CN104403023B - Method for extracting alkali-insoluble glucan from beer waste yeast residue - Google Patents
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Abstract
The invention discloses a method for extracting alkali-insoluble glucan from beer waste yeast residue. The method comprises the following steps: A, using water to wash beer waste yeast residue after cellular content is extracted from beer waste yeast, preparing a suspension a, adding sodium hydroxide and a microbe protease preparation, performing extraction for 1 h-10 h, and then performing centrifugal separation, so as to obtain a precipitate a; B, washing the precipitate a with water, preparing a suspension b, adding a trypsin preparation, performing extraction for 2 h-12 h, and performing centrifugal separation, so as to obtain a precipitate b; and C, washing the precipitate b and drying to obtain glucan powder. Compared with the prior art, the method is simple in operation, also the extraction purity of glucan is high, also resource waste is avoided, cost is reduced, adverse influence of discharge of the beer waste yeast residue in beer industry to environment is reduced, and a new approach for comprehensive utilization of beer waste yeast is opened up, appreciation and synergy of beer waste yeast are realized, and great economic benefit is generated.
Description
Technical field
The present invention relates to a kind of method extracting alkali-insoluble glucan, more particularly, to one kind is from beer waste yeast residue
The method extracting alkali-insoluble glucan.
Background technology
Zymosan is the main component of yeast cell wall, accounts for the 30% about of dry cell weight, beer yeast cells wall has
Three-decker, outer layer is D- mannosan, and centre is protein, and internal layer is D- glucan, and wherein glucan accounts for 30%～40%.
The insoluble glucan of alkali immiscible acid in beer yeast cells wall is with β-(1,3)-D- glucan as main chain, containing β-(1,6) point
The glucan propping up, the material of energy enhance immunity is exactly β-(1,3)-D- glucan, has and divides compared with the glucan of strong biological activity
Son is measured as 100～200KDa.
Glucan can strengthen the non-specific immune systems of animal, and the activity of activating macrophage, by phagocytosis
Absorb, destroy and remove internal pathogenic microorganism, improve the immunologic function of animal.For example glucan is permissible in aquaculture
Reduce the disease of aquatic livestock especially fish, shrimp.Add glucan and can greatly increase aquatic livestock therefore in the feed of animal
Resistance to disease, promotes animal growth.Additionally, glucan can also promote animal intestines and stomach to wriggle, in absorption enteron aisle
Harmful substance, such as mycotoxin；Also the propagation of enteral beneficial bacterium can be promoted.
Due to the unique immunocompetence of yeast dextran, widely should obtain as a kind of feed addictive at present
With.More to the extracting method of yeast cell wall glucans both at home and abroad, mainly have acidity extraction, alkalinity extraction, soda acid one method,
Solvent Extract methods, autolysis-enzyme-alkali method, ultrasonic wave extraction, enzyme process etc..In the industrial production, can according to different production purposes
So that glucan is extracted from yeast cells using different processes, but more than these methods using pure yeast cells it is
Raw material, complex operation, relatively costly.
Beer waste yeast is the accessory substance of Beer Brewage, is brewageing after main fermentation and after fermentation in beer production
Produced after operation, containing abundant nutrient content and functional components.The industrial at present beer that already starts with gives up
Yeast extracts ribonucleic acid, and to improve the value of beer waste yeast, but in the process, beer waste yeast extracts cell
After content, remaining beer waste yeast residue is not but utilized effectively, and the main component of this beer waste yeast residue is broken
Broken beer yeast cells wall, containing abundant glucan, it directly discharges is the waste to resource.
Content of the invention
The purpose of the present invention is intended to overcome above-mentioned deficiency of the prior art, provide a kind of simple to operate, purification rate is high, keep away
Exempt from the method extracting alkali-insoluble glucan from beer waste yeast residue of the wasting of resources.
