CN109912700B - Method for co-producing yeast glucan, mannoprotein and yeast extract by using beta-1, 6 glucanase - Google Patents
Method for co-producing yeast glucan, mannoprotein and yeast extract by using beta-1, 6 glucanase Download PDFInfo
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Abstract
The invention discloses a method for coproducing yeast glucan, mannoprotein and yeast extract by using beta-1, 6 glucanase, which takes yeast cells as raw materials, and obtains the yeast extract by carrying out high-temperature inactivation treatment, centrifugation and supernatant drying on the yeast cells; performing enzymolysis on yeast cell walls by using beta-1, 6 glucanase, centrifuging to obtain an enzymolysis liquid supernatant and an enzymolysis precipitate respectively, and performing water extraction, alcohol precipitation, centrifugal separation and drying on the enzymolysis liquid supernatant to obtain mannoprotein; recovering ethanol, concentrating the rest liquid part, and spray drying to obtain yeast extract; and (4) degreasing, treating by protease and spray drying the enzymolysis precipitate to obtain the yeast glucan. The yeast glucan and mannoprotein prepared by the invention has high purity and yield, the preparation process has low cost, strong acid and strong base reagents are not used, and the environmental pollution is small.
Description
Technical Field
The invention relates to a method for co-producing yeast glucan, mannoprotein and a yeast extract by using beta-1, 6 glucanase.
Background
Yeast is an important food and industrial microorganism, and China has abundant yeast resources. The yeast cell wall is composed of polysaccharides, proteins, and small amounts of lipids, chitin. The two main polysaccharides are, respectively, beta-D-glucan, which accounts for about 50-55% of the dry weight of the cell wall, and is distributed on the inner side of the cell wall to form a cross-linked skeleton structure of the cell wall; mannan proteins are distributed on the outside of the cell wall, accounting for about 40-45% of the dry weight of the cell wall, and confer cellular biological activity and control of cell wall pore size. Both beta-glucan and mannan proteins have abundant biological functions. The yeast beta-glucan has a unique triple helix structure, can be specifically combined with an immune cell receptor of an organism, and is stimulated to be in a high-activity state. The yeast beta-glucan activates macrophages by binding with macrophage receptors, and plays roles in resisting tumors, resisting bacteria, resisting oxidation and healing wounds. The mannoprotein consists of 5-10% of protein and 80-90% of mannan, and has the function of promoting humoral immunity and cellular immunity of animals. Mannoprotein can regulate the balance of intestinal flora, adsorb exogenous pathogenic bacteria and toxin, and has the functions of resisting radiation, resisting oxidation, resisting tumor, etc. In food processing, mannoprotein can effectively reduce the generation of tartaric acid precipitate in the storage process of red wine, maintain the clarity of red wine, weaken the astringency of tannin in the wine and promote the release of aromatic substances of red wine. In addition, the mannoprotein also has a certain emulsification stabilizing effect and the capability of prolonging the shelf life of vegetables and fruits.
Yeast beta-glucan and mannoprotein can be extracted from yeast, and the prior laboratory and industrial preparation processes mainly comprise an acid-base extraction method, a hot water extraction method, an enzyme method and the compound use of the methods. Franziskus K et al (1999) proposed an acid extraction method for mannan, which comprises washing wet yeast, placing in a mixture of ethanol, sulfuric acid and water (2:1:1, v/v/v), heating, centrifuging, collecting supernatant, freeze-drying to obtain mannan, wherein the product contains protein 4.15%, total sugar 90.10%, and mannose 73.2%. CN101117358A discloses an alkaline method for preparing mannan, which comprises treating yeast cell wall with 0.5-1.5mol/L NaOH, removing protein by 12-20% CTAB solution and 0.3-0.8% sodium borate-sodium acetate solution, and drying the supernatant to obtain mannan. The acid-base extraction method mostly uses strong acid and strong base with high concentration, which can not only reduce the activity of the polysaccharide, but also pollute the environment. Liu hong Zhi, etc. (2009) determined the best conditions for preparing yeast mannan by water extraction, using waste saccharomyces cerevisiae as raw material, oscillating and autolyzing for 24h under the conditions of pH6.5, 3% of NaCl by mass fraction, 50 ℃ of temperature and 120r/min of stirring speed; then treating for 3h at 120 ℃; finally, a mannoprotein product with the purity of 92.6 percent is obtained by deproteinization and gel chromatographic column separation. The hot water extraction method has low cost and simple operation, but the obtained product contains soluble amino acid, pigment and other impurities, has low purity and needs to be further purified by adopting ion exchange columns, electrophoresis and other methods.
