CN102268468B - Method for preparing mannan-oligosaccharide by using waste beer yeast - Google Patents
Method for preparing mannan-oligosaccharide by using waste beer yeast Download PDFInfo
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Abstract
The invention discloses a method for preparing mannan-oligosaccharide by using waste beer yeast, and relates to the technical field of feed additives. The invention aims to solve the problem of high production cost in the conventional method for preparing the mannan-oligosaccharide by hydrolyzing vegetable polysaccharide through beta-mannase. The preparation method comprises the following steps of: (1) preparing the waste beer yeast into a yeast cell wall liquid or solid; (2) preparing a crude enzyme liquid of iso-mannase; and (3) adding the crude enzyme liquid of iso-mannase prepared in the step (2) into the yeast cell wall liquid or solid prepared in the step (1), and hydrolyzing to obtain the mannan-oligosaccharide. The method is used for producing the mannan-oligosaccharide serving as an animal and aquatic feed additive.
Description
Technical field
The present invention relates to field of feed additive technology, relate in particular to and a kind ofly utilize the beer waste yeast self-dissolving and add the method that microbial enzyme prepares mannooligo saccharide.
Background technology
The brewing industry of China is through the fast development of nearly several more than ten years, and beer production producer has been developed to family more than 800 at present.Since two thousand one, China has become first beer production state in the world, and beer production every year is also with 5%～7% speed increment.2010, the beer production amount reached 4,483 ten thousand tons.Beer waste yeast is the important by-products in the beer production, and the output of yeast slurry accounts for 2%～3% of beer ultimate production.Nearly 900,000 tons～1,350,000 tons in the waste yeast mud that annual China beer producers dumps.The main component of beer waste yeast is protein, polysaccharide, nucleic acid, VITAMIN, trace element and enzyme, and wherein polysaccharide mainly is present in the yeast cells wall, accounts for 60% of cell walls.At present, the comprehensive utilization great majority of beer waste yeast are concentrated on protein, nucleic acid, VITAMIN, trace element and enzyme aspect to wherein, and to the Application and Development research of its cell wall polysaccharides seldom.
Be that the method that raw material prepares cell wall polysaccharides can be summarized as substantially: chemical broken wall (acidolysis, alkaline hydrolysis), physical wall breaking (liquid shear, solid shearing etc.), biological wall breaking (enzymolysis, self-dissolving) with the beer waste yeast.Wherein, chemical broken wall not only can cause the destruction of some nutritive ingredients, and has increased the difficulty of extracts active ingredients; Though the physical wall breaking method is simple, cost is low, the complete reservation nutritive ingredient of energy, its shell-broken effect is relatively poor; And the enzymolysis process in the biological wall breaking can increase extraction cost, so the preparation method of above-mentioned cell wall polysaccharides all is not easy to industrial widespread use.Autolysis method is that to have the fresh viable yeast of enzymic activity be raw material, utilize the enzyme system of yeast cell itself, under certain condition, a kind of method that the glucide in the yeast body, protein and nucleic acid is decomposed into small-molecule substances such as reducing sugar, amino acid, skin class, Nucleotide and extracts in the yeast cell.Autolysis method technology is easy, is widely used, and production cost is low, and environmental pollution is little, is suitable for industrialization and extracts.
Mannooligo saccharide (MOS, be Mannose-oligosaccharides) be that seminose or seminose and glucose are by α-1,2, α-1,3, α-1,6 or β-1,3, the oligosaccharide of β-1,4 glycosidic link composition has the animal gastrointestinal tract micro-ecological environment of adjusting and the immunoregulatory function of animal body.Have important use is worth at aspects such as food, medicine and animal-feeds.Because advantages such as the gentle and easy controls of enzymolysis process reaction conditions, therefore, adopting mannosans enzymic hydrolysis mannosans to prepare mannooligo saccharide is the main method of producing mannooligo saccharide.The source of mannase is more extensive, comprises bacterium, fungi, actinomycetes, plant and mollusk etc.Wherein, microbe-derived mannase has distinguishing features such as pH best point, temperature range and Substratspezifitaet, it is active high, cost is low, steady sources, extraction are convenient, and has widely than animals and plants and to use, and is the main source that produces mannase.At present, the genus bacillus in the bacterium, pseudomonas, vibrios, the streptomycete in the aspergillus in the fungi, mould, the yeast of wood, mould, pore fungus, sclerotinite and the actinomycetes all is the frequent species that produces mannase.According to its effect substrate difference, mannase can be divided into 'beta '-mannase and different mannase, wherein, the effect substrate of 'beta '-mannase is vegetable polysaccharidess such as Rhizoma amorphophalli powder, locust bean gum, the effect substrate of different mannase is different mannosans, and the different mannosans of this class mainly is present in the microbial cell walls such as yeast.At present, mannooligo saccharide mainly is to be prepared from vegetable polysaccharidess such as beta-mannase enzymic hydrolysis Rhizoma amorphophalli powder, locust bean gums, and production cost is higher.And be raw material with different mannosans, adopt different mannase hydrolysis method to prepare mannooligo saccharide and do not see the document record.
