CN101508966B - Method for preparing manna oligosacchride with zymohydrolysis of konjaku flour - Google Patents
Method for preparing manna oligosacchride with zymohydrolysis of konjaku flour Download PDFInfo
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- CN101508966B CN101508966B CN200910014349XA CN200910014349A CN101508966B CN 101508966 B CN101508966 B CN 101508966B CN 200910014349X A CN200910014349X A CN 200910014349XA CN 200910014349 A CN200910014349 A CN 200910014349A CN 101508966 B CN101508966 B CN 101508966B
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Abstract
The invention relates to a method for preparing mannan oligosaccharide by zymolytic konjaku flour, which belongs to the biological technical field of food. In the invention, beta-mannase is prepared after fermentation by using strain Bacillus sp. MSJ-5 producing beta-mannase and having the preservation number of CCTCC No: M208258, and then mannan oligosaccharide is prepared by using the prepared beta-zymolytic konjaku flour of mannase; and the zymolysis conditions are 50 to 55 DEG C, 50 to 70r/min for stirring and 20 to 30 hours for zymolysis. The method can conveniently and cheaply realize industrial production of mannan oligosaccharide, thereby meeting the requirements of the market on mannan oligosaccharide.
Description
Technical field
The present invention relates to a kind of method of preparing manna oligosacchride with zymohydrolysis of konjaku flour, belong to technical field of food biotechnology.
Background technology
Oligosaccharide (Oligosaccharide) is meant the low polymkeric substance that is connected the straight or branched that forms by 2-10 monose by glycosidic link.According to function, oligosaccharide can be divided into common oligosaccharides and functional oligosaccharide.Common oligosaccharides can be digested and assimilated by animal body, also intestinal microflora growth is had certain promoter action when providing energy and nutrition for body, and common have sucrose, maltose, lactose, trisaccharide maltose, a trehalose etc.Functional oligosaccharide is meant that the digestive enzymes in the animal digestive tract do not have an effect to it, can not be digested, absorb by body, but be the required oligosaccharides of intestinal beneficial bacterium propagation such as bifidus bacillus, comprise mannooligo saccharide, wood oligose, oligochitosan, Nutriflora P, lactulose, galactooligosacchari(es, isomaltose, grape oligosaccharides and wood oligose etc., functional oligosaccharide has become the focus of people's research at present.Because functional oligosaccharide can not be digested and assimilated by body, be also referred to as indigestible property oligosaccharides (Non-digestible oligosaccharides, NDOs).In recent years, oligosaccharides is not only a class functional food, as the new physiologically active substance of a class, at aspects such as Nutrition and health care, medical diagnosis on disease and control, livestock-raising, plant growth regulating and disease resistances great application potential is arranged all especially.The exploitation of functional oligosaccharide has developed into one in the world and has related to subjects such as medical science, chemistry, engineering, is applied to the important industry in food, medicine, feed, each field of agricultural.
The physiological function of functional oligosaccharide mainly comprises the following aspects: (1) is adjusted intestinal microflora, (2) and is reduced enteron aisle pH, (3) nutritional product, (4) and can increase excremental dry weight, (5) alleviate constipation symptom, (6) and suppress dysentery, (7) protection gastrointestinal system, respiratory system and urogenital system and avoid absorption that disease propagation, (8) strengthen all kinds of mineral elements, (9) and carbohydrate metabolism and lipometabolic useful influence, (10) are reduced cancer threaten.Just because of above-mentioned physiological function, functional oligosaccharide is considered to functional food, be known as " a kind of in good time appearance, meet that human health requires, be food ingredient target, that physiological function is arranged with active effect to human body ".In addition, functional oligosaccharide shows the speciality that is better than food fibre, because they only need very low per daily dose, and as long as just can not cause symptoms such as diarrhoea, dysentery according to the recommended dose picked-up.Their sugarinesses are slight, do not have bad quality and taste, and water-soluble fully, have good physical stability, can well be linked together with food and drink processing.