In the enzymatic method for preparing beta-zymosan, the method of destroying cell wall by autolysis or lysozyme, laminarinase and the like is commonly adopted, and then amylase, alkaline protease and neutral protease are applied to further purify zymosan products. Liu et al (2011) propose a method for preparing yeast beta-glucan by mild treatment, which mainly comprises the following steps: inducing autolysis, high-temperature extraction, homogenizing wall breaking, reflux degreasing and biological enzymolysis, and finally obtaining the beta-D-glucan with the yield of 11 percent and the purity of 93 percent. CN102603917 discloses a method for preparing yeast glucan by an enzyme method, which comprises the steps of desalting, degreasing and drying by utilizing alpha-amylase, saccharifying enzyme, mannosidase and neutral protease to obtain high-purity beta-1, 3-glucan. CN 104862355A discloses a method for co-producing glucan and mannoprotein by using yeast cell walls by an enzyme method, which comprises adding alkaline protease to carry out enzymolysis on the yeast cell walls subjected to high-pressure homogenization treatment, drying heavy centrifugal liquid to obtain glucan, filtering light centrifugal liquid by an alkali nanofiltration membrane, and concentrating and drying trapped liquid to obtain the mannoprotein. In addition, CN1940084A is the only proposal in domestic patent to utilize beta-1, 3-glucosaccharase, beta-1, 6-glucosaccharase and chitinase to carry out combined enzymolysis, and prepare wine additive saccharomyces cerevisiae mannoprotein through pyrolysis. The enzyme preparation can keep the natural structure and the activity function of the zymosan, but the product extracted by the enzyme method has high protein content and low purity, needs to be further purified by methods such as an ion exchange column, electrophoresis and the like, and has high enzyme price and high cost.
Based on the main problem of the extraction of the active components of the prior zymosan, the invention further protects the myxobacteria beta-1, 6-glucanase related to CN 105524934A on the comprehensive utilization application of the enzyme in the zymosan cells. The beta-1, 6-glucanase GluM is glucan which is obtained by purifying extracellular supernatant of Corallococcus sp. The specific enzyme activity of the beta-1, 6-glucanase GluM is as high as 24000 U.mg -1 . While the beta-1, 6-glycosidic bond is an important cross-linking structure in the yeast cell wall, long-chain beta-1, 3-glucan and outer mannan are cross-linked into a net structure, and the destruction of the beta-1, 6-glycosidic bond leads to the lysis of the whole cell wall. In addition, beta-1, 6-glucanases of fungal origin may also play a similar role. CN1940084A utilizes three biological enzymes capable of breaking the whole structural connection of yeast cell walls, and the enzymatic activity and cost are unknown.
According to the invention, only beta-1, 6-glucanase which breaks a cross-linked structure is utilized, and papain which is cheap and mature in market products is combined, so that the release of mannoprotein and yeast glucan can be promoted efficiently and pertinently, and meanwhile, yeast cell resources are fully utilized to obtain three important active components, namely yeast glucan, yeast mannoprotein and yeast extract.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a biological enzyme method, namely a method for co-producing yeast glucan, mannoprotein and yeast extract by using beta-1, 6 glucanase.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for co-producing yeast glucan, mannoprotein and yeast extract by using beta-1, 6 glucanase comprises the steps of taking yeast cells as raw materials, performing high-temperature inactivation treatment on the yeast cells, centrifuging and drying supernatant to obtain the yeast extract; performing enzymolysis on yeast cell walls by using beta-1, 6 glucanase, centrifuging to obtain an enzymolysis liquid supernatant and an enzymolysis precipitate respectively, and performing ethanol precipitation, centrifugal separation and drying on the enzymolysis liquid supernatant to obtain mannoprotein; recovering ethanol, concentrating the rest liquid part, and spray drying to obtain yeast extract; and sequentially carrying out degreasing, protease treatment and spray drying on the enzymolysis precipitate to obtain the yeast glucan.
The yeast extract product obtained by the technical scheme of the invention is a light yellow powdery solid; the mannoprotein product is a white loose solid; the yeast glucan product is a grayish brown, powdered solid.
The method for co-producing the yeast glucan, the mannoprotein and the yeast extract by utilizing the beta-1, 6 glucanase preferably comprises the following steps:
(1) taking yeast cells as raw materials, carrying out high-temperature inactivation treatment on the yeast cells, centrifuging, and respectively collecting precipitates and supernate: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(2) adding beta-1, 6-glucanase liquid into the yeast cell wall obtained in the step (1), carrying out enzymolysis reaction, centrifuging and collecting supernatant;
(3) adding ethanol into the supernatant of the enzymolysis liquid obtained in the step (2) for alcohol precipitation, centrifuging, and collecting precipitates;
(4) adding deionized water into the precipitate obtained in the step (3) for dissolving, performing membrane dialysis, and collecting trapped fluid;
(5) spray drying the trapped fluid obtained in the step (4) to obtain mannoprotein;
(6) precipitating the enzymolysis liquid obtained in the step (2), adding petroleum ether, heating, refluxing, centrifuging, and collecting the precipitate;
(7) adding protease into the precipitate obtained in the step (6), performing enzymolysis reaction, centrifuging, and collecting the precipitate;
(8) and (4) carrying out spray drying on the precipitate obtained in the step (7) to obtain the yeast glucan.
(9) And (4) recovering ethanol from the liquid part obtained in the step (3), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
In the step (1), the yeast cells are preferably any one or more of baker's yeast cells, saccharomyces cerevisiae cells and waste industrial saccharomyces cerevisiae cells.
In the step (1), the waste industrial saccharomyces cerevisiae cells need to be pretreated, and the method comprises the following steps: preparing suspension with mass percentage concentration of 3-15% from waste industrial saccharomyces cerevisiae cells, passing through a sieve with the aperture of 80-120 meshes, centrifugally washing for 3-5 times by distilled water, wherein the centrifugal rotation speed is 10000-15000 rpm, and the centrifugal time is 10-15 min.
In the step (1), preferably, the yeast cells are prepared into suspension with the mass percentage concentration of 6-15%, and the suspension is subjected to high-temperature treatment at the temperature of 110-.
In the step (2), the centrifugal rotation speed of the collected enzyme liquid is 10000-.
