CN104630188A - Method for producing low-temperature glucomannanase by fermentation of marine microorganisms - Google Patents

Method for producing low-temperature glucomannanase by fermentation of marine microorganisms Download PDF

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CN104630188A
CN104630188A CN201510084703.1A CN201510084703A CN104630188A CN 104630188 A CN104630188 A CN 104630188A CN 201510084703 A CN201510084703 A CN 201510084703A CN 104630188 A CN104630188 A CN 104630188A
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liquid
low temperature
contribution ratio
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low
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王晓辉
李晓艳
窦少华
于爽
张庆芳
迟乃玉
王强
张旭姣
金连豆
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Dalian University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase

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Abstract

The invention relates to a method for producing low-temperature glucomannanase by fermentation of marine microorganisms. The method specifically comprises the following steps: activating a strain for generating glucomannanase, and carrying out gradual low-temperature domestication, so that the strain grows well in a low-temperature environment; carrying out gradual enlarge cultivation on the bacteria for producing the glucomannanase after low-temperature domestication at 24-30 DEG C; inoculating into a liquid fermentation medium according to the inoculating amount which is 3%-9% of the volume of a fermentation liquid, cultivating at 24-30 DEG C for 72-144 hours to obtain low-temperature glucomannanase; and further concentrating, separating and purifying the collected crude enzyme according to different requirements and use objects, so as to prepare enzyme preparations with different activity, purity and dosage forms. The low-temperature glucomannanase produced by the method has high enzyme activity and high catalysis efficiency at a low temperature; the processing time is shortened; the production technology is simplified; the production cost is reduced; the production efficiency is improved; the product quality is improved; and the enzyme preparation is simple, convenient and fast to apply and operate, and has relatively wide development value and market application advantages.