For solving above-mentioned technical problem, the present invention employs the following technical solutions：
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is extracted remaining beer waste yeast residue after cellular content wash with water, be then made into suspension
Liquid a, adds NaOH, microprotein enzyme preparation, and using stainless steel reaction under conditions of temperature is 30 DEG C～70 DEG C
Kettle extracts 1h～10h, then extract is centrifuged, and takes precipitation a；
B, enzyme process purify again
Precipitation a that step A is obtained is made into suspension b after washing with water, add trypsin inhibitor, and is 30 in temperature
DEG C～70 DEG C, pH be to extract 2h～12h using stainless steel cauldron under conditions of 6.0～10.0, then extract is centrifuged
Separate, take precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, it is dried, that is, obtain yeast dextran powder；
Preferably, in step, beer waste yeast is carried out sieving, de- bitter, deodorization is configured to yeast concentration after processing
It is 1%～20% suspension by weight, adds sodium chloride, make the sodium chloride concentration in suspension be 1% by weight
～20%, then using NaOH, the pH value of this suspension is adjusted to 7.5, be then used by stainless steel cauldron in temperature 50
DEG C～100 DEG C under conditions of extract 2h～12h, the extract obtaining is rapidly cooled to room temperature, then with plate and frame filter press filter,
The filter residue obtaining is beer waste yeast and extracts remaining beer waste yeast residue after cellular content；
Preferably, in step, in described suspension a the concentration of beer waste yeast residue be 5% by weight～
15%th, the concentration of NaOH is 5%～10% by weight；
Preferably, in step, described microprotein enzyme preparation is Validase TSP Concentrate II, enzyme activity with
The ratio of concentration of substrate is 1000U/g～10000U/g；
Preferably, the centrifugation in step A, step B is all using disk plate centrifuge, rotating speed is 5000r/min, separates
Time is 20min；
Preferably, in stepb, it is 5% that precipitation a that step A is obtained is made into concentration by weight after washing with water
～15% suspension b, the enzyme activity of described trypsin inhibitor is 500U/g～5000U/g with the ratio of concentration of substrate；
Preferably, the drying mode in step C is roller drying, inflow temperature is 100 DEG C～200 DEG C, leaving water temperature
For 50 DEG C～90 DEG C, drum rotation speed is 1r/min～50r/min；
Following characteristics are had according to the yeast dextran powder obtained by said method：Proterties is unformed powder to be dried, uniformly
Unanimously, color is canescence, tasteless, is insoluble in water, by weight, its content of ashes≤7%, moisture≤7%, total reducing sugar contains
Amount >=80%.
The present invention is to produce the beer waste yeast after beer to extract the useless ferment of remaining beer after cellular content with brewery
Female residue is raw material, obtains yeast dextran using the extracting of enzyme process twice.First time enzyme-alkali method extrct removes alkali solubility sweet dew
Glycan, extracts, with enzyme process, the albumen removing on cell membrane second.Yeast cell wall is mainly by glucan and the outer layer of internal layer
Mannosan composition, centre is mingled with protein in cell wall matter, and these albumen are also connected to mannosan with covalent bond, and portion greatly
The glucan dividing is insoluble in alkaline solution, but mannosan is then alkali-soluble.The present invention adopts sodium hydroxide solution pair
Yeast cell wall is stripped, and adds protease in extracting, hydrolyzes the protein that is connected with mannosan so that cell
Mannosan on wall extracts more thorough, realizes being kept completely separate of two kinds of polysaccharide, greatly improves the purity of glucan in precipitation.
In extracting at second simultaneously, also add protease, will be attached to remaining proteolysis on cell membrane and become peptide and amino acid, release
It is put in the aqueous solution so that the purity of glucan improves further in precipitation.
The present invention adds microprotein enzymatic reagent to be because that this protease purification cannot not obtain completely when extracting first time
Entirely, also carry the activity of cellulase and dextranase, it can not only hydrolyze the protein being connected to mannosan so that sweet
Dew glycan is easier to discharge from cell membrane, so that mannosan is extracted more thorough, and can also hydroglucan and sweet dew
Connecting key between glycan is so that mannosan and glucan can more thoroughly separate.
Present invention addition trypsin inhibitor when extracting for second is hydrolyzed and is because that it has very strong enzyme activity
And the special hydrolysis ability for yeast cells wall-held protein, can be clean by the proteolysis on cell membrane, thus being discharged into molten
In liquid, improve the purity of glucan in precipitation further.
Brewer's yeast is the industry byproduct of Beer Brewage, and it is safe and reliable as feedstuff, according to side of the present invention
Yeast dextran powder purity obtained by method is higher, and recovery rate >=36%, it is used directly for feed manufacturing.