Summary of the invention
Prepare mannosans at existing with 'beta '-mannase hydrolyzing plant polysaccharide, the problem that production cost is high, the purpose of this invention is to provide a kind of beer waste yeast that utilizes and make different mannosans, and be hydrolyzed in conjunction with different mannase and prepare the method for mannooligo saccharide.
The technical solution adopted for the present invention to solve the technical problems is: a kind of beer waste yeast that utilizes
The method steps of preparation mannooligo saccharide is as follows:
(1) makes the yeast cells wall liquid or solid by beer waste yeast;
(2) the different mannase crude enzyme liquid of preparation;
(3) will make through step (1) through the different mannase crude enzyme liquid adding that step (2) make
Hydrolysis prepares mannooligo saccharide in the yeast cells wall liquid.
The step of preparation yeast cells wall solid is as follows in the described step (1):
(a) beer waste yeast pre-treatment: the beer waste yeast after will washing carries out centrifugation and obtains yeast slurry;
(b) preparation yeast cells wall liquid: be that the yeast slurry of 50g/L～150g/L, acetic acid-sodium-acetate buffer and the distilled water that mass concentration is 400g/L～500g/L mix with mass concentration, the mixed solution that makes is inserted in the sealed vessel, stir evenly the static 0.5h～1h in back, and under 30 ° of C～80 ° C of temperature, heating in water bath 12h～48h; Yeast slurry, acetic acid-sodium-acetate buffer and three kinds of materials of distilled water are pressed the 1:3.5:4.5 proportioning and are formed;
(c) preparation yeast cells wall solid: will carry out centrifugation through the yeast cells wall liquid that step (b) makes, and abandon supernatant liquor, will precipitate part dry 12h～48h under 40 ° of C～80 ° C of temperature, and make the yeast cells wall solid.
Acetic acid-sodium-acetate buffer described in the described step (b) is that mass concentration is respectively the sodium-chlor of sodium-acetate, 20g/L of acetic acid, the 109.2g/ of 2.4g/L and 1L distilled water is mixed, and the pH value of described acetic acid-sodium-acetate buffer is 6.
The step of the different mannase crude enzyme liquid of preparation is as follows in the described step (2):
(a) preparation seed culture based sols and fermentation culture based sols:
Feedstock production seed culture based sols by following mass parts: yeast cells wall 0.5g～20g,
Peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, seed culture medium pH value of solution value is 5.0～8.0.
Feedstock production fermentation culture based sols by following mass parts: yeast cells wall 0.5g～20g,
Peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, fermention medium pH value of solution value is 5.0～8.0.
(b) preparation of fermentation liquid: bacillus subtilis strain is inoculated in the seed culture based sols that step (a) makes, under 25 ° of C～45 ° C of temperature, cultivate 12h～72h, be that the inoculum size of 10g/L～200g/L is inoculated into the fermention medium water culture that makes through step (a) by mass concentration, temperature is that 25 ° of C～45 ° C, oscillation frequency are 100r/min～250r/min in the vibrator, the shaking culture time is 12h～72h, makes fermented liquid.
(c) preparation of different mannase crude enzyme liquid: the fermented liquid that makes through step (b) is carried out centrifugation, get supernatant liquor and make different mannase crude enzyme liquid.
The preparation process of mannooligo saccharide is as follows in the described step (3):
It is that the yeast cells wall liquid of 10g/L～200g/L makes mixed solution that the different mannase crude enzyme liquid of getting mass concentration and be 200g/L～700g/L adds mass concentration, described pH of mixed value is 5～8, hydrolysis temperature is 30 ° of C～80 ° C, hydrolysis time is 2h～10h, the described mixed solution of heating in water bath is to thick, under 30 ° of C～100 ° C, dry to constant weight, pulverize and make the mannooligo saccharide powder.
