrifuging to remove thalli, removing protein in the supernatant by a Sevage reagent, evaporating and concentrating to obtain a fructan solution, adding ethanol into the fructan solution for precipitation, centrifuging to collect the precipitate, dissolving the precipitate with water, and freeze-drying to obtain Levan-type fructan.
4. The method according to claim 3, wherein the cultivation time is 48 to 72 hours.
5. The method according to claim 3, wherein the process conditions for removing the bacterial cells by centrifugation are as follows: the rotating speed is 8000-10000 rpm, and the centrifugation time is 15-20 min.
6. The method according to claim 3, wherein the process conditions for centrifuging and collecting the precipitate are as follows: the rotating speed is 3000-6000 rpm, and the centrifugation time is 5-10 min.
7. The method of claim 2 or 3, wherein the fermentation medium consists of: 20-180 g/L of sucrose and NaH 2 PO 4 1 g/L，CaCl 2 0.07 g/L，MgCl 2 0.2 g/L，FeSO 4 0.0125 g/L，K 2 NO 3 3 g/L，MnSO 4 0.003 g/L，ZnCl 2 0.0075 g/L，pH 6.8~7.2。
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CN102533605A (en) * 2012-01-16 2012-07-04 江南大学 Strain capable of producing levansucrase and method for producing levan by using levansucrase
CN108570431A (en) * 2018-05-08 2018-09-25 云南博仕奥生物技术有限公司 One plant of gel-shaped series bacillus and its application

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103740618B (en) * 2013-12-31 2015-08-05 光明乳业股份有限公司 One Bacillus species novel bacterial and cultural method thereof and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533605A (en) * 2012-01-16 2012-07-04 江南大学 Strain capable of producing levansucrase and method for producing levan by using levansucrase
CN108570431A (en) * 2018-05-08 2018-09-25 云南博仕奥生物技术有限公司 One plant of gel-shaped series bacillus and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Characterization of the levan produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity;Xiaofen Xu 等;《Carbohydrate Polymers》;20181231;第178-186页，参见全文 *
多粘类芽孢杆菌PS04胞外多糖分子结构研究;蹇华丽 等;《食品工业科技》;20140831;第114-121页，参见全文 *

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