CN1683547A - Liquid deep layer fermenting process for producing extracellular polysaccharide using medicinal fungus long root mushroom - Google Patents

Liquid deep layer fermenting process for producing extracellular polysaccharide using medicinal fungus long root mushroom Download PDF

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CN1683547A
CN1683547A CN 200510020385 CN200510020385A CN1683547A CN 1683547 A CN1683547 A CN 1683547A CN 200510020385 CN200510020385 CN 200510020385 CN 200510020385 A CN200510020385 A CN 200510020385A CN 1683547 A CN1683547 A CN 1683547A
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culture
fermentation
dissolved oxygen
cultivated
oudemansiella radicata
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邹祥
胡昌华
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Southwest University
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Southwest University
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Abstract

The deep fermentation process uses the spawn of Oudemansiella radicata from Central-China Agricultural Univ. and includes the following steps: slant spawn culture, shake flask seed culture, 7L fermenting tank culture, and polysaccharide extraction and determination. The present invention selects corn powder and soybean cake powder as main fermentation material and has low production cost. It is found that palmitic acid is key nutritious additive factor capable of raising the yield of extracellular polysaccharide of Oudemansiella radicata up to 30-40 %. The fermentation process is controlled through on-line control of variation in dissolved oxygen concentration. The present invention can realize large scale fermentation with simple technological operation, and is suitable for industrial production.

