CN103484392A

CN103484392A - Application of pseudomonas fluorescens PGM37 strain to produce glucomannan

Info

Publication number: CN103484392A
Application number: CN201210210753.6A
Authority: CN
Inventors: 江晓路; 赵丽
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2012-06-13
Filing date: 2012-06-13
Publication date: 2014-01-01

Abstract

The invention relates to an application of pseudomonas fluorescens PGM37 strain to produce glucomannan. The invention discloses a separated purified bacterial strain, and the bacterial strain has the characteristic of producing glucomannan, and also has the characteristics of simple nutrition requirements, easy culture, stable character, high glucomannan yield and the like. The polysaccharide has a weight-average molecular weight of 3619.6 kDa determined by GPC. Structural analysis shows that the polysaccharide is composed of beta-D-glucose and beta-D-mannose arranged at intervals in a linear chain form with a content ratio of 1:1, and the polysaccharide has a structure of linear beta-D-Glcp(1,4)-beta-D-Manp(1,4)-beta-D-Glcp(1,4)-beta-D-Manp. The yield is up to 10.12 g/L.

Description

Produce konjac glucomanna with Pseudomonas fluorescens PGM37 bacterial strain

Technical field

Biotechnology

Background technology

Konjac glucomanna is the high molecular weight non-ionic polysaccharide be formed by connecting with certain form by glucose and seminose.Konjac glucomanna in the present invention is the complex polysaccharide be combined into by certain glycosidic link by certain mol ratio by D-Glucose (Glc) and D-MANNOSE (Man).Through retrieval, not about relevant report and patent with this patent Related Bacteria generation konjac glucomanna.Konjac glucomanna has unique physico-chemical property and stronger strengthening immunity, reduces blood fat, Green Tea Extract oxidation, radioprotective, the biological activity that delays senility, and good moisture-absorbing moisture-keeping performance is arranged, be widely used in the industry such as food medical science, pharmacy, makeup, textiles and food, therefore, the research of konjac glucomanna had great importance and be worth.At present both at home and abroad the plant tissue that the konjaku stem tuber is representative is only take in the source of the konjac glucomanna of report, contain abundant Rhizoma amorphophalli glucomannan (simple KGM) in the konjaku stem tuber, having the effect that poison is attacked in reducing blood-fat, serum courage fat and detumescence, is also important additive and industrial chemicals.Usually after fresh konjaku being processed into to konjaku powder, use, the main component of konjaku powder is KGM, and it is the stocking property polysaccharide of konjaku, is also a kind of polyose half fiber.In the konjaku plant, contained konjac glucomanna is with the ratio of 1: 1.6, by the mixed polysaccharide that β-(Isosorbide-5-Nitrae) glycosidic link connects into by β-D-Glucose and β-D-MANNOSE.Microorganism is the important sources that polysaccharide is produced, but the microorganism that produces konjac glucomanna is not also reported at present, with the plant konjac glucomanna, compare, that microbe-derived konjac glucomanna has is with short production cycle, output is high, cost is low, good stability, not climate and geographical environmental condition restriction and the advantage such as physiologically active is strong, can supplement konjac glucomanna only has the limitation of this single source of konjaku, has the stronger market competitiveness and vast potential for future development.The microbial polysaccharide that has dropped into suitability for industrialized production in China only has dextran (Glucan), xanthan gum (Xanthan gum), gelling gum (Gellan gum), the solidifying polysaccharide (Curdlan) of heat and hyaluronic acid (Hyaluronic acid, a few kind such as HA), and underproduce China market demand 1/3, most of need to rely on import.Therefore, the good bacterial strain of sugared proterties is produced in screening, and developing the new microbial polysaccharide with suitability for industrialized production potentiality is the key that promotes China's polysaccharide industrial development.

Summary of the invention

The purpose of this invention is to provide a kind of method that can produce the microorganism Pseudomonas fluorescens PGM37 of konjac glucomanna and utilize this bacterial strain production konjac glucomanna.