In the step (2), the beta-1, 6-glucanase is preferably beta-1, 6-glucanase shown in GenBank No. MH747076, GenBank No. NP596461 or GenBank No. XP024773174.
In the step (2), the beta-1, 6-glucanase is preferably selected from the group consisting of beta-glucanase and beta-glucanase with a preservation number of CCTCC NO: (ii) Corallococcus sp. EGB from M2012528 (GenBank No. MH747076 for. beta. -1, 6-glucanase), or fermentation supernatant from Schizosaccharomyces pombe (GenBank No. NP596461 for. beta. -1, 6-glucanase) or Trichoderma harzianum (Trichoderma harzianum, GenBank No. 024773174 for. beta. -1, 6-glucanase); more preferably, the gene is derived from a collection number of CCTCC NO: (ii) Corallococcus sp. EGB from M2012528 (accession No. GenBank No. MH747076 for. beta. -1, 6-glucanase).
In the step (2), the beta-1, 6-glucanase is derived from a strain with a preservation number of CCTCC NO: and (3) inoculating the Corallococcus sp.EGB of M2012528 into a VY/4 liquid enzyme production culture medium, performing fermentation culture, and centrifuging to collect a supernatant so as to obtain the beta-1, 6 glucanase in the step (2).
CorallococcEGB in VY/4 liquid medium formula: 0.40% yeast cell wall, 0.1% CaCl 2 0.1 percent yeast extract (w/v), pH7.0, the rotating speed is 150-250rpm, the fermentation temperature is 20-40 ℃, the culture time is 2-5 days, the thalli are removed by centrifugation, and the obtained supernatant is the beta-1, 6-glucanase enzyme liquid which is used for the subsequent yeast cell treatment.
The method utilizes the sequence derived from CCTCC NO: the enzymolysis conditions of the beta-1, 6 glucanase of the Corallococcus sp.EGB of M2012528 for the enzymolysis of the yeast cell wall are as follows: the enzyme dosage is 2-10U/g yeast, the pH value of the enzymolysis system is 6-8, the time is 12-24h, the temperature is 30-50 ℃, and the stirring speed is 150-250 rpm.
In the step (3), preferably, the dosage of the ethanol is two to four times of the volume of the obtained supernatant, the temperature is 0-4 ℃, and the mixture stays overnight; and (3) centrifuging and collecting the precipitate, wherein the centrifugation rotation speed is 6000-10000 rpm, and the centrifugation time is 5-10min, or performing filter pressing on the precipitate by using a plate frame to obtain the precipitate.
In the step (4), preferably, the precipitate obtained in the step (3) is dissolved by adding deionized water to prepare a solution to be dialyzed with the mass concentration of 2-8%, and the cut-off molecular weight of the dialysis membrane is 5-10kDa, preferably 7kDa, and the temperature is 0-4 ℃.
In the step (6), preferably, the using amount of the petroleum ether is 5-40mL/g, the reflux temperature is 60-95 ℃, and the reflux time is 2-4 h.
The enzymolysis conditions in the step (7) are preferably as follows: selecting papain, wherein the dosage of the papain is 5000-60000U/g, the time is 5-15h, the temperature is 50-70 ℃, and the stirring speed is 150-250 rpm.
In one embodiment of the present invention, the yeast extract product obtained in step (1) is a light yellow, powdery solid, and the yield is 56.5% (based on the dry weight of the yeast cells, the same applies below).
In one embodiment of the present invention, the mannoprotein product obtained in step (6) is a white, loose solid with a yield of 11.1% and a purity of 90.66%.
In one embodiment of the present invention, the yeast glucan product obtained in step (9) is a dark brown, powdered solid, with a yield of 17.4% and a purity of 85.36%.
The beta-1, 6-glucanase in the Corallococcus sp EGB fermentation liquor has the capacity of cracking the cell wall of yeast. The invention simultaneously selects beta-1, 6-glucanase from different microbial sources and beta-1, 6-glucanase prepared by different preparation methods, such as Schizosaccharomyces pombe (S
Santero E et al, 2010)), Trichoderma harzianum (T.harzianum (Manuel Montero et al, 2005)) to perform heterologous expression or direct culture and purification separation on the beta-1, 6-glucanase, observing the cracking condition of the beta-1, 6-glucanase on the yeast cell wall, finding that the beta-1, 6-glucanase of different sources and different preparation methods can crack the yeast cell wall to realize the function of preparing yeast mannoprotein and yeast glucan, so the practical application of the beta-1, 6-glucanase of different sources and different preparation methods also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following advantages and effects:
1. beta-1, 6-glucan is an important cross-linking structure in yeast cell walls, long-chain beta-1, 3-glucan and outer mannan are cross-linked into a network structure, and the destruction of beta-1, 6-glucan will result in the lysis of the entire cell wall. Therefore, the beta-1, 6-glucanase with high specific enzyme activity is utilized to act on the beta-1, 6-glycosidic bond in the yeast cell wall, the yeast cell wall can be efficiently and specifically enzymolyzed, the release of yeast glucan and mannoprotein on the cell wall is promoted, and the yield of the yeast glucan and mannoprotein is improved.
2. The invention adopts beta-1, 6-glucanase to treat yeast cells to extract yeast mannoprotein, can avoid the damage of protease treatment to the yeast mannoprotein caused by long-time high-temperature water treatment, and simultaneously, the water extraction and alcohol precipitation method can simply and quickly obtain the yeast mannoprotein, avoid the equipment and operation complexity caused by the technology of using a nanofiltration membrane and the like, and improve the purity of the yeast mannoprotein.