Description

Utilize medicinal fungus long root mushroom to produce the deep liquid fermentation process of exocellular polysaccharide
Technical field:
The invention belongs to technical field of bioengineering, be the cultivation that utilizes organism, fermentation and prepare known, or new organism, or the technology of its metabolite.Be specifically related to a kind of deep liquid fermentation process that utilizes medicinal fungus long root mushroom to produce exocellular polysaccharide.
Background technology
Oudemansiella Radicata (Oudemansiella radicata) is called long root oudemansiella radicata, the careless bacterium of big hair, long root money bacterium etc.Be subordinate to Basidiomycotina, Hymenomycetes, Agaricales, white mushroom section, oudemansiella radicata genus.Oudemansiella Radicata is top grade in edible, and endoplasm delicacy, handle are crisp good to eat, rich in proteins, amino acid, fat, carbohydrate, VITAMIN and trace element components, and the edibleness height is superior rare edible and medicinal fungi.People such as the heavy Yuan of the Japan big Zhuo of scholar studies confirm that the Oudemansiella Radicata polysaccharide had obvious restraining effect to small white mouse sarcoma S-180 in 1977, and inhibiting rate reaches more than 95%, and ehrlich carcinoma reaches 90%, and can improve body immune function, has very high pharmaceutical use.
Oudemansiella Radicata is a novel medicinal fungi of introducing cultivation in recent years at home; be in experiment in cultivation and popularization stage at present; because solid state cultivation exists the cycle long; yield poorly; shortcomings such as weather effect; limiting its large-scale development uses; and adopt the liquid submerged fermentation culture technique is the new production method of present deep development medicinal fungi active polysaccharide resource; and it is successful glossy ganoderma; mushroom; Grifola frondosa; rare medicinal fungus such as Agaricus blazei Murrill is accomplished scale production; both at home and abroad Oudemansiella Radicata liquid submerged fermentation Study on Technology is started late; particularly on reactor to aspect the engineering science research of Oudemansiella Radicata fermentative production active polysaccharide, also do not see relevant report at present as yet.
Summary of the invention:
Purpose of the present invention mainly is to consider from the angle of suitability for industrialized production, substratum and processing condition to Oudemansiella Radicata fermentation influence have been carried out systematic research, screening obtains reducing the optimum medium composition and the processing condition of industrial production cost, proposes to produce at the horizontal Oudemansiella Radicata liquid submerged fermentation of 7L fermentor tank the zymotechnique of exocellular polysaccharide.
Below be used to realize the concrete technical solution of the object of the invention
Bacterial classification: the bacterial classification that the present invention uses is No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
The Oudemansiella Radicata strain identification: spore is colourless, and is smooth, and oval is to wide circle, 13～18 μ m * 10～15 μ m.The nearly fusiformis of utricule, 75～175 μ m * 10～29 μ m.Pleat edge utricule is colourless, nearly fusiformis, and the top is blunt slightly, 87～100 μ m * 10～25 μ m, appraisal basis (fourth of the twelve Earthly Branches morning mist chief editor: China's economic fungi, in February, 1998, the 1st edition)
The overall procedure of technology of the present invention comprises: slant strains cultivation → shake-flask seed cultivation → 7L fermentor cultivation → polysaccharide extraction and determination
1, slant strains is cultivated: add 5% wheat bran juice with the PDA substratum, cultivate slant strains in vitro, and culture temperature 25-30 ℃, incubation time 7-10 days, pH 6.5-7.0.
2, shake-flask seed is cultivated:
Liquid seed culture medium (g/L): Semen Maydis powder 10-30, peptone 2-5, soybean cake powder 3-5, sucrose 10-30, KH 2PO 42-4, MgSO 47H 2O 1-3, VB 10.1-0.3
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, inoculation 2-3 piece is in liquid seed culture medium, in 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 160-200rev/min, culture temperature 26-30 ℃, pH 6.3-6.8, incubation time 7-10 days.
3,7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 10-40, soybean cake powder 2-5, KH 2PO 42-4, MgSO 47H 2O 1-3, palmitinic acid 0-3, VB 10.1-0.3, pH 6.0-6.5;
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 160-300rev/min, leavening temperature 26-30 ℃, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.Fermentation time is during to 108-115 hour, and dissolved oxygen drops to 35-40%, can stop fermentation.
The palmitinic acid that adds 0-3g/L in above-mentioned fermention medium helps the Oudemansiella Radicata mycelial growth and promotes the synthetic of Oudemansiella Radicata exocellular polysaccharide, and best to add concentration be 1.5g/L, and exocellular polysaccharide is raising 30-40% when not adding.
The fermenting process oxygen update rate changes and the variation of exocellular polysaccharide synthesis rate reaches unanimity, when the exocellular polysaccharide resultant quantity reaches maximum value (2.80-2.87g/l) about 110 hours, dissolved oxygen drops to Schwellenwert (about 35%), the exocellular polysaccharide resultant quantity reduces after 113 hours, dissolved oxygen begins to rise, therefore can online dissolved oxygen concentration be control strategy, judge fast whether fermenting process finishes, when dropping to Schwellenwert when constant, can stop fermentation, this is best fermentation period, and the Oudemansiella Radicata exopolysaccharides also reaches maximum value.
4, exocellular polysaccharide extracts and measures
With the centrifugal supernatant liquor that gets of fermented liquid, in supernatant liquor, add 95% ethanol to 60%, 4 ℃ of refrigerator overnight of concentration, the centrifugal crude extracellular polysaccharide that gets, dissolved in distilled water after the washing is measured polysaccharide content with the phenolsulfuric acid method.
The advantage that the present invention had