Konjaku (glucomannoglycan) is the edibility vegetable jelly that extensively distributes in the area, Asia.Particularly in China, konjaku is used as the hemicellulose based food very long history.But up to the present, this abundant and cheap hemicellulose resource except part as all not obtaining sufficient development and utilization the foodstuff additive of low value.Utilize the beta-mannase enzymic hydrolysis to contain the mannosans vegetable jelly, can generate and have the functional oligosaccharide that different monose are molecular, character is good---mannooligo saccharide.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of bacterial strain of product 'beta '-mannase of new screening is provided, utilize this bacterial strain, the fermentative preparation 'beta '-mannase, and the method for utilizing this 'beta '-mannase preparing manna oligosacchride with zymohydrolysis of konjaku flour is provided.
Summary of the invention
The present invention utilizes the bacterial strain bacillus sp.MSJ-5 that produces 'beta '-mannase to carry out the fermentative preparation 'beta '-mannase, utilize prepared 'beta '-mannase preparing manna oligosacchride with zymohydrolysis of konjaku flour again, can make things convenient for the suitability for industrialized production that realizes mannooligo saccharide cheaply, satisfy the demand of market mannooligo saccharide.
Detailed Description Of The Invention
Technical scheme of the present invention is as follows:
One, produces the bacterial strain of 'beta '-mannase
The bacterial strain of product 'beta '-mannase of the present invention is bacillus sp.MSJ-5, sieves from konjaku ground, Sichuan soil sample, and preserving number CCTCC NO:M208258, depositary institution is Chinese typical culture collection center.
Two, preparing manna oligosacchride with zymohydrolysis of konjaku flour
A kind of method of preparing manna oligosacchride with zymohydrolysis of konjaku flour, step is as follows:
(1) bacterial strain slant culture
Produce the bacterial strain bacillus sp.MSJ-5 of 'beta '-mannase, preserving number CCTCC NO:M208258, bacterial strain slant medium: 1.0～1.2 parts of carob bean gum, (NH
4)
2SO
40.4～0.6 part, K
2HPO
43H
20.8～1.0 part of O, 1.2～1.5 parts in agar, 100 parts in water more than is weight part.
(2) liquid seeds is cultivated
Liquid seed culture medium: 2.0～2.5 parts of yeast powders, 1.0～1.2 parts in wheat bran, K
2HPO
43H
20.8～1.0 part of O, 100 parts in water are weight part, down together.Sterilization, cooling, 5～10 parts of the bacteria suspensions of the slant strains of the bacillus sp.MSJ-5 that inoculation step (1) makes, 28～30 ℃, shake 100～120 rev/mins of bottle rotating speeds, cultivated 20～24 hours, get the liquid seeds of bacillus sp.MSJ-5, stand-by.
(3) fermentative preparation of 'beta '-mannase liquid
The liquid seeds of the bacillus sp.MSJ-5 that step (2) is made by weight 5～8 parts join to produce in the enzyme substratum and ferment, fermentation condition is 30～32 ℃, 27～34 hours.
Described product enzyme substratum is: 2.0～2.5 parts of Rhizoma amorphophalli powders, 1.3～1.5 parts of yeast powders, 1.0～1.2 parts of dregs of beans, K
2HPO
43H
2O0.4～0.5 part, pH 7.0～7.2.The work of 'beta '-mannase enzyme is 651 ± 37U/ml in the 'beta '-mannase liquid that fermentation makes.
Above-mentioned fermenting enzyme liquid has higher enzymic activity, Zulkovsky starch is only had very low enzymolysis activity, Mierocrystalline cellulose and xylan are not had hydrolytic activity mannosans, illustrates in this fermenting enzyme liquid mainly based on mannase.This mannase is to β-1,4-mannosans (Rhizoma amorphophalli powder, carob bean gum) has activity, and to α-1,6-mannosans (yeast mannosans) does not have activity, therefore, the mannase of bacillus sp.MSJ-5 fermentative production is a 'beta '-mannase.With the carob bean gum is substrate, optimal pH 5.5, and pH6～9,50 ℃ following insulation 1 hour still can keep the enzyme more than 70% to live.With the carob bean gum is substrate, and this enzyme is 50～60 ℃ to the suitableeest enzyme of carob bean gum temperature alive, 35～65 ℃, be incubated 1 hour, and still can keep the enzymic activity more than 50%.