3. The invention can realize the full enzymolysis of the yeast cell wall by utilizing beta-1, 6 glucanase secreted by Corallococcus sp.
4. The invention combines high-temperature inactivation, biological enzymolysis and spray drying technology, and the adopted equipment can meet the requirement of industrial production, does not relate to strong acid, strong alkali and other reagents, has no pollution to the environment, and is easy for industrial large-scale production.
5. The invention takes the yeast cells as raw materials, coproduces three important yeast active components of yeast glucan, mannoprotein and yeast extract, and fully exerts the additional value of the yeast cells.
In conclusion, the beta-1, 6 glucanase is utilized for the first time to effectively promote the release of yeast glucan and mannoprotein in the yeast cell wall, and the mannoprotein and the yeast glucan with high purity and high yield are efficiently prepared. Meanwhile, the enzyme method has simple preparation process, avoids complex process flow, saves cost, avoids using strong acid and strong alkali, reduces pollution to the environment, and plays an important role in improving the added value of yeast cell products, driving the development of the brewing industry and improving the economic benefits of the large-scale enzyme application industry.
Drawings
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a high performance liquid chromatogram of the product yeast glucan obtained in the invention after acid hydrolysis;
FIG. 3 is a high performance liquid chromatogram of the mannoprotein product obtained by the present invention after acid hydrolysis;
FIG. 4 is an infrared spectrum of the yeast glucan product obtained in the present invention;
FIG. 5 is an infrared spectrum of mannoprotein of the product of the invention;
FIG. 6 is a scanning electron microscope image of the yeast glucan product of the present invention.
FIG. 7 is a scanning electron microscope image of the yeast glucan product of the present invention.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, which are carried out in the light of the technical solutions of the present invention, and it should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.
Example 1 a method for co-producing yeast glucan, mannoprotein and yeast extract using beta-1, 6 glucanase, using baker's yeast as raw material, comprising the following steps (as shown in figure 1):
(1) bread yeast is used as a raw material, yeast cells are prepared into 9% suspension by mass percentage, high-temperature inactivation treatment is carried out at the temperature of 110 ℃, the time of 20min and the pressure of 0.1 Mpa, the suspension is centrifuged at the rotating speed of 8000rpm for 10min, and precipitates and supernatant are respectively collected: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(2) inoculation of Corallococcus sp. EGB in VY/4 liquid enzyme production Medium (0.40% Yeast cell wall, 0.1% CaCl 2 0.1 percent yeast extract (w/v), pH7.0), fermenting and culturing for 2 days, centrifuging and collecting enzyme liquid, wherein the centrifugal rotating speed is 12000 rpm, and the centrifugal time is 15min, so as to obtain beta-1, 6-glucanase enzyme liquid;
(3) adding the yeast cell wall obtained in the step (1) into the beta-1, 6 glucanase liquid obtained in the step (2), and carrying out enzymolysis under the conditions that the enzyme dosage is 8U/g yeast, the enzymolysis system pH is 7, the time is 24h, the temperature is 37 ℃, and the stirring speed is 220 rpm. After enzymolysis, centrifuging and collecting supernatant, wherein the centrifugation speed is 8000rpm, and the centrifugation time is 10 min;
(4) adding ethanol with the volume being three times that of the supernatant of the enzymolysis liquid obtained in the step (3) to carry out alcohol precipitation at the temperature of 4 ℃, standing overnight, centrifuging and collecting precipitates, wherein the centrifugal speed is 8000rpm, and the centrifugal time is 10 min;
(5) adding deionized water into the precipitate obtained in the step (4) to prepare a solution with the mass fraction of 5%, performing membrane dialysis by using a membrane with the molecular weight cutoff of 7kDa at the temperature of 4 ℃, and collecting the trapped fluid after 2 days;
(6) and (5) spray drying the trapped fluid obtained in the step (5) to obtain the yeast mannoprotein.
(7) Adding 20mL/g of petroleum ether into the enzymatic hydrolysate precipitate obtained in the step (3), heating and refluxing for 3h at the temperature of 60 ℃, centrifuging and collecting the precipitate at the rotation speed of 8000rpm for 10 min;
(8) adding 40000U/g of papain into the precipitate obtained in the step (7), carrying out enzymolysis for 10 hours at 65 ℃ and at a stirring speed of 180rpm, and carrying out centrifugal collection on the precipitate at a rotation speed of 8000rpm for 10 min;
(9) and (5) carrying out spray drying on the precipitate obtained in the step (8) to obtain the yeast beta-glucan.
(10) And (4) recovering ethanol from the liquid part obtained in the step (4), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
The yeast extract obtained in this example was a pale yellow, powdery solid with an yield of 56.5%; the mannoprotein is white and loose solid, the yield of the obtained yeast mannoprotein is 11.1 percent, and the purity is 90.66 percent; yeast glucan is a grayish brown powdery solid with a yield of 17.4% and a purity of 85.36%.