The invention provides based on 7L fermentor tank level, utilize medicinal fungus long root mushroom to produce the exocellular polysaccharide deep liquid fermentation process, lay a good foundation for realizing large-scale industrial production, optimize the cheap raw material Semen Maydis powder that screening obtains helping reducing the industrial production cost, soybean cake powder is main fermentation raw material, and find, it is that the factor is added in crucial nutrition that acquisition can significantly improve the palmitinic acid that the Oudemansiella Radicata exopolysaccharides reaches 30-40%, fermenting process is changed to control strategy with online dissolved oxygen concentration, technological operation control is simple in the time of can realizing large scale fermentation, workable characteristics are amplified and are applied thereby help further industrialization.
Embodiment
Embodiment 1 (adding palmitinic acid, 110 hours periods of fermentation)
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
(1) test tube slant is cultivated: cultivate slant strains in vitro
Substratum: the PDA substratum adds the wheat bran juice of 5% (weight percent)
Culture condition: 26 ℃ of culture temperature, incubation time 8 days, pH 6.7.
(3) shake-flask seed is cultivated
Seed culture medium (g/L): Semen Maydis powder 10, peptone 2, soybean cake powder 3, sucrose 20, KH 2PO 42, MgSO 47H 2O 2, VB 10.1;
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, and inoculate the 2-3 piece in liquid seed culture medium, 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 180rev/min, 26 ℃ of culture temperature, pH 6.5, incubation time 7 days.
(4) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 20, soybean cake powder 3, KH 2PO 43, MgSO 47H 2O 2, palmitinic acid 1.5, VB 10.1mg pH 6.2;
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 180rev/min, 26 ℃ of leavening temperatures, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.In the time of about fermentation time to 110 hour, dissolved oxygen drops to about 35%, finishes fermentation, and exocellular polysaccharide is measured 2.80-2.87g/L.
Embodiment 2 (not adding palmitinic acid technology, 110 hours periods of fermentation)
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
(1) test tube slant is cultivated: cultivate slant strains in vitro,
Substratum: the PDA substratum adds the wheat bran juice of 5% (weight percent)
Culture condition: 28 ℃ of culture temperature, incubation time 10 days, pH 6.7.
(2) shake-flask seed is cultivated
Seed culture medium (g/L): Semen Maydis powder 20, peptone 2, soybean cake powder 3, sucrose 20, KH 2PO 42, MgSO 47H 2O 1, VB 10.1.
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, and inoculate the 2-3 piece in liquid seed culture medium, 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 200rev/min, 26 ℃ of culture temperature, pH 6.5, incubation time 7 days.
(3) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 20, soybean cake powder 4, KH 2PO 43, MgSO 47H 2O 2, VB 10.1 pH 6.0;
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 200rev/min, 26 ℃ of leavening temperatures, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.In the time of about fermentation time to 110 hour, finish fermentation, this moment exocellular polysaccharide content 2.1-2.2g/L.
Embodiment 3 (add palmitinic acid, the fermentation period is 113 hours)
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
(1) test tube slant is cultivated: cultivate slant strains in vitro
Substratum: the PDA substratum adds the wheat bran juice of 5% (weight percent)
Culture condition: 26 ℃ of culture temperature, incubation time 8 days, pH 6.5.
(2) shake-flask seed is cultivated seed culture medium (g/L): Semen Maydis powder 10, peptone 2, soybean cake powder 3, sucrose 20, KH 2PO 42, MgSO 47H 2O1, VB 10.1;
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, and inoculate the 2-3 piece in liquid seed culture medium, 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 160rev/min, 26 ℃ of culture temperature, pH 6.5, incubation time 8 days.
(3) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 20, soybean cake powder 3, KH 2PO 43, MgSO 47H 2O 2, palmitinic acid 1.5, VB 10.1 pH 6.0.
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 180rev/min, 26 ℃ of leavening temperatures, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.In the time of about fermentation time to 113 hour, dissolved oxygen drops to about 38%, finishes fermentation, and measuring exocellular polysaccharide content is 2.76-2.84g/L.
Embodiment 4
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
(1) test tube slant is cultivated: cultivate slant strains in vitro
Substratum: the PDA substratum adds the wheat bran juice of 5% (weight percent)
Culture condition: 26 ℃ of culture temperature, incubation time 8 days, pH 6.8.
(2) shake-flask seed is cultivated
Seed culture medium (g/L): Semen Maydis powder 20, peptone 2, soybean cake powder 4, sucrose 20, KH 2PO 44, MgSO 47H 2O2, VB 10.1;
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, and inoculate the 2-3 piece in liquid seed culture medium, 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 200rev/min, 28 ℃ of culture temperature, pH 6.5, incubation time 8 days.
(3) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 30, soybean cake powder 4, KH 2PO 43, MgSO 47H 2O 2, palmitinic acid 1.5, VB 10.1 pH 6.5.
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 180rev/min, 26 ℃ of leavening temperatures, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.In the time of about fermentation time to 110 hour, dissolved oxygen drops to about 35%, finishes fermentation, and measuring exocellular polysaccharide content is 2.65-2.80g/L.
Embodiment 5
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center.
(1) test tube slant is cultivated: cultivate slant strains in vitro
Substratum: the PDA substratum adds the wheat bran juice of 5% (weight percent)
Culture condition: 26 ℃ of culture temperature, incubation time 8 days, pH 6.7.
(2) shake-flask seed is cultivated
Seed culture medium (g/L): Semen Maydis powder 20, peptone 3, soybean cake powder 3, sucrose 10, KH 2PO 42, MgSO 47H 2O1, VB 10.1;
Culture condition: will cut into the mycelia piece of soya bean size with the activatory slant strains, and inoculate the 2-3 piece in liquid seed culture medium, 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 180rev/min, 28 ℃ of culture temperature, pH 6.5, incubation time 9 days.
(3) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 40, soybean cake powder 3, KH 2PO 42, MgSO 47H 2O 1, palmitinic acid 1.5, VB 10.1 pH 6.5.
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank (emerging BIOTECH-7BGZ is protected in Shanghai) of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 180rev/min, 28 ℃ of leavening temperatures, air flow 3L/min, fermenting process dissolved oxygen (DO) is worth by the online detection of dissolved oxygen electrode.In the time of about fermentation time to 110 hour, dissolved oxygen drops to about 37%, finishes fermentation, and measuring exocellular polysaccharide content is 2.54-2.65g/L.