The pH value of the mixed solution of described different mannase crude enzyme liquid and yeast cells wall liquid is
5～8; The hydrolysis temperature of described mixed solution is 40-70 ° of C, and hydrolysis time is 4-6h.
The bacillus subtilis strain that adopts in the seed liquor preparation also can substitute with security bacillus cereus or other safe microorganisms that secretes different mannase in actual applications.
The effect of this patent is: utilize the beer waste yeast autolysis method to prepare yeast cells wall, utilizing himself enzyme system to add water under optimum conditions gets final product, reduce unnecessary cost, added the microbe-derived different mannase preparation mannooligo saccharide that is hydrolyzed again.Be the pollution of a solution brewing industry, turn waste into wealth from social benefit, improve the effective measure of environment; From economic benefit, produce mannosans with the residue recycling behind the waste beer yeast extraction bioprotein, improved the added value of waste beer yeast comprehensive utilization.Product stability is good, productive rate is high, and its technology is simple, zero pollution, and has reduced production cost.
Embodiment
Embodiment 1: the method for utilizing beer waste yeast to prepare mannooligo saccharide of the present invention may further comprise the steps:
One, the preparation of yeast cells wall
1) beer waste yeast pre-treatment: beer waste yeast is taken out the back wash with tap water from fermentor tank, water temperature generally is no more than 10 ° of C, to prevent yeast autolysis, waste yeast after the washing is carried out centrifugation obtain yeast slurry, centrifuge speed 3000r/min, the centrifugation time is 30min, and the yeast slurry of handling well is the canescence butteriness, cryopreservation.
2) preparation yeast juice: mass concentration is respectively the acetic acid of 2.2g/L～2.6g/L, the sodium-acetate of 99.2g/L～119.2g/, sodium-chlor and the mixed acetic acid-sodium-acetate buffer that gets of distilled water of 10g/L～30g/L, and the pH value of acetic acid-sodium-acetate buffer is 5～8.Then, be that the yeast slurry of 50g/L～150g/L, acetic acid-sodium-acetate buffer that mass concentration is 400g/L～500g/L and distilled water are formed the preparation yeast juice by the 1:3.5:4.5 proportioning by mass concentration, the yeast juice that makes is inserted in the sealing stainless steel cask, stir evenly the static 0.5h～1h in back, and under 30 ° of C～80 ° C of temperature, heating in water bath 12h～48h.
3) extract yeast cells wall: yeast juice is carried out centrifugation, and centrifuge speed is 1000r/min～9000r/min, and the centrifugation time is 5min～30min.Abandon supernatant liquor, will precipitate part and transfer on the culture dish and put into drying baker, dry 12h～48h under 40 ° of C～80 ° C of temperature makes the yeast cells wall solid, and the yield of yeast cells wall is 99.56%.
Two, the preparation of different mannase
According to its effect substrate difference, mannase can be divided into 'beta '-mannase and different mannase, wherein, the effect substrate of 'beta '-mannase is vegetable polysaccharidess such as Rhizoma amorphophalli powder, locust bean gum, this class mannosans is by β-1, the straight or branched polymerization sugar that 4-D-mannopyranose glycosidic bond is formed by connecting by the monose molecules such as seminose, glucose and semi-lactosi of different quantities.The effect substrate of different mannase is different mannosans, this class mannosans mainly is present in the microbial cell walls such as yeast, be hyperbranched polymer, with α-1, the 6-D-seminose is skeletal chain, wherein most of even whole residue has α-1,2-or-1, the side chain that contains 2～5 mannose residues that 3-connects.Different mannase among the present invention is to utilize subtilis to prepare, and specifically may further comprise the steps:
1) preparation seed culture based sols and fermentation culture based sols: the feedstock production seed culture medium of pressing following mass parts: yeast cells wall 0.5g～20g, peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, the pH value of solution value is 5.0～8.0; Feedstock production fermention medium by following mass parts: yeast cells wall 0.5g～20g, peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, pH value of solution value 5.0～8.0.
2) preparation of seed culture based sols: bacterial strain (subtilis) is inoculated in the test tube of seed culture medium, under 25 ° of C～45 ° C of temperature, cultivate 12h～72h, be that the inoculum size of 10g/L～200g/L is inoculated into the fermention medium water culture by mass concentration, temperature is that 25 ° of C～45 ° C, oscillation frequency are 100r/min～250r/min in the vibrator, the shaking culture time is 12h～72h, makes fermented liquid.
