CN111909872B

CN111909872B - Paenibacillus ZX1905, extracellular polysaccharide Lubcan produced by same and application of extracellular polysaccharide Lubcan

Info

Publication number: CN111909872B
Application number: CN202010766268.1A
Authority: CN
Inventors: 张建法; 孙夏青
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing Nangyuan Biotechnology Co ltd
Priority date: 2020-08-03
Filing date: 2020-08-03
Publication date: 2022-05-20
Anticipated expiration: 2040-08-03
Also published as: CN111909872A

Abstract

The invention discloses a bacillus like ZX1905, an extracellular polysaccharide Lubcan produced by the same and application thereof. The collection number of the paenibacillus ZX1905 is CCTCC NO: M2020320. The structure of the extracellular polysaccharide Lubcan is as follows:

the paenibacillus ZX1905 does not secrete pigment in the growth process, the culture operation of the strain is simple, the components of the culture medium are clear, the cost is low, and the invention is suitable for industrial production. The extracellular polysaccharide Lubcan produced by the paenibacillus ZX1905 has good moisturizing and moisture absorption performances, has a moisturizing effect equivalent to that of hyaluronic acid, can replace hyaluronic acid, and is applied to the fields of biological medicine materials, cosmetics and the like as a moisturizing component.

Description

Paenibacillus ZX1905, extracellular polysaccharide Lubcan produced by same and application of extracellular polysaccharide Lubcan

Technical Field

The invention belongs to the technical field of microorganisms and microbial extracellular polysaccharide, and relates to a Paenibacillus sp.ZX1905, extracellular polysaccharide Lubcan produced by the same, and application of the same in preparation of cosmetics.

Background

The microbial polysaccharide has complex chemical structure and biological functions, such as functions of resisting tumors, resisting oxidation, regulating immunity, reducing blood sugar, preserving moisture, absorbing moisture and the like, and is widely applied to the industries of pharmacy, food, cosmetics and the like. Various strains have been used for producing polysaccharides, such as Xanthomonas campestris NRRL B-1459 for producing xanthan gum, Sphingomonas palmiobacter ATCC 31461 for producing gellan gum, Sinorhizobium meliloti Rm41 or Agrobacterium sp.ZCCC3656 for producing succinoglycan, Agrobacterium sp.ZX09 for producing Sorana gum, and Streptococcus sp.ID9102 for producing hyaluronic acid.

Hyaluronic acid is widely used in the cosmetic field due to its unique moisturizing ability. Hyaluronic acid may be extracted from animal tissue or produced by fermentation of streptococci. The separation cost of extracting high purity and high molecular weight hyaluronic acid from animal tissues is very high, and further application of animal-derived biochemical reagents is limited due to the risk of cross-species viruses and other uncertain factors. The major problems with hyaluronic acid production by Streptococcus strains are high cost and limited yield, e.g.the yield of hyaluronic acid produced by Streptococcus sp.ID9102 in medium containing 40g/L glucose, 7.5g/L yeast extract and 10g/L casein peptone is 6.94g/L (J.Im, J.Song, J.Kang, D.Kang, Optimization of medium components for high-molecular-weight hyaluronic acid production by Streptococcus sp.ID9102 a static approach, J.Ind.biol.Biotechnol.36 (11) (2009) 1347.). To solve this problem, many scholars have screened for microorganisms capable of producing polysaccharides having moisturizing ability. Polysaccharides produced by Pseudomonas fluorescens PGM37 have better moisturizing ability than glycerol, but inferior to hyaluronic acid (L.ZHao, F.Fan, P.Wang, X.Jiang, Culture medium optimization of a new bacterial polysaccharide with excellent moisture retention activity, applied.Microbiol. Biotechnol.97(7) (2013) 2841-2850.); the polysaccharides produced by Zungwangia profunda SM-A87 both have lower moisture and moisture absorbing capacity than hyaluronic acid (M.L.Sun, S.B.Liu, L.P.Qiao, et al, A novel exopolysaccharide from bacterium fundamenta profunda SM-A87: low-cost transfer, mobility transfer, and antioxidant activities, applied. Microbiol. Biotechnol. 98(17 (2014)) 7437-45.). Therefore, it is important to find an economical and high-yield natural polysaccharide having a moisturizing property equivalent to that of hyaluronic acid.