Description

The method of marine microorganism fermentative production low temperature zonal contribution ratio
Technical field
The present invention relates to the fields such as microbiology, biological chemistry, enzyme engineering, fermentation engineering, be specifically related to a kind of method of low temperature zonal contribution ratio marine microorganism fermentative production.
Background technology
Zonal contribution ratio (Glucomannanase) be a kind of konjac glucomanna can be degraded to oligosaccharides can secrete enzyme (Dong Guiqing, Guangxi light industry, 2007) outside inducing cell.Oligosaccharides belongs to functional oligose, there is extremely significant effect for reducing fat, can as hyperlipidemia, diabetes assisting therapy product (Hsiao-Ling Chen, Journal ofthe American College ofNutrition, 2003); Because of generation and absorption that it can reduce ammonia in intestines, the removing toxic substances burden (Chen Li, Chinese biochemical drug magazine, 2003) of liver to blood ammonia can be alleviated; Oligosaccharides can improve superoxide dismutase activity in body, enhancing body resistance of oxidation (Yang Yanyan, herbal medicine, 2001) simultaneously.But relevant oligosaccharides product, only has and appears at foreign market on a small quantity at present, domestic substantially without this series products (Zhou Haiyan, Chinese biological engineering magazine, 2005).
The domestic research for zonal contribution ratio is still in the starting stage, mainly comprises (Zhou Haiyan, hubei agricultural science, 2005 such as strain improvement, optimization culture conditions, enzyme purification, zymologic property, gene clone; Dong Guiqing, Guangxi light industry, 2007; Xu Jian, genomics and applied biology, 2012; ).The industrialization of marine microorganism fermentative production low temperature zonal contribution ratio, large-scale production and application have not been reported.Marine microorganism fermentative production low temperature zonal contribution ratio has very large application advantage compared with medium and high temperature zonal contribution ratio: cold-adapted enzyme has high enzymatic activity and high catalytic efficiency at low temperatures, greatly can shorten the treatment time and save expensive heating or refrigeration costs; Also sizable advantage is had in energy-conservation; The vigor of cold-adapted enzyme can be made to lose through gentle thermal treatment, and low temperature or thermophilic process can not affect the quality (Chen Xiulan etc., Chinese agriculture science and technology Leader, 2007) of product.These characteristics will contribute to popularization and the use of marine microorganism fermentative production low temperature zonal contribution ratio.The application of low temperature zonal contribution ratio produces far-reaching influence by traditional food, medicine and other fields.Therefore, marine microorganism fermentation zonal contribution ratio suitability for industrialized production can create good economic benefit and social benefit (Zhou Haiyan, Chinese biological engineering magazine, 2005).
Summary of the invention
The object of this invention is to provide the method for marine microorganism fermentative production low temperature zonal contribution ratio, the method mainly marine microorganism after domestication by low temperature, fermentative production low temperature zonal contribution ratio in 24 ~ 30 DEG C of liquid nutrient mediums, the low temperature zonal contribution ratio crude enzyme liquid activity that this production method obtains can reach 3587U/mL, as again through abstraction and purification, the zymin of different concns and purity can be obtained.
The method of marine microorganism fermentative production low temperature zonal contribution ratio of the present invention specifically comprises the following steps:
(1) actication of culture of zonal contribution ratio can be produced, then through domestication by low temperature step by step, acclimation temperature from high to low, 30 DEG C → 28 DEG C → 26 DEG C → 24 DEG C, makes its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures; The domestication by low temperature substratum of this bacterial classification comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o 0.001 ~ 0.004%, agar 1.8 ~ 2.0%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
The described bacterial classification that can produce zonal contribution ratio derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), bacterium numbering: 1.2244 or 1.504 or 1.775 or 5.809;
(2) according to a conventional method by the zonal contribution ratio producing strains after domestication by low temperature in step (1) on liquid seed culture medium in 24 ~ 30 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, yeast extract paste 0.4 ~ 0.9%, peptone 0.3 ~ 1.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o 0.001 ~ 0.004%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
(3) pressed by liquid two stage seed in 3 ~ 9% inoculum size access liquid fermentation mediums of fermentating liquid volume, when cultivating 72 ~ 144h for 24 ~ 30 DEG C, namely marine microorganism fermentative production low temperature zonal contribution ratio terminates; Described liquid fermentation medium comprises by weight percentage: konjaku powder 0.4 ~ 4.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o 0.001 ~ 0.004%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
(4) by the fermented liquid of step (3) 4,000 ~ 8,000rpm collected by centrifugation supernatant liquor, the liquid collected is crude enzyme liquid;
(5) different with use object according to difference needs, the crude enzyme liquid (4) obtained is concentrated, separation and purification further, is prepared into the zymin of different activities, purity and formulation.
Further, the activation medium that step (1) produces zonal contribution ratio bacterial classification comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, yeast extract paste 0.4 ~ 0.9%, peptone 0.3 ~ 1.0%, NaCl 0.3 ~ 0.7%, agar 1.8 ~ 2.0%, tap water 100mL, in 0.1MPa, 115 DEG C of steam sterilizing 15min.
The bacterial classification initial activation that the present invention uses and the explanation that growth conditions provides by China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) are carried out.Bacterial strain first activates, step by step after domestication by low temperature, by condition of enzyme production fermentative production low temperature zonal contribution ratio of the present invention, bacterial strain after domestication by low temperature can preserve 2 months in 4 DEG C of environment, and the bacteria suspension made with 25 ~ 50vt% glycerine can long term storage under-80 DEG C of conditions.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the present invention utilizes marine microorganism fermentative production low temperature zonal contribution ratio, the vigor of cold-adapted enzyme can be made to lose through gentle thermal treatment, and low temperature or thermophilic process can not affect the quality of product, particularly there is high enzymatic activity and high catalytic efficiency at low temperatures, improve and improve the quality of products;
(2) this invention simplifies production technique, shorten process period, have great advantage in energy-conservation;
(3) the low temperature zonal contribution ratio of production that obtains of the inventive method is simple to operate, convenient, fast in application, konjac glucomanna can be degraded to the functional oligosaccharides of different polymerization degree, this oligose has hypoglycemic, reducing blood-fat, the physiological function such as anti-oxidant, there is larger using value in food, medicine etc., low temperature zonal contribution ratio can also improve raw material availability and production efficiency, reduce costs, it has a extensive future.