Example 2
About 20mg of the yeast glucan and mannoprotein obtained in example 1 were weighed, 10mL of trifluoroacetic acid (4M) was added, and hydrolysis was carried out at 100 ℃ for 4 hours. The hydrolysate was rotary evaporated at 80 ℃ and adjusted to neutrality with 4M NaOH. Absorbing 150 mu L of hydrolysate, adding 150 mu L of 0.6M NaOH, adding 200 mu L of 0.5M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution, and carrying out water bath reaction at 70 ℃ for 90 min. After the reaction is finished, the reaction solution is cooled to room temperature, 0.3M HCl is added into the reaction solution to adjust the reaction solution to be neutral, and deionized water is added to make up to 1 mL. PMP was extracted by adding 1mL of chloroform, centrifuged at 12000 rpm for 1min, and extracted repeatedly three times. The upper aqueous phase solution passes through a 0.22 mu m aqueous phase filter membrane, and 20 mu L of the upper aqueous phase solution is taken out and put on a machine. The derived sample is subjected to C18 chromatographic column and ultraviolet detector OD 230 The mobile phase was a mixed solution of PBS and acetonitrile (PBS 0.02M, pH6.7, PBS: acetonitrile volume ratio 83:17), flow rate was 1.0ml/min, column temperature was 30 ℃.
As can be seen from FIG. 2, the HPLC chromatogram of the standard shows that the retention time of the mannose standard is 25.5min, the retention time of the glucose standard is 41.3min, and the peak of the chromatogram with the retention time of 18.3min represents PMP, a monosaccharide-derived solvent. The HPLC chromatogram of yeast glucan shows chromatographic peaks of only two monosaccharides, the chromatographic peak with a retention time of 25.5min is mannose generated after hydrolysis of the yeast glucan product, the chromatographic peak with a retention time of 41.1min is glucose generated after acid hydrolysis of the yeast glucan product, but the peak area of glucose accounts for 94.92% of the total peak area, and the peak area of mannan accounts for 5.07% of the total peak area. Therefore, the yeast glucan product prepared by the method has higher purity. As can be seen from FIG. 3, the HPLC chromatogram of the sample showed a peak of only one monosaccharide, and the peak of the chromatogram with a retention time of 25.5min was the peak of mannose produced by acid hydrolysis of the mannoprotein product of example 1. Therefore, the mannoprotein product prepared by the method has high purity and no other heterosaccharide.
Example 3
The content of each component of the yeast glucan and mannoprotein obtained in example 1 was determined. The measurement of crude protein and ash content was carried out by AOAC method (1990); the total sugar is determined by adopting an anthrone-sulfuric acid method; the determination of yeast glucan and mannoprotein is carried out in example 2, using external standard method for quantification.
As a result, as shown in Table 1, the yeast cells were composed mainly of polysaccharides and proteins, and had a total sugar content of 34.32% (wherein glucan was 20.56% and mannan was 13.32% of the dry weight of the cells) and a protein content of 59.31%. The yield of the mannoprotein prepared by the method is 11.1 percent, and the purity is 90.66 percent; the yield of yeast glucan was 17.44% and the purity was 85.36%.
TABLE 1 analysis of Yeast and Yeast polysaccharide components
Example 4
Infrared spectroscopy was used to measure the yeast glucan and mannoprotein obtained in example 1, and 5mg of the anhydrous sample was placed under the probe and BIO-RAD Win-IR was used at 500-4500cm -1 Scanning is performed over the range.
As shown in FIG. 4, 1076cm -1 、1427/1460cm -1 The absorption peak of (1) is a characteristic peak of the polysaccharide; 890cm -1 The characteristic peaks in the vicinity proved to be characteristic absorption peaks of the angle-varying oscillation of beta-D-pyranose C-H, TableThe component contains beta-D-glucopyranose ring, and molecules are connected by beta-glycosidic bond; 1255/1252cm -1 、1375/1372cm -1 、2923cm -1 The characteristic peak of (A) indicates that it is a glucan having a β - (1-3) bond. As shown in FIG. 5, 3400/3435cm -1 The absorption peak of (A) is that-O-H on the polysaccharide forms intermolecular and intramolecular hydrogen bonds; 2900/2930cm -1 The absorption peak of (A) is C-H stretching vibration, which is a characteristic peak of the saccharides; 1645/1652cm -1 For amide characteristic absorption peaks, it can be concluded that the protein in the sample is likely to be a glycobinding protein; 810/820cm -1 The absorption peak of (A) is a characteristic peak of alpha-mannose pyran configuration. The results show that the tested sample has characteristic absorption peaks of mannoprotein. The results show that the tested sample has a characteristic absorption peak of the yeast glucan.
Example 5
The yeast extract obtained in example 1 was a yellow, powdery solid. The determination of each physical and chemical index of the yeast extract is carried out according to the method of the national standard GB/T23530-2009.
TABLE 2 physical and chemical indexes of Yeast extract
Example 6
A small amount of dried yeast glucan and mannan sample is adhered to a sample table, and is placed in a vacuum spraying degree instrument to be plated with a conductive film (gold), and then the observation is carried out by using a scanning electron microscope.
As shown in figures 6 and 7, the yeast cells are subjected to high-temperature inactivation, beta-1, 6 glucanase enzymolysis, and enzymolysis supernatant is subjected to water extraction, alcohol precipitation, membrane dialysis and spray drying to obtain mannoprotein; the scanning electron microscope image of the yeast glucan obtained by degreasing, protease treatment and spray drying of the enzymolysis precipitate shows that the polysaccharide is in a characteristic crosslinking and porous state.