3) preparation of different mannase crude enzyme liquid: fermented liquid is carried out centrifugation, and centrifuge speed is 4000r/min～13000r/min under the low temperature, and centrifugation time is 5min～20min, gets supernatant liquor and is different mannase crude enzyme liquid.
Wherein, peptone is organic compound, it be with meat, casein or gelatin with acid or protease hydrolysis after the dry outward appearance that forms be flaxen pulvis.Peptone is rich in organic nitrogen compound and some VITAMIN and carbohydrate, can be used as the main raw material of microbiological culture media.Bacillus subtilis strain in the step 2 also can be the security bacillus cereus or other secrete the safe microorganism of different mannase.
Three, the preparation of mannooligo saccharide
It is the yeast cells wall liquid of 10g/L～200g/L that the different mannase crude enzyme liquid of getting mass concentration and be 200g/L～700g/L adds mass concentration, it is 5.0～8.0 that the pH value is surveyed in the mixed solution sampling, hydrolysis temperature is 30 ° of C～80 ° C, hydrolysis time is 2h～10h, the heating in water bath mixed solution is to thick, under 30 ° of C～100 ° C, dry to constant weight, pulverize and make the mannooligo saccharide powder.
The preparation of embodiment 2:1kg mannooligo saccharide
Get the 1kg beer waste yeast, to the acetic acid-sodium-acetate buffer that wherein adds 4.375L distilled water and 5.625L (pH=6.0), under 50 ° of C of temperature, water-bath 24h, carry out centrifugation with the mixed solution of rotating speed 3000r/min, centrifugation time is 10min, abandons supernatant liquor, to precipitate part and transfer on the culture dish and put into drying baker, make the yeast cells wall solid behind the dry 24h under 60 ° of C.Get yeast cells wall solid 1kg, add different mannase crude enzyme liquid 6.875L and distilled water 5.625L, regulating the pH value is 6.0, under 55 ° of C of temperature, take out oven dry behind the water-bath hydrolysis 4.5h, and pulverize and make the mannooligo saccharide that weight is 1kg, the effective content of mannooligo saccharide is 6.06%.
The preparation of embodiment 2:1000kg mannooligo saccharide
Get the 1000kg beer waste yeast, to the acetic acid-sodium-acetate buffer that wherein adds 4375L distilled water and 5625L (pH=6.0), under 50 ° of C of temperature, water-bath 24h, carry out centrifugation with the mixed solution of rotating speed 3000r/min, centrifugation time 10min abandons supernatant liquor, to precipitate part and transfer to drying baker, make the yeast cells wall solid behind the dry 24h under 60 ° of C.Get yeast cells wall solid 1000kg, add different mannase crude enzyme liquid 6875L and distilled water 5625L, regulating the pH value is 6.0, under 55 ° of C of temperature, take out oven dry behind the water-bath hydrolysis 4.5h, and grinding makes the mannooligo saccharide that weight is 1000kg.
The preparation of embodiment 3:20 ton mannooligo saccharide
Get 25 tons of beer waste yeasts, to the acetic acid-sodium-acetate buffer that wherein adds 87500L distilled water and 112500L (pH=6.0), under 50 ° of C of temperature, water-bath 24h, carry out centrifugation with the mixed solution of rotating speed 3000r/min, centrifugation time 10min abandons supernatant liquor, to precipitate part and transfer to drying baker, make the yeast cells wall solid behind the dry 24h under 60 ° of C.Get 20 tons of yeast cells wall solids, add different mannase crude enzyme liquid 137500L and distilled water 112500L, regulating pH value is 6.0, under 55 ° of C of temperature, takes out oven dry behind the water-bath hydrolysis 4.5h, and pulverizing to make weight be 20 tons mannooligo saccharide.
To utilize this product of above-mentioned technology acquisition as fodder additives, consumption with 5g/kg adds in the feed of Sarotherodon sp, the rate of body weight gain of its postlarva and fingerling has improved 14.3% and 19.2% respectively, improved serum superoxide dismutases (SOD), N,O-Diacetylmuramidase and alkaline phosphatase activities.Studies show that, add the mannooligo saccharide of 0.50%～0.75% this patent preparation in the feed, can significantly improve the tilapia growth performance, improve the enteron aisle structure function, improve nutrient digestion rate and body non-specific immune function, the suitable addition of the mannooligo saccharide in the Sarotherodon sp feed is recommended as 0.50%.