Separation and purification of the present invention goes out a strain and derives from bacterium Pseudomonas fluorescens PGM37 in whole meal flour, and it has the characteristic that produces konjac glucomanna.Adopt the preliminary evaluation of the morphological observations such as gramstaining, pod membrane, flagellum, mobility observation as bacterial classification, then according to " uncle Jie Shi Bacteria Identification handbook the 9th edition " with " common bacteria system identification handbook is carried out Physiology and biochemistry and tested and do further evaluation.By 16SrDNA gene sequencing and systematic evolution tree analysis, qualification result is Pseudomonas fluorescens bacterium.Naming this bacterial strain is Pseudomonas fluorescens PGM37.Culture presevation unit: Chinese Typical Representative culture collection center.Address: Wuhan, China, Wuhan University.Preservation date is on 05 27th, 2012, and preserving number is M2012183.Obtaining this polysaccharide weight-average molecular weight through GPC mensuration is 3619.6kDa, the exocellular polysaccharide that adopts the methods such as vapor-phase chromatography, infrared spectroscopy, ESI-LC-MS to produce Pseudomonas fluorescens PGM37 carries out component and structural analysis, determine that this sugar is spaced with linear form by β-D-Glucose and β-D-MANNOSE, content was than 1: 1, for linear β-D-Glcp-(1,4)-β-D-Manp-(Isosorbide-5-Nitrae)-β-D-Glcp-(Isosorbide-5-Nitrae)-β-D-Manp structure.

The accompanying drawing explanation

Fig. 1: the molecular weight integral distribution curve of Pseudomonas fluorescens PGM37 konjac glucomanna

Fig. 2: the gas-chromatography proximate analysis collection of illustrative plates of Pseudomonas fluorescens PGM37 konjac glucomanna

Embodiment

The present invention realizes by following technological line:

1, strain separating is identified:

Get 1g whole meal flour sample, join in the triangular flask that the 25mL enrichment medium is housed, 28 ℃, 150r/min shake-flask culture 48h.Then, after enrichment culture liquid being diluted to 10 times by stroke-physiological saline solution, get 0.1mL coating PDA culture medium flat plate.Be inverted for 28 ℃ and cultivate 48h, obtain the bacterium colony that the thickness transparent appearance is arranged on flat board, the bacterium that obtains producing exocellular polysaccharide, preserve under 4 ℃ of conditions.Enrichment medium is by sucrose 20g, yeast extract paste 3g, NaCl 2g, K ₂hPO ₄1g, MgSO ₄0.5g, (NH ₄) ₂8O ₄0.5g form, adding distil water 1000mL, adjust pH7.0.According to a conventional method bacterial strain is carried out to the 16SrDNA gene sequencing, the sequence of Pseudomonas fluorescens in sequencing result and the Genebank similarity of comparing is the highest.By phylogenetic analysis, in conjunction with this strain morphology and physiological and biochemical property, be defined as Pseudomonas fluorescens Pseudomonas fluorescens, therefore naming this bacterial strain is Pseudomonas fluorescens PGM37.This culture presevation unit: Chinese Typical Representative culture collection center.Address: Wuhan, China, Wuhan University.Preservation date is on 05 27th, 2012, and preserving number is M2012183.

2, the production method of Pseudomonas fluorescens Pseudomonas fluorescens PGM37 exocellular polysaccharide: the structure that it is characterized in that nutrient media components, fermentation condition, preparation method and gained exocellular polysaccharide:

Produce sugar culture-medium component (g/L): sucrose 40, yeast extract paste 3, NaCl 1, CaCl ₂0.2, K ₂hPO ₄1, pH 7.0.

Fermentation condition: liquid amount is the bottled 100mL substratum of every 250mL triangle, and inoculum size is 5% (v/v), and initial pH value is 7.0,28 ℃, and 150r/min cultivates 48h.

Production stage:

(1) actication of culture: will preserve bacterial classification and be connected in the PDA slant medium, and after 28 ℃ of constant temperature culture 24h, be kept at 4 ℃ of refrigerators standby.

(2) inoculation culture prepares seed liquor: the seed liquor nutrient media components is (g/L): sucrose 20, and yeast extract paste 2.5, NaCl 1, K ₂hPO ₄1, pH 7.0, the every 250mL shaking flask dress of liquid amount 100mL substratum.PDA slant strains in step (1) is inoculated in to the seed liquor substratum, in 28 ℃ of shaking tables, cultivates 2d, the shaking table revolution is 150r/min.

(3) produce sugar culture-medium and produce the sugar cultivation: producing sugar culture-medium component and content is (g/L): sucrose 40, and yeast extract paste 3, NaCl 1, CaCl ₂0.2, K ₂hPO ₄1, pH 7.0.The seed liquor that step (2) is fermented is inoculated in the product sugar culture-medium with 5% (v/v) inoculum size, it is characterized in that fermentation culture conditions is: liquid amount is the bottled 100mL substratum of 250mL triangle, inoculum size is 5% (v/v), and 28 ℃, 150r/min cultivates 48h.