Compared with prior art, the invention has the beneficial effects as follows：
The inventive method extracts alkali glucan using enzymatic isolation method twice from beer waste yeast residue, not only simple to operate,
And the DNA purity of glucan is high, the waste of resource can also be avoided, cost-effective, reduce beer waste yeast in brewing industry
The adverse effect to environment for the discharge of residue；Meanwhile, the inventive method can be with the side extracting ribonucleic acid with beer waste yeast
Method is used in combination, and is that the comprehensive utilization of beer waste yeast opens up a new way, realizes appreciating and enlarging of beer waste yeast,
Extend and widen industry chain (supply chain), produce huge economic benefit.
Specific embodiment
In order to be able to preferably understand to technical scheme, carry out in detail below by specific embodiment
Explanation：
Embodiment one
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is extracted remaining beer waste yeast residue after cellular content wash with water, be then made into weight
Gauge concentration is 10% suspension a, adds NaOH, Validase TSP Concentrate II, in suspension a, NaOH is dense
Degree is 7.5% by weight, and the enzyme activity of Validase TSP Concentrate II is 5500U/g with the ratio of concentration of substrate, then in temperature
Spend for extracting 5.5h using stainless steel cauldron under conditions of 50 DEG C, then with disk plate centrifuge with the rotating speed of 5000r/min to taking out
Extract is centrifuged 20min, takes precipitation a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 10% after washing with water, add pancreas egg
White enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 2500U/g, is then 50 in temperature
DEG C, pH be to extract 7h using stainless steel cauldron under conditions of 8.0, then with disk plate centrifuge with the rotating speed of 5000r/min to taking out
Extract is centrifuged 20min, takes precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 150 DEG C, leaving water temperature be 70 DEG C, cylinder turns
Speed carries out roller drying under conditions of 25r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 5.6%, and moisture is 5.1%, and total sugar content (with glucose meter) is
84.7%.
Embodiment two
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is extracted remaining beer waste yeast residue after cellular content wash with water, be then made into weight
Gauge concentration is 15% suspension a, adds NaOH, microprotein enzymatic reagent, the concentration of NaOH in suspension a
It is 10% by weight, the enzyme activity of microprotein enzymatic reagent is 10000U/g with the ratio of concentration of substrate, is then in temperature
Extract 1h using stainless steel cauldron under conditions of 70 DEG C, then with the rotating speed of 5000r/min, extract is entered with disk plate centrifuge
Row centrifugation 20min, takes precipitation a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 15% after washing with water, add pancreas egg
White enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 5000U/g, is then 70 in temperature
DEG C, pH be to extract 2h using stainless steel cauldron under conditions of 10.0, then with disk plate centrifuge with the rotating speed of 5000r/min to taking out
Extract is centrifuged 20min, takes precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 200 DEG C, leaving water temperature be 90 DEG C, cylinder turns
Speed carries out roller drying under conditions of 50r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 6.9%, and moisture is 6.3%, and total sugar content (with glucose meter) is
82.1%.
Embodiment three
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is extracted remaining beer waste yeast residue after cellular content wash with water, be then made into weight
Gauge concentration is 5% suspension a, adds NaOH, Validase TSP Concentrate II, in suspension a, NaOH is dense
Degree is 5% by weight, and the enzyme activity of Validase TSP Concentrate II is 1000U/g with the ratio of concentration of substrate, then in temperature
For under conditions of 30 DEG C using stainless steel cauldron extract 10h, then with disk plate centrifuge with the rotating speed of 5000r/min to extract
It is centrifuged 20min, take precipitation a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 5% after washing with water, add tryptose
Enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 500U/g, then temperature be 30 DEG C,
PH be 6.0 under conditions of using stainless steel cauldron extract 12h, then with disk plate centrifuge with the rotating speed of 5000r/min to extracting
Thing is centrifuged 20min, takes precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 100 DEG C, leaving water temperature be 50 DEG C, cylinder turns
Speed carries out roller drying under conditions of 1r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 7%, and moisture is 7%, and total sugar content (with glucose meter) is
80%.