(4) 'beta '-mannase preparing manna oligosacchride with zymohydrolysis of konjaku flour
Making Rhizoma amorphophalli powder content with deionized water preparation Rhizoma amorphophalli powder liquid is 10～15mg/ml, the 'beta '-mannase liquid that step (3) is made adds in the Rhizoma amorphophalli powder liquid pH7.0,50～70 rev/mins of stirrings by 'beta '-mannase 5～10U/mg Rhizoma amorphophalli powder, 50～55 ℃, enzymolysis 20-30 hour.Enzymolysis finishes, and heats to 100 ℃, and insulation 5～10min makes enzyme-deactivating, after the cooling, and 10000rpm, the centrifugal 5～10min of room temperature, supernatant is mannooligo saccharide liquid.
Do not limit in detail among the above preparation method, all get final product by state of the art.
Three, the analysis of enzymolysis product
With the enzymolysis product mannooligo saccharide liquid of above-mentioned preparation is the nitrocellulose membrane filtration of 0.22 μ m with the aperture, adopt high performance liquid chromatography (High performance liquid chromatography, HPLC) monose in the mensuration enzymolysis solution and the amount of oligosaccharides, analytical column adopts Aminex HPX-42C (Bio-rad, 300mm * 7.8mm), detector is a RID-10A type differential refraction detector, with distilled water (ddH
2O) be moving phase, flow velocity is 0.4ml/min, and column temperature is 75 ℃.With available from the sweet dew 2-6 sugar of Sigma company as standard substance, detect oligosaccharides product degree of polymerization (as Fig. 1, Fig. 2).Enzymolysis analysis is the result show, 'beta '-mannase produces the polymerization degree in the process of efficient zymohydrolysis of konjaku flour be the oligosaccharides of 2-6, and wherein trisaccharide is a primary product.Best enzymatic hydrolysis condition: concentration of substrate, 10mg/ml, enzyme concn 10U/mg Rhizoma amorphophalli powder.
The excellent results of method of the present invention is as follows:
1, the bacterial strain of product 'beta '-mannase provided by the invention---bacillus sp.MSJ-5 (preserving number CCTCCNO:M208258) has higher product enzyme activity, and the Rhizoma amorphophalli powder that is rich in mannosans can be induced this bacterial strain secretion 'beta '-mannase.The Rhizoma amorphophalli powder that utilization is cheap etc. is a raw material, and the work of 'beta '-mannase enzyme is 651 ± 37U/ml in the 'beta '-mannase liquid that fermentation makes.
2, the present invention utilizes the 'beta '-mannase that bacillus sp.MSJ-5 fermentation makes, efficient zymohydrolysis of konjaku flour, and the polymerization degree of the oligosaccharides that produces in the process of efficient zymohydrolysis of konjaku flour is the oligosaccharides of 2-6, and wherein trisaccharide is a primary product, and the monose seminose seldom.
3, konjaku (glucomannoglycan) is the edibility vegetable jelly that extensively distributes in the area, Asia.In China, this abundant and cheap hemicellulose resource of konjaku except part as all not obtaining sufficient development and utilization the foodstuff additive of low value, the present invention satisfies the demand of market to mannooligo saccharide for the konjaku resource provides the approach of the high-valued mannooligo saccharide of High-efficient Production.
Description of drawings
Fig. 1 is high performance liquid chromatography (HPLC) the assay determination result of the Rhizoma amorphophalli powder of 'beta '-mannase enzymolysis different concns.Use ddH
2O prepares 1,5 respectively, the Rhizoma amorphophalli powder of 10mg/ml, adds the 'beta '-mannase of 5U/mg, according to the condition enzymolysis in the inventive method step (4).X-coordinate is the time, unit: minute; Ordinate zou is a diopter, unit: volt.Curve is zymolyte, the standard substance mixing solutions HPLC elution curve of Rhizoma amorphophalli powder content 10mg/ml, 5mg/ml, 1mg/ml from top to bottom successively among the figure.M1-M6 wherein is a sugared peak, disaccharides peak, trisaccharide peak, tetrose peak, pentasaccharides peak, six sugared peaks successively.The result shows, the Rhizoma amorphophalli powder of 10mg/ml is a concentration of substrate preferably.