Disclosure of Invention

One of the objects of the present invention is to provide a Paenibacillus (Paenibacillus sp. ZX1905) producing the extracellular polysaccharide Lubcan having moisturizing and moisture absorbing ability comparable to hyaluronic acid.

The inventor separates a strain from soil of farmland in Henan province, and the strain is identified as a Paenibacillus strain (Paenibacillus sp.) through molecular biology and named as Paenibacillus sp.ZX1905. The strain is preserved in China Center for Type Culture Collection (CCTCC) at 7 months and 15 days in 2020, the preservation address is Wuhan university of Lojia mountain eight roads in Wuchang district in Wuhan city, Hubei province in China, and the preservation number is CCTCC NO: M2020320.

The second purpose of the invention is to provide the exopolysaccharide Lubcan produced by the paenibacillus ZX 1905.

The extracellular polysaccharide Lubcan has the following structure:

consists of glucuronic acid, glucose, mannose, galactose and rhamnose in a molar ratio of 2:3:1:2: 2.

The invention also aims to provide a production method of the extracellular polysaccharide Lubcan, which comprises the following steps:

step 1, inoculating a paenibacillus ZX1905 seed solution into a fermentation culture medium, and fermenting to obtain a fermentation liquid;

step 2, adding water into the fermentation liquor for dilution, adding 0.1% NaOH, uniformly mixing, boiling in a boiling water bath, centrifuging and collecting supernate;

and 3, adding sodium acetate and ethanol into the supernatant, centrifuging, collecting the precipitate, and then washing the precipitate with ethanol to obtain the Lubcan polysaccharide pure product.

In the invention, the fermentation medium is as follows: KH (Perkin Elmer)₂PO₄ 0.6g/L，Na₂HPO₄ 1.2g/L，CaCl₂ 0.1g/L， MgCl₂·6H₂O 0.3g/L，FeSO₄ 0.0125g/L，MnSO₄ 0.003g/L，ZnCl₂0.01g/L, 4g/L of peptone and 20-30 g/L of soluble starch are dissolved in water, and the pH value is 8.0.

In the invention, the fermentation conditions of the paenibacillus ZX1905 are as follows: the fermentation temperature is 28 ℃, the rotation speed is 230rpm, and the fermentation time is 28-32 h.

In a specific embodiment of the invention, in step 2, the fermentation broth is diluted with an equal volume of water.

In the embodiment of the present invention, in the step 2, the boiling water bath boiling time is 10 min.

In the specific embodiment of the present invention, in step 2, the centrifugation speed is 8000rpm and the centrifugation time is 10 min.

In a specific embodiment of the present invention, in step 3, sodium acetate was added in an amount of 1g/L and ethanol was added in an amount of 2 times the volume of the supernatant.

The invention also aims to provide the application of the extracellular polysaccharide Lubcan in preparing moisturizing products.

The moisturizing product is a product with moisturizing function suitable for hyaluronic acid, and comprises but is not limited to biological medical materials or cosmetics.

The paenibacillus ZX1905 is a strain capable of secreting extracellular polysaccharide Lubcan equivalent to the moisture retention and moisture absorption performance of hyaluronic acid. The growth process of the paenibacillus ZX1905 does not secrete pigment, the culture operation of the strain is simple, and compared with the production of hyaluronic acid, the culture medium has definite components and low cost, and is suitable for industrial production. The exopolysaccharide Lubcan produced by the paenibacillus ZX1905 is acidic polysaccharide, and compared with the purification process of hyaluronic acid, the extraction and purification method is simple and the production cost is low. The exopolysaccharide Lubcan has the moisture retention and moisture absorption capacity equivalent to hyaluronic acid, can replace hyaluronic acid, and is applied to the field of biological medicine materials or cosmetics as a moisture retention component.