Example IV
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is carried out sieving, de- bitter, deodorization is configured to the suspension that yeast concentration is 1% by weight after processing
Liquid, adds sodium chloride, makes the sodium chloride concentration in suspension be 1% by weight, then using NaOH, this is suspended
The pH value of liquid is adjusted to 7.5, is then used by stainless steel cauldron and extracts 12h under conditions of temperature 50 C DEG C, by taking out of obtaining
Extract is rapidly cooled to room temperature, then is filtered with plate and frame filter press, and the filter residue obtaining is beer waste yeast and extracts cellular content
Remaining beer waste yeast residue afterwards；
Again the beer waste yeast obtaining residue is washed with water, is then made into the suspension a that concentration by weight is 6%,
Add NaOH, microprotein enzymatic reagent, in suspension a, the concentration of NaOH is 5% by weight, microprotein
The enzyme activity of enzymatic reagent is 5000U/g with the ratio of concentration of substrate, then using stainless steel reaction under conditions of temperature is 60 DEG C
Kettle extracts 10h, then is centrifuged 20min with the rotating speed of 5000r/min to extract with disk plate centrifuge, takes precipitation a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 10% after washing with water, add pancreas egg
White enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 1000U/g, is then 55 in temperature
DEG C, pH be to extract 12h using stainless steel cauldron under conditions of 8.5, then with disk plate centrifuge with the rotating speed of 5000r/min to taking out
Extract is centrifuged 20min, takes precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 150 DEG C, leaving water temperature be 70 DEG C, cylinder turns
Speed carries out roller drying under conditions of 20r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 5.5%, and moisture is 7%, and total sugar content (with glucose meter) is
83.4%.
Embodiment five
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is carried out sieving, de- bitter, deodorization be configured to after processing yeast concentration be by weight 10% outstanding
Supernatant liquid, adds sodium chloride, makes the sodium chloride concentration in suspension be 10% by weight, is then hanged this using NaOH
The pH value of supernatant liquid is adjusted to 7.5, is then used by stainless steel cauldron and extracts 7h under conditions of 75 DEG C of temperature, by the extracting obtaining
Thing is rapidly cooled to room temperature, then is filtered with plate and frame filter press, after the filter residue obtaining is beer waste yeast extraction cellular content
Remaining beer waste yeast residue；
Again the beer waste yeast obtaining residue is washed with water, is then made into the suspension a that concentration by weight is 8%,
Add NaOH, Validase TSP Concentrate II, in suspension a, the concentration of NaOH is 6% by weight, withered grass gemma
The enzyme activity of Bacillus protease is 3000U/g with the ratio of concentration of substrate, then using stainless steel under conditions of temperature is 60 DEG C
Reactor extracts 10h, then is centrifuged 20min with the rotating speed of 5000r/min to extract with disk plate centrifuge, takes precipitation
a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 13% after washing with water, add pancreas egg
White enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 2000U/g, is then 55 in temperature
DEG C, pH be to extract 10h using stainless steel cauldron under conditions of 8.5, then with disk plate centrifuge with the rotating speed of 5000r/min to taking out
Extract is centrifuged 20min, takes precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 140 DEG C, leaving water temperature be 50 DEG C, cylinder turns
Speed carries out roller drying under conditions of 30r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 5.9%, and moisture is 6.4%, and total sugar content (with glucose meter) is
81.7%.
Embodiment six
A kind of method extracting alkali-insoluble glucan residue from beer waste yeast, it comprises the following steps：
A, enzyme-alkali method purification
Beer waste yeast is carried out sieving, de- bitter, deodorization be configured to after processing yeast concentration be by weight 20% outstanding
Supernatant liquid, adds sodium chloride, makes the sodium chloride concentration in suspension be 20% by weight, is then hanged this using NaOH
The pH value of supernatant liquid is adjusted to 7.5, is then used by stainless steel cauldron and extracts 2h under conditions of 100 DEG C of temperature, by taking out of obtaining
Extract is rapidly cooled to room temperature, then is filtered with plate and frame filter press, and the filter residue obtaining is beer waste yeast and extracts cellular content
Remaining beer waste yeast residue afterwards；
Again the beer waste yeast obtaining residue is washed with water, is then made into the suspension a that concentration by weight is 12%,
Add NaOH, Validase TSP Concentrate II, in suspension a, the concentration of NaOH is 9% by weight, withered grass gemma
The enzyme activity of Bacillus protease is 7000U/g with the ratio of concentration of substrate, then using stainless steel under conditions of temperature is 45 DEG C
Reactor extracts 8h, then is centrifuged 20min with the rotating speed of 5000r/min to extract with disk plate centrifuge, takes precipitation
a；
B, enzyme process purify again
The precipitation a that step A is obtained is made into the suspension b that concentration by weight is 8% after washing with water, add tryptose
Enzyme preparation, in suspension b, the ratio of the enzyme activity of trypsin inhibitor and concentration of substrate is 4000U/g, then temperature be 60 DEG C,
PH be 8.0 under conditions of using stainless steel cauldron extract 6h, then with disk plate centrifuge with the rotating speed of 5000r/min to extract
It is centrifuged 20min, take precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, inflow temperature be 160 DEG C, leaving water temperature be 60 DEG C, cylinder turns
Speed carries out roller drying under conditions of 35r/min, that is, obtain yeast dextran powder.