Those skilled in the art will recognize that; above-mentioned embodiment is exemplary; be in order to make those skilled in the art can better understand this patent content; should not be understood as the restriction to this patent protection domain; change or modify so long as disclose spiritual any being equal to of doing according to this patent, all fall into this patent protection domain.
Claims (2)
1. a method of utilizing beer waste yeast to prepare mannooligo saccharide comprises the steps:
(1) makes the yeast cells wall liquid or solid by beer waste yeast; Concrete treatment process comprises: beer waste yeast pre-treatment, preparation yeast cells wall liquid and preparation yeast cells wall solid three steps; Wherein:
(a) described beer waste yeast pre-treatment: be beer waste yeast to be taken out the back wash with tap water from fermentor tank, water temperature is no more than 10 ° of C, to prevent yeast autolysis, waste yeast after the washing is carried out centrifugation obtain yeast slurry, centrifuge speed 3000r/min, the centrifugation time is 30min, and the yeast slurry of handling well is the canescence butteriness, cryopreservation;
(b) described preparation yeast cells wall liquid: be to be that the yeast slurry of 50g/L～150g/L, acetic acid-sodium-acetate buffer that mass concentration is 400g/L～500g/L and distilled water mix with mass concentration, the mixed solution that makes is inserted in the sealed vessel, stir evenly the static 0.5h～1h in back, and under 30 ° of C～80 ° C of temperature, heating in water bath 12h～48h; Yeast slurry, acetic acid-sodium-acetate buffer and three kinds of materials of distilled water are pressed the 1:3.5:4.5 volume proportion and are formed, described acetic acid-sodium-acetate buffer is respectively the sodium-chlor of sodium-acetate, 20g/L of acetic acid, the 109.2g/L of 2.4g/L and 1L distilled water by mass concentration mixed, and the pH value of described acetic acid-sodium-acetate buffer is 6;
(c) described preparation yeast cells wall solid: be that the yeast cells wall liquid that will make through step (b) carries out centrifugation, abandon supernatant liquor, will precipitate part dry 12h～48h under 40 ° of C～80 ° C of temperature, make the yeast cells wall solid;
(2) the different mannase crude enzyme liquid of preparation; The step of the different mannase crude enzyme liquid of described preparation is as follows:
(a) preparation seed culture based sols and fermentation culture based sols; Press following feedstock production seed culture based sols: yeast cells wall 0.5g～20g, peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, seed culture medium pH value of solution value is 5.0～8.0; Press following feedstock production fermentation culture based sols: yeast cells wall 0.5g～20g, peptone 1g～40g, sodium-chlor 0.5g～20g, water 1000ml, fermention medium pH value of solution value is 5.0～8.0;
(b) preparation of fermentation liquid: bacillus subtilis strain is inoculated in the seed culture medium that step (a) makes, under 25 ° of C～45 ° C of temperature, cultivate 12h～72h, be that the inoculum size of 10g/L～200g/L is inoculated into the fermention medium that makes through step (a) and cultivates by mass concentration, temperature is that 25 ° of C～45 ° C, oscillation frequency are 100r/min～250r/min in the vibrator, the shaking culture time is 12h～72h, makes fermented liquid;
(c) preparation of different mannase crude enzyme liquid: the fermented liquid that makes through step (b) is carried out centrifugation, centrifuge speed is 4000r/min～13000r/min under the low temperature, centrifugation time is 5min～20min, gets supernatant liquor and makes different mannase crude enzyme liquid;
(3) will add in the yeast cells wall liquid or solid that step (1) makes through the different mannase crude enzyme liquid that step (2) make, the preparation mannooligo saccharide is hydrolyzed; The concrete preparation process of mannooligo saccharide is as follows: it is that the yeast cells wall liquid of 10g/L～200g/L makes mixed solution that the different mannase crude enzyme liquid of getting mass concentration and be 200g/L～700g/L adds mass concentration, described pH of mixed value is 5～8, hydrolysis temperature is 30 ° of C～80 ° C, hydrolysis time is 2h～10h, the described mixed solution of heating in water bath is to thick, under 30 ° of C～100 ° C, dry to constant weight, pulverize and make the mannooligo saccharide powder.
2. a kind of method of utilizing beer waste yeast to prepare mannooligo saccharide according to claim 1, it is characterized in that: the hydrolysis temperature of the mixed solution of described step (3) is 30-70 ° of C, and hydrolysis time is 3-7h.
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