Drawings

FIG. 1 is a plate colony of Paenibacillus ZX1905 strain.

FIG. 2 is a liquid chromatogram of the Lubcan component assay. Wherein A is a standard monosaccharide profile; b is the monosaccharide composition analysis pattern of Lubcan. The labels in the figure are: man-mannose, Rha-rhamnose, GlcA-glucuronic acid, Glc-glucose, Gal-galactose.

FIG. 3 is a one-dimensional nuclear magnetic resonance spectrum of Lubcan. A is¹H NMR; b is¹³C NMR。

FIG. 4 is a two-dimensional nuclear magnetic resonance spectrum of Lubcan. A is COSY; b is HSQC; c is HMBC; d is TOCSY; e is NOESY.

FIG. 5 is a block diagram of Lubcan.

Fig. 6 is a graph comparing the moisturizing and hygroscopic properties of Lubcan and hyaluronic acid and glycerol. A is the hygroscopicity of the sample at 43% relative humidity; b is the hygroscopicity of the sample at a relative humidity of 81%; c is the moisture retention of the sample at 43% relative humidity.

The Paenibacillus ZX1905(Paenibacillus sp. ZX1905) of the invention has been preserved in China Center for Type Culture Collection (CCTCC) in 7, 15 and 2020, the strain preservation number is CCTCC NO: M2020320, and the preservation address is Wuhan university at Lopa of Wuhan city, Hubei China.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings.

Example 1

1. Screening and isolation of Paenibacillus sp.ZX1905:

taking soil of a Henan farmland, adding 1g of a soil sample into a prepared liquid culture medium, culturing for 2 days at the temperature of 28 ℃ and the rotating speed of 230rpm, then taking 1ml of bacterial liquid to dilute and coat the bacterial liquid into a solid culture medium, culturing for 2-3 days at the temperature of 28 ℃, after bacterial colonies grow out, selecting single bacterial colonies with viscous polysaccharide on the surface to dilute and coat the single bacterial colonies on a new solid culture medium according to the morphological characteristics of the bacterial colonies, culturing at the temperature of 28 ℃, selecting the single bacterial colonies to dilute and coat the single bacterial colonies after the bacterial colonies grow out, and repeating the process until the bacterial colonies grow out on the solid culture medium have a single morphology.

The screening culture medium is as follows: KH (Perkin Elmer)₂PO₄ 1g/L，CaCl₂ 0.1g/L，MgCl₂·6H₂O 0.3g/L，FeSO₄ 0.0125g/L， KNO₃2g/L, sucrose 20g/L, pH 7.2, and agar 15 g/L. The medium was sterilized at 121 ℃ for 20 min.

2. Identification of strains

The strain is subjected to morphological, physiological and biochemical tests. Determining the 16S rRNA gene sequence of the strain, comparing the 16S rRNA gene sequence of the strain with the gene sequences in a GenBank database in a homology mode, analyzing the result, and determining the species of the strain on the molecular biology level.

(1) Morphological characteristics: after culturing for 2-3 days at 28 ℃ on a solid plate culture medium, the colony is circular and smooth in surface, and the strain secretes a large amount of extracellular polysaccharide and does not produce pigments. FIG. 1 shows colonies on a plate medium.

(2) The 16S rDNA sequence of the Paenibacillus is shown in a sequence table (SEQ ID NO.1), the similarity of the sequence of the Paenibacillus bacillus physalsphaerae strain SM26 is up to 97.97 percent through Blast comparison, the similarity of the sequence of the Paenibacillus physalsphaerae strain AS7 is 97.89 percent, the similarity of the sequence of the Paenibacillus physalsphaerae strain RC11 is 97.82 percent, the Paenibacillus is named AS Paenibacillus sp.ZX1905.