Embodiment
Below by embodiment, the present invention will be further described, but do not limit the present invention.If no special instructions, the present invention is raw materials used all commercially, and preferably, konjaku powder is purchased from the Qingjian River, Wuhan City konjak products company limited.
Embodiment 1
1) medium preparing
1. strain activation and culture base (wt%): konjaku powder 0.4%, yeast extract paste 0.4%, peptone 0.3%, NaCl 0.5%, agar 1.8%, pH nature, tap water 100mL, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2. bacterial classification domestication by low temperature substratum (wt%): konjaku powder 0.4%, NaNO 30.2%, MgSO 47H 2o 0.3%, KCl 0.3%, K 2hPO 40.1%, FeSO 47H 2o 0.001%, agar 1.8%, tap water 100mL, pH 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
3. liquid seed culture medium (wt%): konjaku powder 0.4%, yeast extract paste 0.4%, peptone 0.3%, NaNO 30.2%, MgSO 47H 2o 0.3%, KCl 0.3%, K 2hPO 40.1%, FeSO 47H 2o 0.001%, tap water 100mL, pH 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
4. liquid fermentation medium (wt%): konjaku powder 0.4%, NaNO 30.2%, MgSO 47H 2o 0.3%, KCl 0.3%, K 2hPO 40.1%, FeSO 47H 2o 0.001%, tap water 100mL, pH6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2) will produce the bacterial classification of zonal contribution ratio, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
3) bacterial classification activated domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 30 DEG C → 28 DEG C → 26 DEG C → 24 DEG C, is tamed about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth in low temperature environment;
4), after according to a conventional method the zonal contribution ratio producing strains after domestication by low temperature being cultivated 48 ~ 72h in 24 ~ 26 DEG C in liquid seed culture medium, carry out enlarged culturing step by step by the inoculum size of 5vt%, be prepared into liquid first order seed and secondary seed;
5) by 4) in the secondary seed the prepared liquid fermentation medium by the inoculum size access 5L of fermentating liquid volume 3 ~ 5%, when cultivating 120 ~ 144h for 24 ~ 26 DEG C, namely marine microorganism fermentative production low temperature zonal contribution ratio terminates;
6) by 5) fermented liquid 4,000 ~ 8,000rpm collected by centrifugation supernatant liquor, the liquid collected is crude enzyme liquid; Crude enzyme liquid activity can reach 3500U/mL;
7) need according to difference different with use object, by 6) crude enzyme liquid that obtains concentrates further, separation and purification, is prepared into the low temperature konjac glucomanna zymin of different activities, purity and formulation.
Embodiment 2
1) medium preparing
1. strain activation and culture base (wt%): konjaku powder 0.6%, yeast extract paste 0.5%, peptone 0.6%, NaCl 0.6%, agar 2.0%, pH nature, tap water 100mL, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2. bacterial classification domestication by low temperature substratum (wt%): konjaku powder 0.6%, NaNO 30.5%, MgSO 47H 2o 0.4%, KCl 0.5%, K 2hPO 40.2%, FeSO 47H 2o 0.003%, agar 2.0, tap water 100mL, pH 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
3. liquid seed culture medium (wt%): konjaku powder 0.6%, yeast extract paste 0.5%, peptone 0.6%, NaNO 30.5%, MgSO 47H 2o 0.4%, KCl 0.3%, K 2hPO 40.2%, FeSO 47H 2o 0.003%, tap water 100mL, pH 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
4. liquid fermentation medium (wt%): konjaku powder 2.0%, NaNO 30.5%, MgSO 47H 2o 0.4%, KCl 0.5%, K 2hPO 40.2%, FeSO 47H 2o 0.003%, tap water 100mL, pH6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2) will produce the bacterial classification of zonal contribution ratio, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
3) bacterial classification activated domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 30 DEG C → 28 DEG C → 26 DEG C → 24 DEG C, is tamed about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth in low temperature environment;
4) according to a conventional method the zonal contribution ratio producing strains after domestication by low temperature is cultivated 48 ~ 72h in 26 ~ 28 DEG C in liquid seed culture medium, carry out enlarged culturing step by step by the inoculum size of 5vt%, be prepared into liquid first order seed and secondary seed;
5) by 4) in the secondary seed the prepared liquid fermentation medium by the inoculum size access 50L of fermentating liquid volume 5 ~ 7%, when cultivating 96 ~ 120h for 26 ~ 28 DEG C, namely marine microorganism fermentative production low temperature zonal contribution ratio terminates;
6) by 5) fermented liquid 4,000 ~ 8,000rpm collected by centrifugation supernatant liquor, the liquid collected is crude enzyme liquid; Crude enzyme liquid activity can reach 3587U/mL;
7) need according to difference different with use object, by 6) crude enzyme liquid that obtains concentrates further, separation and purification, is prepared into the low temperature konjac glucomanna zymin of different activities, purity and formulation.
Embodiment 3
1) medium preparing
1. strain activation and culture base (wt%): konjaku powder 1.0%, yeast extract paste 0.9%, peptone 1.0%, NaCl 0.7%, agar 2.0%, pH nature, tap water 100mL, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2. bacterial classification domestication by low temperature substratum (wt%): konjaku powder 1.0%, NaNO 30.7%, MgSO 47H 2o0.5%, KCl 0.5%, K 2hPO 40.3%, FeSO 47H 2o 0.004%, agar 2.0, tap water 100mL, pH 6.0, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
3. liquid seed culture medium (wt%): konjaku powder 1.0%, yeast extract paste 0.9%, peptone 1.0%, NaNO 30.7%, MgSO 47H 2o 0.5%, KCl 0.5%, K 2hPO 40.3%, FeSO 47H 2o 0.004%, tap water 100mL, pH 6.0, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
4. liquid fermentation medium (wt%): konjaku powder 4.0%, NaNO 30.7%, MgSO 47H 2o 0.5%, KCl 0.5%, K 2hPO 40.3%, FeSO 47H 2o 0.004%, tap water 100mL, pH 6.0, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
2) will produce the bacterial classification of zonal contribution ratio, the bacterial strain provided by China General Microbiological culture presevation administrative center (CGMCC) illustrates and carries out initial activation;
3) bacterial classification activated domestication by low temperature step by step in domestication by low temperature substratum, acclimation temperature from high to low, 30 DEG C → 28 DEG C → 26 DEG C → 24 DEG C, is tamed about 8 days, make its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth in low temperature environment;
4) according to a conventional method the zonal contribution ratio producing strains after domestication by low temperature is cultivated 48 ~ 72h in 28 ~ 30 DEG C in liquid seed culture medium, carry out enlarged culturing step by step by the inoculum size of 5vt%, be prepared into liquid first order seed and secondary seed;
5) by 4) in the secondary seed the prepared liquid fermentation medium by the inoculum size access 500L of fermentating liquid volume 7 ~ 9%, when cultivating 72 ~ 96h for 28 ~ 30 DEG C, namely fermentable is produced low temperature zonal contribution ratio and is terminated;
6) by 5) fermented liquid 4,000 ~ 8,000rpm collected by centrifugation supernatant liquor, the liquid collected is crude enzyme liquid; Crude enzyme liquid activity can reach 3499U/mL;
7) need according to difference different with use object, by 6) crude enzyme liquid that obtains concentrates further, separation and purification, is prepared into the low temperature konjac glucomanna zymin of different activities, purity and formulation.