Claims (1)

1, utilize medicinal fungus long root mushroom to produce the deep liquid fermentation process of exocellular polysaccharide,
Bacterial classification: No. 1, Oudemansiella Radicata (Oudemansiella radicata), available from Hua Zhong Agriculture University bacterial classification experimental center;
Carry out according to the following steps:
(1) slant strains is cultivated: add 5% wheat bran juice with the PDA substratum, cultivate slant strains in vitro, and culture temperature 25-30 ℃, incubation time 7-10 days, pH6.5-7.0;
(2) shake-flask seed is cultivated:
Liquid seed culture medium (g/L): Semen Maydis powder 10-30, peptone 2-5, soybean cake powder 3-5, sucrose 10-30, KH 2PO 42-4, MgSO 47H 2O 1-3, VB 10.1-0.3;
Culture condition: the activatory slant strains cuts into the mycelia piece of soya bean size, and inoculation 2-3 piece is in liquid seed culture medium, in 500ml triangular flask liquid amount 100ml, rotary shaking table is cultivated, rotating speed 160-200rev/min, culture temperature 26-30 ℃, pH6.3-6.8, incubation time 7-10 days;
(3) 7L fermentor cultivation
Fermention medium (g/L): Semen Maydis powder 10-40, soybean cake powder 2-5, KH 2PO 42-4, MgSO 47H 2O1-3, palmitinic acid 0-3, VB 10.1-0.3, pH6.0-6.5;
Culture condition: the cultured seed substratum is equipped with in the full-automatic 7L fermentor tank of 5L fermention medium by the inoculum size access of 10% (v/v), mixing speed 160-300rev/min, leavening temperature 26-30 ℃, air flow 3L/min, fermenting process dissolved oxygen (DO) value is by the online detection of dissolved oxygen electrode, fermentation time is during to 108-115 hour, and dissolved oxygen drops to 35-40%, can stop fermentation;
(4) exocellular polysaccharide extracts and measures.
CN 200510020385 2005-02-18 2005-02-18 Liquid deep layer fermenting process for producing extracellular polysaccharide using medicinal fungus long root mushroom Pending CN1683547A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215591B (en) * 2008-01-08 2011-03-30 汪维云 Method for preparing lentinan from mushroom cultivating rod
CN102823937A (en) * 2012-08-16 2012-12-19 湖北中烟工业有限责任公司 Extracting method of submerged fermentation mycelium extractive of lepista nuda and application thereof in cigarettes
CN104541966A (en) * 2014-12-25 2015-04-29 赵晶晶 Production method for extracting agrocybe aegirit mycelium of agrocybe cylindracea polysaccharide
CN105062899A (en) * 2015-08-26 2015-11-18 海南大学 Hymenopellis raphanipes bacterial strain and extraction and application of crude polysaccharide in same
CN110627919A (en) * 2019-11-06 2019-12-31 安徽大学 Intestinal prebiotics black skin termitomyces albuminosus polysaccharide ORP-1 and preparation method and application thereof
CN110973599A (en) * 2019-12-23 2020-04-10 天津实发中科百奥工业生物技术有限公司 Method for preparing oudemansiella radicata nutrition powder by oudemansiella radicata fermentation method
KR102363474B1 (en) * 2021-06-29 2022-02-15 농업회사법인주식회사힘찬 Method of cultivation of oudemansiella raphanipes and oudemansiella raphanipes thereof

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215591B (en) * 2008-01-08 2011-03-30 汪维云 Method for preparing lentinan from mushroom cultivating rod
CN102823937A (en) * 2012-08-16 2012-12-19 湖北中烟工业有限责任公司 Extracting method of submerged fermentation mycelium extractive of lepista nuda and application thereof in cigarettes
CN102823937B (en) * 2012-08-16 2014-12-10 湖北中烟工业有限责任公司 Extracting method of submerged fermentation mycelium extractive of lepista nuda and application thereof in cigarettes
CN104541966A (en) * 2014-12-25 2015-04-29 赵晶晶 Production method for extracting agrocybe aegirit mycelium of agrocybe cylindracea polysaccharide
CN104541966B (en) * 2014-12-25 2017-06-09 绩溪县徽菜宝生物科技有限公司 It is a kind of for extracting the mycelial production method of the agrocybe of Methods of Polysaccharide From Agrocybe Chaxingu
CN105062899A (en) * 2015-08-26 2015-11-18 海南大学 Hymenopellis raphanipes bacterial strain and extraction and application of crude polysaccharide in same
CN105062899B (en) * 2015-08-26 2017-12-05 海南大学 One plant of two spore intends the extraction and application of Aode mushroom bacterial strain and its Thick many candies
CN110627919A (en) * 2019-11-06 2019-12-31 安徽大学 Intestinal prebiotics black skin termitomyces albuminosus polysaccharide ORP-1 and preparation method and application thereof
CN110973599A (en) * 2019-12-23 2020-04-10 天津实发中科百奥工业生物技术有限公司 Method for preparing oudemansiella radicata nutrition powder by oudemansiella radicata fermentation method
KR102363474B1 (en) * 2021-06-29 2022-02-15 농업회사법인주식회사힘찬 Method of cultivation of oudemansiella raphanipes and oudemansiella raphanipes thereof

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