Example 2

The production and purification method of extracellular polysaccharide Lubcan produced by paenibacillus ZX1905 comprises the following specific steps:

(1) preparing a culture medium: mixing KH with water₂PO₄ 0.6g/L，Na₂HPO₄ 1.2g/L，CaCl₂ 0.1g/L，MgCl₂·6H₂O 0.3g/L，FeSO₄ 0.0125g/L，MnSO₄ 0.003g/L，ZnCl₂0.01g/L of peptone, 4g/L of peptone and 20-30 g/L of soluble starch are dissolved in water, and the pH value is 8.0; sterilizing the culture medium at 121 deg.C for 20 min;

(2) adding a plate single colony into the sterilized culture medium obtained in the step (1), and culturing at the temperature of 28 ℃ and the rotation speed of 230rpm for 12-18h to obtain a seed culture solution;

(3) adding the seed culture solution obtained in the step (2) into the sterilized culture medium in the step (1), and fermenting for 28-32 hours at the temperature of 28 ℃ and the rotating speed of 230rpm to obtain fermentation liquor;

(4) adding water with the same volume to the fermentation liquor obtained in the step (3) to dilute the fermentation liquor, adding 0.1% NaOH, uniformly mixing, boiling in a boiling water bath for 10min, centrifuging at 8000rpm for 10min, and collecting supernatant;

(5) and (4) adding 1g/L of sodium acetate and 2 times of ethanol in volume into the supernatant obtained in the step (4), and washing the precipitate with a small amount of ethanol to obtain the Lubcan polysaccharide pure product.

Example 3

The structure of the extracellular polysaccharide Lubcan produced by the paenibacillus ZX1905 is analyzed as follows:

(1) the molecular weight of Lubcan was determined to be 3270KDa by gel permeation chromatography.

(2) Lubcan was subjected to complete acid hydrolysis with trifluoroacetic acid, PMP derivatization, chloroform extraction and aqueous phase filtration for high performance liquid phase analysis. FIG. 2 is a liquid chromatogram for monosaccharide composition determination. Wherein, the A picture is the peak appearance of standard monosaccharide, the B picture is the peak appearance after Lubcan complete acid hydrolysis, the same component appears in the same position. As can be seen from the figure, Lubcan is an acidic heteropolysaccharide composed of glucuronic acid, glucose, mannose, galactose and rhamnose in a molar ratio of 2:3:1:2: 2.

(3) Lubcan polysaccharide and reduced-Lubcan polysaccharide after reducing uronic acid were subjected to methylation analysis, and the results are shown in Table 1.

TABLE 1 methylation analysis of Lubcan

^aPartially methylated sugar alcohol acetate.

^bThe results are expressed as molar ratios relative to 1,4-linked-Manp residues.

^cDetermined by comparative analysis of lubcan and reduced-lubcan.

From Table 1, it can be seen that Lubcan is composed of 1,3-linked Rhap, 1,3-linked Glcp, 1,4-linked Manp, 1,4-linked GlcAp, 1,3-linked Galp and 1,4,6-linked Glcp, and the molar ratio of each glycoside is consistent with the monosaccharide analysis result.

(4) Partial acid hydrolysis of Lubcan with trifluoroacetic acid and D₂O was prepared at 80mg/ml, and NMR analysis was performed using TMSP as an internal standard. The one-dimensional nuclear magnetic spectrum of the polysaccharide is shown in FIG. 3. Wherein, A is¹H NMR; b is as shown in¹³C NMR. The two-dimensional nuclear magnetic spectrum of the polysaccharide is shown in FIG. 4. Wherein, A is COSY; b is HSQC; the diagram C is HMBC; FIG. D is TOCSY; and E is NOESY.