Claims (2)

1. a method for marine microorganism fermentative production low temperature zonal contribution ratio, is characterized in that, specifically comprise the following steps:
(1) actication of culture of zonal contribution ratio can be produced, then through domestication by low temperature step by step, acclimation temperature from high to low, 30 DEG C → 28 DEG C → 26 DEG C → 24 DEG C, makes its well-grown in low temperature environment, when namely bacterial classification can tame end by stable growth at low temperatures; The domestication by low temperature substratum of this bacterial classification comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o0.001 ~ 0.004%, agar 1.8 ~ 2.0%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
The described bacterial classification that can produce zonal contribution ratio derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), bacterium numbering: 1.2244 or 1.504 or 1.775 or 5.809;
(2) according to a conventional method by the zonal contribution ratio producing strains after domestication by low temperature in step (1) on liquid seed culture medium in 24 ~ 30 DEG C of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed; Described liquid seed culture medium comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, yeast extract paste 0.4 ~ 0.9%, peptone 0.3 ~ 1.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o 0.001 ~ 0.004%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
(3) pressed by liquid two stage seed in 3 ~ 9% inoculum size access liquid fermentation mediums of fermentating liquid volume, when cultivating 72 ~ 144h for 24 ~ 30 DEG C, namely marine microorganism fermentative production low temperature zonal contribution ratio terminates; Described liquid fermentation medium comprises by weight percentage: konjaku powder 0.4 ~ 4.0%, NaNO 30.2 ~ 0.7%, MgSO 47H 2o 0.3 ~ 0.5%, KCl 0.3 ~ 0.5%, K 2hPO 40.1 ~ 0.3%, FeSO 47H 2o 0.001 ~ 0.004%, tap water 100mL, pH value is 6.0 ~ 6.5, in 0.1MPa, 115 DEG C of steam sterilizing 15min;
(4) by the fermented liquid of step (3) 4,000 ~ 8,000rpm collected by centrifugation supernatant liquor, the liquid collected is crude enzyme liquid;
(5) different with use object according to difference needs, the crude enzyme liquid (4) obtained is concentrated, separation and purification further, is prepared into the zymin of different activities, purity and formulation.
2. the method for marine microorganism fermentative production low temperature zonal contribution ratio according to claim 1, it is characterized in that, the activation medium that step (1) produces zonal contribution ratio bacterial classification comprises by weight percentage: konjaku powder 0.4 ~ 1.0%, yeast extract paste 0.4 ~ 0.9%, peptone 0.3 ~ 1.0%, NaCl 0.3 ~ 0.7%, agar 1.8 ~ 2.0%, tap water 100mL, in 0.1MPa, 115 DEG C of steam sterilizing 15min.
CN201510084703.1A 2015-02-16 2015-02-16 Method for producing low-temperature glucomannanase by fermentation of marine microorganisms Pending CN104630188A (en)

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CN110791489A (en) * 2018-08-02 2020-02-14 中国农业大学 Efficient and stable α -galactosidase, and coding gene and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060148A (en) * 2017-12-12 2018-05-22 福建农林大学 A kind of method of Ascosphaera apis fermenting and producing zonal contribution ratio
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CN110791489A (en) * 2018-08-02 2020-02-14 中国农业大学 Efficient and stable α -galactosidase, and coding gene and application thereof
CN110791489B (en) * 2018-08-02 2021-08-24 中国农业大学 Efficient and stable alpha-galactosidase and coding gene and application thereof

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