Example 7
A method for co-producing yeast glucan, mannoprotein and yeast extract by utilizing beta-1, 6 glucanase takes waste industrial saccharomyces cerevisiae as a raw material, and comprises the following steps:
(1) waste industrial saccharomyces cerevisiae cells are used as raw materials, the yeast cells are prepared into suspension with the mass percentage concentration of 5%, the suspension passes through a screen with the aperture of 100 meshes, and is centrifugally washed for 5 times by distilled water, the rotating speed is 10000 rpm, and the centrifugal time is 10 min.
(2) Preparing the cleaned and impurity-removed yeast cells into 9% suspension, performing high-temperature inactivation treatment at 120 ℃, 30min and 0.1 Mpa, centrifuging the suspension for 15min at 8000rpm, and respectively collecting precipitate and supernatant: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(3) inoculating Corallococcus sp.EGB into a VY/4 liquid enzyme production culture medium, fermenting and culturing for 3 days, centrifuging and collecting enzyme liquid, wherein the centrifugal rotation speed is 10000 rpm, and the centrifugal time is 10min, so as to obtain beta-1, 6 glucanase liquid;
(4) adding the yeast cell wall obtained in the step (1) into the beta-1, 6 glucanase obtained in the step (2), and carrying out enzymolysis under the conditions that the enzyme dosage is 10U/g yeast, the enzymolysis system pH7.5, the time is 24h, the temperature is 40 ℃, and the stirring speed is 250 rpm. After enzymolysis, centrifuging and collecting supernatant, wherein the centrifugal speed is 9000 rpm, and the centrifugal time is 15 min;
(5) adding ethanol with the volume being three times of that of the supernatant of the enzymolysis liquid obtained in the step (3) to carry out alcohol precipitation at the temperature of 4 ℃, standing overnight, centrifuging and collecting precipitates, wherein the centrifugal speed is 10000 rpm, and the centrifugal time is 15 min;
(6) adding deionized water into the precipitate obtained in the step (4) to prepare a solution with the mass fraction of 8%, performing membrane separation by using a membrane with the molecular weight cutoff of 7kDa at the temperature of 4 ℃, and collecting the cutoff solution after 1 day;
(7) and (5) spray drying the trapped fluid obtained in the step (5) to obtain the mannoprotein.
(8) Adding 40mL/g of petroleum ether into the enzymatic hydrolysate precipitate obtained in the step (3), heating and refluxing for 2h at 65 ℃, centrifuging and collecting the precipitate, wherein the rotating speed is 8000rpm, and the time is 10 min;
(9) adding the sediment obtained in the step (7) into papain 20000U/g, carrying out enzymolysis for 15h at 40 ℃, stirring at 220rpm, centrifuging, and collecting the sediment at 8000rpm for 10 min;
(10) and (4) carrying out spray drying on the precipitate obtained in the step (8) to obtain the yeast glucan.
(11) And (5) recovering ethanol from the liquid part obtained in the step (5), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
The yeast extract obtained in this example is a light yellow, powdery solid, with a yield of 58.9%; the mannoprotein is white and loose solid, the yield of the obtained mannoprotein is 9.5 percent, and the purity is 89.24 percent; yeast glucan is a gray brown powdery solid with a yield of 16.3% and a purity of 81.32%.
Example 8a process for co-production of yeast glucan, mannoprotein and yeast extract using a chestnut wine schizosaccharomyces s.pombe derived β -1,6 glucanase.
Heterologous expression and isolation and purification of beta-1, 6 glucanase (NM001922380.2) from schizosaccharomyces pombe s.pombe, methods referred to CN 105524934A. P. pomb genome as template, beta-1, 6 glucanase gene full length PCR amplification and pMD19-T Vector overnight enzyme linked. E.coli DH5 alpha competent cells were transformed with the enzyme-linked product, and the monoclonals were selected for culture and were sequenced to verify that the sequences were correct. The recombinant plasmid extracted above and pET-29a (+) are subjected to double enzyme digestion by EcorI and HindIII, and enzyme linkage. Correct pET-29a (+) -exg was transformed into E.coli BL21(DE3) competent cells, expression strains were constructed, and single clones were selected for induction culture. Collecting thalli, carrying out ultrasonic treatment to break thalli cells, and centrifuging to obtain a supernatant, namely the crude enzyme liquid of the beta-1, 6-glucanase. And carrying out ammonium sulfate fractional precipitation on the generated crude beta-1, 6-glucanase liquid, purifying the recombinant beta-1, 6-glucanase by combining purification means such as adsorption and desorption, carrying out dialysis treatment on the purified beta-1, 6-glucanase, and storing for later use.
Baker's yeast is taken as a raw material, and the heterologously expressed beta-1, 6 glucanase from S.pombe is utilized to co-produce mannoprotein, yeast glucan and yeast extract, comprising the following steps:
(1) using baker's yeast as raw material, preparing yeast cell into 9% suspension, inactivating at 115 deg.C for 20min under 0.1 Mpa, centrifuging the yeast suspension at 8000rpm for 15min, and collecting precipitate and supernatant: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(2) adding the yeast cell wall obtained in the step (1) into the beta-1, 6 glucanase from the s.pommbe source, and carrying out enzymolysis under the conditions that the enzyme dosage is 12U/g yeast, the enzymolysis system pH is 7, the time is 24h, the temperature is 37 ℃, and the stirring speed is 220 rpm. After enzymolysis, centrifuging and collecting supernatant, wherein the centrifugation speed is 8000rpm, and the centrifugation time is 10 min;
(3) adding ethanol with the volume being three times that of the supernatant of the enzymolysis liquid obtained in the step (4) to carry out alcohol precipitation at the temperature of 4 ℃, standing overnight, centrifuging and collecting precipitates, wherein the centrifugal speed is 8000rpm, and the centrifugation time is 10 min;
(4) adding deionized water into the precipitate obtained in the step (5) to prepare a solution with the mass fraction of 5%, performing membrane separation on a membrane with the molecular weight cutoff of 7kDa at the temperature of 4 ℃, and collecting the cutoff liquid after 2 days;
(5) and (4) spray drying the trapped fluid obtained in the step (6) to obtain the yeast mannoprotein.