Fig. 2 is high performance liquid chromatography (HPLC) the assay determination result of 'beta '-mannase zymohydrolysis of konjaku flour.Use ddH
2The Rhizoma amorphophalli powder of O configuration 10mg/ml adds the 'beta '-mannase of 10U/mg, according to the condition enzymolysis in the inventive method step (4), takes a sample during respectively at 20min, 1h, 2h, 12h, 24h and 48h, and HPLC measures the amount of product.X-coordinate is the time, unit: minute; Ordinate zou is a diopter, unit: volt.Curve is the Rhizoma amorphophalli powder enzymolysis product of different enzymolysis times and the HPLC elution curve of standard substance mannooligo saccharide mixing solutions among the figure.M1-M6 wherein is a sugared peak, disaccharides peak, trisaccharide peak, tetrose peak, pentasaccharides peak, six sugared peaks successively.'beta '-mannase produces the polymerization degree in the process of efficient zymohydrolysis of konjaku flour be the oligosaccharides of 2-6, and wherein trisaccharide is a primary product, can infer from the retention time at trisaccharide peak, and this trisaccharide is based on the grape mannotriose, with a spot of mannotriose.Also comprise a certain amount of mannobiose and very a spot of seminose in the product simultaneously.
Embodiment
The present invention will be further described below in conjunction with embodiment, but not prior to this.Used bacterial strain is bacillus sp.MSJ-5 among the embodiment, preserving number CCTCC NO:M208258; Used Rhizoma amorphophalli powder is available from Chongqing Li Mao development of agricultural products company limited.
Embodiment: the method for preparing manna oligosacchride with zymohydrolysis of konjaku flour, step is as follows:
(1) bacterial strain slant culture
Produce the bacterial strain bacillus sp.MSJ-5 of 'beta '-mannase, preserving number CCTCC NO:M208258, bacterial strain slant medium: 1.1 parts of carob bean gum, (NH
4)
2SO
40.5 part, K
2HPO
43H
2O, 0.9 part, 1.3 parts in agar, 100 parts in water is weight part.Streak inoculation 5%wt.Slant strains is bacteria suspension by prior art arrangement, and is stand-by.
(2) liquid seeds is cultivated
Liquid seed culture medium: 2.2 parts of yeast powders, 1.2 parts in wheat bran, K
2HPO
43H
2O, 100 parts of 0.9 parts, water are weight part, down with.Sterilization, cooling, 7 parts of the bacteria suspensions of the slant strains of inoculation bacillus sp.MSJ-5,28～30 ℃, shake 100～120 rev/mins of bottle rotating speeds, cultivated 22 hours, stand-by as liquid seeds.
(3) fermentation of 'beta '-mannase
The liquid seeds of the bacillus sp.MSJ-5 that step (2) is made by weight 5 parts join to produce in the enzyme substratum and ferment, fermentation condition is 30～32 ℃, 27～34 hours.Producing the enzyme substratum is: 2.2 parts of Rhizoma amorphophalli powders, 1.4 parts of yeast powders, 1.1 parts of dregs of beans, K
2HPO
43H
20.45 part of O, pH 7.0～7.2.The 'beta '-mannase enzyme work that makes under this fermentation condition is 651 ± 37U/ml.
'beta '-mannase enzyme activity determination method: (pH 6.0 to draw 0.9ml 5g/L (W/V) carob bean gum solution, the 20mmol/L phosphoric acid buffer), in 50 ℃ of insulation 5min, add the suitably enzyme liquid of dilution of 0.1ml, and 1ml 20mmol/L phosphoric acid buffer (pH 6.0), behind 50 ℃ of water-bath 10min, the DNS method is surveyed the content of reducing sugar in the enzymolysis solution.Enzyme activity unit is defined as: under above-mentioned reaction conditions, it is enzyme unit (U) alive that per minute discharges the required enzyme amount of 1 μ mol seminose.