¹In H NMR, chemical shifts between delta 5 and 6ppm are alpha anomeric protons, chemical shifts between delta 4 and 5ppm are beta anomeric protons, and chemical shifts between delta 1.27 and 1.28ppm are assigned to the-CH of rhamnose₃Delta 1.45ppm is assigned to the-CH group of pyruvic acid₃。¹³In C NMRAnd 10 signals are between delta 96 and 106ppm, which indicates that Lubcan has 10 anomeric carbons, and 10 anomeric signal cross peaks can be observed on the HSQC spectrum.¹³Chemical shift delta 19.44 ppm in C NMR attributable to-CH for rhamnose₃Delta 28ppm assigned to the-CH of pyruvic acid₃Delta 178.29 and 178.90ppm are assigned to-COOH, glucuronic and pyruvic acids, respectively. COSY and TOCSY were used to accurately assign proton chemical shifts for each glycoside,¹³c and HSQC were used to assign the chemical shifts of the carbons, HMBC and NOESY were used to determine the order of attachment of the sugar residues. According to one-dimensional and two-dimensional NMR of Lubcan and previous research results, the method of Lubcan¹H NMR and¹³a summary of the chemical shifts of the C NMR is summarized in Table 2. The bold chemical shifts in table 2 are sugar residue substitution sites.

TABLE 2 Lubcan¹H NMR and¹³c NMR chemical shifts (ppm)

(5) In combination with the above analysis, it was determined that the structure of the extracellular polysaccharide Lubcan produced by Paenibacillus ZX1905 is shown in FIG. 5.

Example 4

The test of the moisture retention and moisture absorption performance of the exopolysaccharide Lubcan, hyaluronic acid and glycerol specifically comprises the following steps:

prior to hygroscopicity testing, the polysaccharide samples were pulverized into a fine powder. The samples were freeze dried for 24 h. The dried sample (0.1g) was placed in a closed moisture-retaining container and the temperature was maintained at 25 ℃. Saturated K₂CO₃Maintenance of 43% relative humidity (RH 43%), saturation (NH)₄)₂SO₄Maintain 81% relative humidity (RH 81%). The moisture absorption capacity was evaluated by the weight increase of the samples after 1, 2, 4,6, 8, 10, 12 and 24 h. The moisture absorption rate (R) of the sample was calculated by the following formula_a)：

W₀And W_tIs the weight of the sample before and after it is placed in the closed container.

In the moisture retention test of the sample, firstly, the sample (0.1g) after being frozen and dried for 24h is put into a closed container containing a certain volume of distilled water, the sample is kept for 24h at 25 ℃, and then the sample is transferred to saturated K₂CO₃(RH 43%) the moisture holding capacity of the samples was evaluated by weight loss after 1, 2, 4,6, 8, 10, 12 and 24h in a closed container. The moisture retention rate (R) of the sample was calculated by the following formula_h)：

W₀And W_tIs the weight of water in the sample before and after being placed in the RH 43% closed container.

Fig. 6 is a graph comparing the moisturizing and hygroscopic properties of Lubcan and hyaluronic acid and glycerol. Panel A shows the hygroscopicity of the sample at RH 43%, the R of Lubcan and hyaluronic acid over the entire detection time_aAre all higher than glycerol, and at 24h, R of Lubcan_a(16.98%) R with hyaluronic acid_a(17.28%) are almost identical and higher than glycerol (11.26%). Panel B is the hygroscopicity of the sample at RH 81%, R of Lubcan at 24h_aThe value was 40.41%, hyaluronic acid 41.20%, glycerol 70.48%. C diagram is the moisture retention at RH 43%, R of Lubcan and hyaluronic acid of the sample_hShows a sustained decline over the first 8h and then reaches a steady value, while the R of glycerol_hThe decline continued throughout the test time. R of Lubcan, hyaluronic acid and glycerol after dehydration for 24h_hThe values were 29.34%, 25.16% and 17.21%, respectively. In summary, Lubcan has comparable water uptake and retention capacity to hyaluronic acid.