It is that unformed powder is dried according to the yeast dextran powder that this method obtains, uniformity, color is canescence, no
Taste, is insoluble in water, and by weight, its content of ashes is 6.2%, and moisture is 6.6%, and total sugar content (with glucose meter) is
80.7%.
Although reference be made herein to invention has been described for the explanatory embodiment of the present invention, and above-described embodiment is only this
Bright preferably embodiment, embodiments of the present invention are simultaneously not restricted to the described embodiments it should be appreciated that people in the art
Member can be designed that a lot of other modifications and embodiment, all made within the spirit and principles in the present invention any repair
Change, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (7)
1. extract the method for alkali-insoluble glucan a kind of residue from beer waste yeast it is characterised in that it includes following step
Suddenly：
A, enzyme-alkali method purification
Beer waste yeast is extracted remaining beer waste yeast residue after cellular content wash with water, is then made into suspension a,
Add NaOH, microprotein enzyme preparation, and taken out using stainless steel cauldron under conditions of temperature is 30 DEG C～70 DEG C
Carry 1h～10h, then extract is centrifuged, take precipitation a；
B, enzyme process purify again
Precipitation a that step A is obtained is made into suspension b after washing with water, add trypsin inhibitor, and temperature be 30 DEG C～
70 DEG C, pH be to extract 2h～12h using stainless steel cauldron under conditions of 6.0～10.0, then extract is centrifuged,
Take precipitation b；
C, drying
After precipitation b that step B is obtained washes with water, it is dried, that is, obtain yeast dextran powder；
In step, described microprotein enzyme preparation is Validase TSP Concentrate II；
All using disk plate centrifuge, rotating speed is 5000r/min for centrifugation in step A, step B, and disengaging time is 20min.
2. according to claim 1 extract alkali-insoluble glucan method it is characterised in that in step, by beer
Waste yeast carries out sieving, de- bitter, deodorization is configured to the suspension that yeast concentration is 1%～20% by weight after processing, then plus
Enter sodium chloride, make sodium chloride concentration in suspension be 1%～20% by weight, then using NaOH by this suspension
PH value adjust to 7.5, be then used by stainless steel cauldron and extract 2h～12h under conditions of temperature 50 C～100 DEG C, will
To extract be rapidly cooled to room temperature, then with plate and frame filter press filter, the filter residue obtaining be beer waste yeast extract cell
Remaining beer waste yeast residue after content.
3. the method extracting alkali-insoluble glucan according to claim 1 is it is characterised in that in step, described outstanding
In supernatant liquid a the concentration of beer waste yeast residue be by weight 5%～15%, concentration of NaOH be 5% by weight～
10%.
4. the method extracting alkali-insoluble glucan according to claim 1 is it is characterised in that in step, described micro-
Bioprotein enzyme preparation, enzyme activity is 1000U/g～10000U/g with the ratio of concentration of substrate.
5. according to claim 1 extract alkali-insoluble glucan method it is characterised in that in stepb, by step A
The precipitation a obtaining is made into, after washing with water, the suspension b that concentration by weight is 5%～15%, described trypsin inhibitor
Enzyme activity is 500U/g～5000U/g with the ratio of concentration of substrate.
6. the method extracting alkali-insoluble glucan according to claim 1 is it is characterised in that drying mode in step C
For roller drying, inflow temperature is 100 DEG C～200 DEG C, and leaving water temperature is 50 DEG C～90 DEG C, and drum rotation speed is 1r/min～50r/
min.
7. the yeast dextran obtained by the method for extraction alkali-insoluble glucan according to any one of claim 1-6
Powder is it is characterised in that it has following characteristics：Proterties is that unformed powder is dried, uniformity, and color is canescence, tasteless, difficult
It is dissolved in water, by weight, its content of ashes≤7%, moisture≤7%, total sugar content >=80%.
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