(4) preparing manna oligosacchride with zymohydrolysis of konjaku flour
Making Rhizoma amorphophalli powder content with deionized water preparation Rhizoma amorphophalli powder liquid is 10mg/ml, the 'beta '-mannase liquid that step (3) is made adds in the Rhizoma amorphophalli powder liquid pH7.0,50～70 rev/mins of stirrings by 'beta '-mannase 12U/mg Rhizoma amorphophalli powder, 53 ± 1 ℃, enzymolysis 24 hours.Enzymolysis finishes, and heats to 100 ℃, and insulation 8min makes enzyme-deactivating, after the cooling, and 10000rpm, the centrifugal 8min of room temperature, supernatant is mannooligo saccharide liquid.
Claims (2)
1. produce the bacterial strain of 'beta '-mannase, this bacterial strain is genus bacillus (Bacillus sp.) MSJ-5, preserving number CCTCC NO:M208258, depositary institution: Chinese typical culture collection center.
2. the method for a preparing manna oligosacchride with zymohydrolysis of konjaku flour, step is as follows:
(1) bacterial strain slant culture
Produce bacterial strain genus bacillus (Bacillus sp.) MSJ-5 of 'beta '-mannase, preserving number CCTCC NO:M208258, bacterial strain slant medium: 1.0～1.2 parts of carob bean gum, (NH
4)
2SO
40.4～0.6 part, K
2HPO
43H
20.8～1.0 part of O, 1.2～1.5 parts in agar, 100 parts in water is weight part;
(2) liquid seeds is cultivated
Liquid seed culture medium: 2.0～2.5 parts of yeast powders, 1.0～1.2 parts in wheat bran, K
2HPO
43H
20.8～1.0 part of O, 100 parts in water are weight part, down together; Sterilization, cooling, 5～10 parts of the bacteria suspensions of the slant strains of the bacillus sp.MSJ-5 that inoculation step (1) makes, 28～30 ℃, shake 100～120 rev/mins of bottle rotating speeds, cultivated 20～24 hours, get the liquid seeds of genus bacillus (Bacillus sp.) MSJ-5, stand-by;
(3) fermentative preparation of 'beta '-mannase liquid
The liquid seeds of genus bacillus (Bacillus sp.) MSJ-5 that step (2) is made by weight 5～8 parts join to produce in the enzyme substratum and ferment, fermentation condition is 30～32 ℃, 27～34 hours;
Producing the enzyme substratum is: 2.0～2.5 parts of Rhizoma amorphophalli powders, 1.3～1.5 parts of yeast powders, 1.0～1.2 parts of dregs of beans, K
2HPO
43H
2O0.4～0.5 part, pH 7.0～7.2; The work of 'beta '-mannase enzyme is 651 ± 37U/ml in the 'beta '-mannase liquid that fermentation makes;
(4) 'beta '-mannase preparing manna oligosacchride with zymohydrolysis of konjaku flour
Making Rhizoma amorphophalli powder content with deionized water preparation Rhizoma amorphophalli powder liquid is 10～15mg/ml, the 'beta '-mannase liquid that step (3) is made adds in the Rhizoma amorphophalli powder liquid pH7.0,50～70 rev/mins of stirrings by 'beta '-mannase 10～15U/mg Rhizoma amorphophalli powder, 50～55 ℃, enzymolysis 20-30 hour; Enzymolysis finishes, and heats to 100 ℃, and insulation 5～10min makes enzyme-deactivating, after the cooling, and 10000rpm, the centrifugal 5～10min of room temperature, supernatant is mannooligo saccharide liquid.
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| CN106119317A (en) * | 2016-06-30 | 2016-11-16 | 成都永安制药有限公司 | A kind of preparation method of the Rhizoma amorphophalli Oligomeric manna sugar powder improving immunity |
| CN106520727A (en) * | 2016-11-08 | 2017-03-22 | 蔡河齐 | Method for producing beta-mannase by adopting lactic acid bacteria cultured by taking konjaku flour as carbon source |
| CN112029751B (en) * | 2019-06-03 | 2022-05-03 | 中国农业大学 | Production method and application of thermophilic fungus mannase |
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