Sequence listing

<110> Nanjing university of science and technology

<120> Paenibacillus ZX1905, exopolysaccharide Lubcan produced by same and application of exopolysaccharide Lubcan

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1377

<212> DNA

<213> Paenibacillus (Paenibacillus)

<400> 1

gttagcggcg gacgggtgag taacacgtag gtaacctgcc tgtaagaccg ggataacatt 60

cggaaacgaa tgctaatacc ggatatgcgg tttgctcgca tgagcgaatc gggaaagacg 120

gtgcaagctg tcacttacag atggacctgc ggcgcattag ctagttggtg gggtaacggc 180

tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 240

cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 300

gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgccaggg 360

aagaacgagt gggagagtaa ctgctcctgc tatgacggta cctgagaaga aagccccggc 420

taactacgtg ccagcagccg cggtaatacg tagggggcaa gcgttgtccg gaattattgg 480

gcgtaaagcg cgcgcaggcg gttttgtaag tcaggtgttt aatctcgggg ctcaaccccg 540

attcgcatct gaaactgcaa gacttgagtg cagaagagga aagtggaatt ccacgtgtag 600

cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgacttt ctgggctgta 660

actgacgctg aggcgcgaaa acgtggggaa caaacaggat tagataccct ggtagtccac 720

gccgtaaacg atgaattcta ggtgttaggg gtttcgatac ccttggttcc gaagttaaca 780

cattaaacat tccgcctggg gagtacgctc ccaagagtga aactcaaagg aattggcggg 840

gacccgccca aacagtggag tatgtggttt aattcgaaac aacccgaaga accttaccag 900

gtcttgacat ccctctgaat cccctagaga taggggcggc ccttcgggga cagaggagac 960

aggtggtgca tggttgtcgt caactcgtgt cgtgagatgt tgggttaagt cccccaacga 1020

acccaaccct tgattttagt ttccaacact ttaaggtggg cactctagaa tgactgccgg 1080

tgacaaaccg gaggaaggcg gggatgacgt caaatcatca tgccccttat gacctgggct 1140

acacacgtac tacaatggcc gttacaacgg gaagcgaaag agcgatctgg agcgaatcct 1200

aaaaaggcgg tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag tcggaattgc 1260

tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggtcttgta cacaccgccc 1320

gtcacaccac gagagtttac aacacccgaa gccggtgggg taaccgcaag gagccag 1377

Claims

1. Paenibacillus sp.ZX1905 with the preservation number of CCTCC NO: M2020320.

2. The exopolysaccharide Lubcan produced by Paenibacillus ZX1905 of claim 1, which has the following structure:

3. The production method of the exopolysaccharide Lubcan according to claim 2, characterized by comprising the following steps:

and step 3, adding sodium acetate and ethanol into the supernatant, centrifuging, collecting the precipitate, and then washing the precipitate with ethanol to obtain the Lubcan polysaccharide pure product.

4. The method of claim 3, wherein the fermentation medium is: KH (Perkin Elmer)₂PO₄0.6g/L，Na₂HPO₄ 1.2g/L，CaCl₂ 0.1g/L，MgCl₂·6H₂O 0.3g/L，FeSO₄ 0.0125g/L，MnSO₄0.003g/L，ZnCl₂0.01g/L, 4g/L of peptone and 20-30 g/L of soluble starch are dissolved in water, and the pH value is 8.0.

5. The production method according to claim 3, wherein the fermentation conditions of the Paenibacillus ZX1905 are as follows: the fermentation temperature is 28 ℃, the rotation speed is 230rpm, and the fermentation time is 28-32 h.

6. The method of claim 3, wherein in step 2, the fermentation broth is diluted with an equal volume of water.

7. The production method according to claim 3, wherein in the step 2, the boiling-water bath boiling time is 10min, the centrifugation speed is 8000rpm, and the centrifugation time is 10 min.

8. The production method according to claim 3, wherein in the step 3, sodium acetate is added in an amount of 1g/L and ethanol is added in an amount of 2 times the volume of the supernatant.

9. Use of the exopolysaccharide Lubcan according to claim 2 in the preparation of a moisturizing product.

10. The use according to claim 9, wherein the moisturizing product is a biomedical material or a cosmetic with a moisturizing function.