(6) Adding 30mL/g of petroleum ether into the enzymatic hydrolysate precipitate obtained in the step (2), heating and refluxing for 3h at the temperature of 60 ℃, centrifuging and collecting the precipitate at the rotation speed of 8000rpm for 10 min;
(7) adding 40000U/g of papain into the precipitate obtained in the step (6), carrying out enzymolysis for 10 hours at 65 ℃ and at a stirring speed of 180rpm, and carrying out centrifugation to collect the precipitate at a rotation speed of 8000rpm for 10 min;
(8) and (4) carrying out spray drying on the precipitate obtained in the step (7) to obtain the yeast glucan.
(9) And (4) recovering ethanol from the liquid part obtained in the step (3), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
The yeast extract obtained in this example was a pale yellow, powdery solid with an yield of 54.3%; the mannoprotein is white and loose solid, and the yield of the obtained yeast mannoprotein is 10.9 percent; yeast glucan is a grayish brown powdery solid, and the yield is 16.5%.
Example 9a process for co-producing yeast glucan, mannoprotein and yeast extract using a trichoderma harzianum (t. harzianum) derived β -1,6 glucanase.
Fermentation culture of a crude beta-1, 6 glucan enzyme solution derived from Trichoderma harzianum (T.harzianum). Inoculating Harzianum to PDA liquid culture medium, fermenting and culturing at 28 deg.C for 48 h. Then the bacteria are washed by sterile water and transferred to an enzyme production culture medium (formula: 0.5 g/LMgSO) 4 ，0.01g/LFeSO 4 ，0.425g/L KCl，0.115g/L MgCl 2 ，2.1g/L NH 4 Cl，0.92g/LNaH 2 PO 4 5g/L yeast cell wall), and fermenting and culturing for 48h at 28 ℃ to obtain the beta-1, 6 glucanase enzyme derived from T.harzianum.
Bread yeast is taken as a raw material, beta-1, 6 glucanase enzyme liquid from trichoderma harzianum (T.harzianum) is utilized to treat yeast cell walls to co-produce mannoprotein, yeast glucan and yeast extract, and the method comprises the following steps:
(1) using baker's yeast as raw material, preparing yeast cell into 9% suspension, inactivating at 115 deg.C for 20min under 0.1 Mpa, centrifuging the yeast suspension at 8000rpm for 10min, and collecting precipitate and supernatant: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(2) adding the yeast cell wall obtained in the step (1) into the beta-1, 6 glucanase liquid from the obtained T.harzianum source, and carrying out enzymolysis under the conditions that the enzyme dosage is 40U/g yeast, the enzymolysis system pH is 7, the time is 24h, the temperature is 37 ℃, and the stirring speed is 220 rpm. After enzymolysis, centrifuging and collecting supernatant, wherein the centrifugation speed is 8000rpm, and the centrifugation time is 10 min;
(3) adding ethanol with the volume being three times that of the supernatant of the enzymolysis liquid obtained in the step (4) to carry out alcohol precipitation at the temperature of 4 ℃, standing overnight, centrifuging and collecting precipitates, wherein the centrifugal speed is 8000rpm, and the centrifugation time is 10 min;
(4) adding deionized water into the precipitate obtained in the step (5) to prepare a solution with the mass fraction of 5%, performing membrane separation on a membrane with the molecular weight cutoff of 7kDa at the temperature of 4 ℃, and collecting the cutoff solution after 2 days;
(5) and (4) spray drying the trapped fluid obtained in the step (6) to obtain the yeast mannoprotein.
(6) Precipitating the enzymolysis liquid obtained in the step (2), adding 20mL/g of petroleum ether, heating and refluxing for 3h at the temperature of 60 ℃, at the centrifugal rotation speed of 8000rpm for 10min, and collecting the precipitate;
(7) adding 50000U/g of papain into the precipitate obtained in the step (6), degreasing, precipitating, carrying out enzymolysis for 10 hours at 65 ℃, stirring at the speed of 180rpm, centrifuging at the speed of 8000rpm for 10min, and collecting the precipitate;
(8) and (4) carrying out spray drying on the precipitate obtained in the step (7) to obtain the yeast glucan.
(9) And (4) recovering ethanol from the liquid part obtained in the step (3), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
The yeast extract obtained in this example was a pale yellow, powdery solid with an yield of 52.5%; the mannoprotein is white and loose solid, and the yield of the obtained yeast mannoprotein is 10.5 percent; the yeast beta-glucan is a gray brown powdery solid, and the yield is 21.3%.
The results show that the beta-1, 6-glucanase of different sources and different preparation methods can crack the yeast cell wall, and the function of mildly preparing the yeast mannoprotein and the yeast glucan by the enzyme method is realized.