(4) preparation of polysaccharide: after the centrifugal removal thalline of the centrifugal 5min of fermenting culture 3600rpm that step (3) is obtained, fermented liquid is concentrated into to 1/2 of original volume, again pH is adjusted to 8, finally by 2 times of 95% ethanol precipitation, standing over night in 4 ℃ of refrigerators, 3600rpm is centrifugal, and 10min obtains white precipitate, precipitates water-soluble, the centrifugal 10min of redissolution liquid 3600rpm by obtaining, remove insolubles.The liquid that redissolves precipitates polysaccharide again, and precipitation washes twice with dehydrated alcohol, obtains the konjac glucomanna that Pseudomonas fluorescensPGM37 produces.

3, polysaccharide is identified:

(1) purity of polysaccharide is identified: take the Crude polysaccharides sample 20mg after freeze-drying, make its dissolving with 1mL distilled water, the centrifugal 10min of 3600r/min, get supernatant liquor 0.5mL at every turn and cross Sephacryl S-300HR gel chromatography column, use the ultrapure water wash-out, flow velocity 1mL/2min.After Fraction Collector is collected automatically, each chromatography component detects sugar cloth by the phenolsulfuric acid method, and the pipe sequence number of take is X-coordinate OD _490nmvalue is the ordinate zou mapping.Result shows that product is the homogeneous mesokurtosis.

(2) polysaccharide molecule flow measurement: water GPC (Gel Permeation Chromatography) method, the serial blue dextran (Sigma) of different sized molecules amounts (T5, T10, T20, T50, T100) of take is standard, be mixed with aseptic ultrapure water the standardized solution that concentration is 5mg/mL, take elution volume as X-coordinate, lgM (molecular weight logarithmic value) is ordinate zou, is depicted as typical curve.The same standard substance of sample operation method, calculating weight-average molecular weight by sample appearance time and typical curve is 3619.6kDa (seeing accompanying drawing 1).

Operational condition: use Agilent1100 type high performance liquid chromatograph to measure; SB-804HQ type gel column; Na with 1g/L ₂sO ₄for elutriant, flow velocity is 0.5mL/min; Differential detector (RI); Service temperature is 40 ℃; Sample size is 20 μ L.

(3) polysaccharide structures is identified: the konjac glucomanna polysaccharide sample that PGM37 is produced derives and carries out gas chromatographic analysis by alditol acetate method after trifluoroacetic acid hydrolysis, determine the composition of proportions (seeing accompanying drawing 2, peak area ratio 1: 1) with 1: 1 by glucose and seminose of this sugar.Determine the β-pyranose that is configured as of this polysaccharide by Infrared spectroscopy, and formed by β-D-Glucose and β-D-MANNOSE.Polysaccharide after enzymolysis by liquid phase-electro spraying ionization-mass spectrometry technology obtain degrading molecular weight fragment of oligosaccharides, the specificity of desmoenzyme and degraded substrate determines that the mode of connection of polysaccharide is β-(1,4) glycosidic link, and be the linear pattern polysaccharide, mode of connection is β-D-Glcp-(1,4)-β-D-Manp-(Isosorbide-5-Nitrae)-β-D-Glcp-(Isosorbide-5-Nitrae)-β-D-Manp.

Claims

1. a Pseudomonas fluorescens PGM37 bacterial strain, this culture presevation unit: Chinese Typical Representative culture collection center.Address: Wuhan, China, Wuhan University.Preservation date is on 05 27th, 2012, and preserving number is M2012183.

2. the konjac glucomanna that a Pseudomonas fluorescens PGM37 bacterial strain produces, its weight-average molecular weight is 3619.6kDa.

3. the konjac glucomanna that a Pseudomonas fluorescens PGM37 bacterial strain produces, this sugar is spaced with linear form by β-D-Glucose and β-D-MANNOSE, its glucose: seminose=1: 1.

4. a bacterial classification Pseudomonas fluorescens PGM37 who produces konjac glucomanna, the genetic marker of bacterial strain be the 16SrDNA nucleotide sequence as PGM37.SEQ, comprise the nucleic acid that there is the nucleotide sequence of at least 90% identity with the nucleotide sequence shown in PGM37.SEQ.