Claims (11)
1. A method for utilizing beta-1, 6 glucanase to coproduce yeast glucan, mannoprotein and yeast extract is characterized in that yeast cells are taken as raw materials, and the yeast extract is obtained by carrying out high-temperature inactivation treatment, centrifugation and drying supernatant on the yeast cells; performing enzymolysis on yeast cell walls in the precipitate by using beta-1, 6 glucanase, centrifuging to obtain an enzymolysis liquid supernatant and an enzymolysis precipitate respectively, and performing ethanol precipitation, centrifugal separation and drying on the enzymolysis liquid supernatant to obtain mannoprotein; recovering ethanol, concentrating the rest liquid part, and spray drying to obtain yeast extract; sequentially carrying out degreasing, protease treatment and spray drying on the enzymolysis precipitate to obtain yeast glucan; the beta-1, 6-glucanase is selected from beta-1, 6-glucanase shown in GenBank number MH747076, GenBank number NP596461 or GenBank number XP 024773174.
2. The method of claim 1, comprising the steps of:
(1) taking yeast cells as raw materials, carrying out high-temperature wall breaking treatment on the yeast cells, centrifuging, and respectively collecting precipitates and supernate: precipitating into processed yeast cell walls; concentrating the supernatant, and spray drying to obtain yeast extract;
(2) adding beta-1, 6-glucanase liquid into the yeast cell wall obtained in the step (1), carrying out enzymolysis reaction, centrifuging, and respectively collecting supernatant and enzymolysis precipitate of enzymolysis liquid;
(3) adding ethanol into the supernatant of the enzymolysis liquid obtained in the step (2) for alcohol precipitation, centrifuging, and collecting precipitates;
(4) dissolving the precipitate obtained in the step (3) in deionized water, performing 5-10kDa membrane dialysis, and collecting trapped fluid;
(5) spray drying the trapped fluid obtained in the step (4) to obtain mannoprotein;
(6) adding petroleum ether into the enzymolysis precipitate obtained by the centrifugation in the step (2), heating and refluxing, centrifuging, and collecting the precipitate;
(7) adding protease into the precipitate obtained in the step (6), performing enzymolysis reaction, centrifuging, and collecting the precipitate;
(8) spray drying the precipitate obtained in the step (7) to obtain yeast glucan;
(9) and (4) recovering ethanol from the liquid part obtained in the step (3), concentrating the residual liquid after recovery, and performing spray drying to obtain the yeast extract.
3. The method of claim 2, wherein in the step (1), the yeast cells are selected from any one or more of saccharomyces cerevisiae cells, baker's yeast cells and waste industrial saccharomyces cerevisiae cells.
4. The method of claim 3, wherein the step (1) of pre-treating the waste industrial Saccharomyces cerevisiae cells comprises: preparing the waste industrial saccharomyces cerevisiae cells into suspension with the mass concentration of 3-15%, passing through a screen with the aperture of 80-120 meshes, centrifugally washing for 3-5 times by distilled water, wherein the centrifugal rotation speed is 10000-15000 rpm, and the centrifugal time is 10-15 min.
5. The method as claimed in claim 2, wherein in the step (1), the yeast cells are prepared into a suspension with a mass concentration of 6-15%, and the high-temperature wall breaking treatment is performed at a temperature of 110-.
6. The method according to claim 2, wherein in step (2), the beta-1, 6-glucanase is derived from a strain with a accession number of CCTCC NO: of M2012528Corallococcus sp, EGB, the conditions of enzymatic hydrolysis are: the enzyme dosage is 2-10U/g yeast, the pH value of the enzymolysis system is 6-8, the time is 12-24h, the temperature is 30-50 ℃, and the stirring speed is 150-250 rpm.
7. The method of claim 6, wherein said step of removing said metal oxide layer is performed by a chemical vapor deposition processCorallococcus sp and EGB are inoculated in a VY/4 liquid enzyme production culture medium, fermentation culture and centrifugation are carried out to collect supernatant, thus obtaining the beta-1, 6 glucanase in the step (2).
8. The method according to claim 2, wherein in the step (3), the ethanol is used in an amount of two to four times the volume of the obtained supernatant, and the temperature is 0-4 ℃ overnight; and (4) centrifuging and collecting the precipitate, wherein the centrifugation rotation speed is 6000-10000 rpm, and the centrifugation time is 5-10min or obtaining the precipitate through plate and frame filter pressing.
9. The method according to claim 2, wherein in the step (4), the precipitate obtained in the step (3) is dissolved by adding deionized water to prepare a solution to be dialyzed with a mass concentration of 2-8%, and the dialysis membrane has a molecular weight cut-off of 5-10kDa and a temperature of 0-4 ℃.
10. The method as claimed in claim 2, wherein the amount of petroleum ether used in step (6) is 5-40mL/g, the reflux temperature is 60-95 ℃, and the reflux time is 2-4 h.
11. The method according to claim 2, wherein the enzymolysis conditions in the step (7) are as follows: selecting papain, wherein the dosage of the papain is 20000-one 60000U/g, the time is 5-15h, the temperature is 30-70 ℃, and the stirring speed is 150-one 250 rpm.
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| CN102351943A (en) * | 2011-09-28 | 2012-02-15 | 南京同凯兆业生物技术有限责任公司 | Method for coproducing glucan and mannoprotein by using industrial waste yeast |
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