WO2020192496A1

WO2020192496A1 - Method for co-production of yeast glucan, mannose protein and yeast extract using beta-1,6-glucanase

Info

Publication number: WO2020192496A1
Application number: PCT/CN2020/079724
Authority: WO
Inventors: 崔中利; 李周坤; 乔燕; 叶现丰; 曹慧; 方晓东
Original assignee: 南京农业大学
Priority date: 2019-03-22
Filing date: 2020-03-17
Publication date: 2020-10-01
Also published as: CN109912700B; CN109912700A

Abstract

Disclosed is a method for co-production of yeast glucan, mannose protein and yeast extract using beta-1,6-glucanase. The method comprises the following steps: taking yeast cells as a raw material, subjecting the yeast cells to a high-temperature deactivation treatment, performing centrifugation and drying the supernatant to obtain a yeast extract; performing enzymolysis on yeast cell walls by means of beta-1,6-glucanase, centrifuging same to obtain an enzymatic hydrolysate supernatant and an enzymolysis precipitate, and subjecting the enzymatic hydrolysate supernatant to water extraction, alcohol precipitation, centrifugal separation and drying to obtain a mannose protein; after the recovery of ethanol, subjecting residual liquid to concentration and spray drying to obtain a yeast extract; and subjecting the enzymolysis precipitate to degreasing, protease treatment and spray drying to obtain yeast glucan. The yeast glucan and the mannose protein prepared in the present invention are high in purity and yield, and the preparation processes therefor are low in cost and low in environmental pollution due to the avoidance of the consumption of strong acid and strong base agents.

Description

Method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6-glucanase

Technical field

The invention relates to a method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6 glucanase.

Background technique

Yeast is an important food and industrial microorganism. my country has abundant yeast resources. Yeast cell walls are composed of polysaccharides, proteins, and a small amount of lipids and chitin. The two main polysaccharides are β-D-glucan, which accounts for about 50-55% of the dry weight of the cell wall. It is distributed on the inner side of the cell wall and constitutes the cross-linked skeleton structure of the cell wall; the mannan protein is distributed on the outer side of the cell wall. , About 40-45% of the dry weight of the cell wall, endows the cell with biological activity and controls the cell wall pore size. Both β-glucan and mannan protein have abundant biological functions. Yeast β-glucan has a unique triple helix structure, which can specifically bind to the body's immune cell receptors and stimulate it to be in a highly active state. Yeast β-glucan binds to macrophage receptors and activates macrophages to play a role in anti-tumor, anti-bacterial, antioxidant and wound healing. Mannoprotein is composed of 5-10% protein and 80-90% mannan, and has the ability to promote animal humoral immunity and cellular immunity. Mannoprotein can adjust the balance of intestinal flora, combine to adsorb foreign pathogenic bacteria and toxins, and has anti-radiation, anti-oxidation, anti-tumor and other active functions. In terms of food processing, mannoprotein can effectively reduce the precipitation of tartaric acid during the storage of red wine, maintain the clarity of red wine, weaken the astringency of tannins in wine, and promote the release of red wine aromatic substances. In addition, mannose protein also has a certain emulsification stabilizing effect and the ability to extend the shelf life of vegetables and fruits.

Yeast β-glucan and mannoprotein can be extracted from yeast. The current laboratory and industrial preparation processes mainly include acid-base extraction, hot water extraction, enzymatic method, and a combination of the above methods. Franziskus K et al. (1999) proposed the acid extraction of mannans. The washed wet yeast was placed in a mixture of ethanol, sulfuric acid and water (2:1:1, v/v/v), heated and centrifuged, and the supernatant was frozen. Mannan is obtained by drying. The product contains 4.15% protein and 90.10% total sugars, of which 73.2% mannose. CN 101117358 A discloses a method for preparing mannan by alkaline method, which includes treating yeast cell wall with 0.5-1.5 mol/L NaOH, passing 12-20% CTAB solution and 0.3-0.8% sodium borate-sodium acetate solution The protein is removed, and the supernatant is dried to obtain mannan. Acid-base extraction methods mostly use high concentrations of strong acids and bases, which will not only reduce the activity of polysaccharides, but also pollute the environment. Liu Hongzhi et al. (2009) determined the optimal conditions for preparing yeast mannan by water extraction method, using waste Saccharomyces cerevisiae as raw material, pH 6.5, 3% NaCl, temperature 50℃, stirring rate 120r/min Under the conditions of oscillating and autolyzed for 24h; then treated at high temperature of 120℃ for 3h; finally, deproteinized and separated by gel chromatography column to obtain a mannoprotein product with a purity of 92.6%. The hot water extraction method has low cost and simple operation, but the product obtained contains soluble amino acids, pigments and other impurities, and the purity of the product is low. It needs to be further purified by methods such as ion exchange columns and electrophoresis.

In the enzymatic preparation of β-zymosan, it is commonly used to destroy the cell wall by autolysis or lysozyme, laminarinase and other methods, and then use amylase, alkaline protease, and neutral protease to further purify the zymosan product. Liu et al. (2011) proposed a gentle process to prepare yeast β-glucan. The main steps include: induction of autolysis, high temperature extraction, homogeneous wall breaking, reflux degreasing and biological enzymatic hydrolysis, and finally β-D- The yield of dextran reached 11% and the purity was as high as 93%. CN 102603917 discloses a method for preparing yeast glucan by an enzymatic method, which includes using α-amylase, glucoamylase, mannase and neutral protease to obtain high-purity β-1,3 after desalting, degreasing and drying -Glucan. CN 104862355 A discloses a method for co-producing glucan and mannose protein by using yeast cell wall by enzymatic method, which includes adding alkaline protease to enzymatically hydrolyze the yeast cell wall subjected to high-pressure homogenization, centrifuging heavy liquid and drying to obtain glucan. The liquid is filtered through an alkaline nanofiltration membrane, and the retentate is concentrated and dried to obtain mannose protein. In addition, CN1940084A is the only domestic patent that proposes the use of β-1,3-glucosidase, β-1,6-glucosidase, and chitinase combined enzymatic hydrolysis, and the preparation of wine additive Saccharomyces manna through pyrolysis Glycoprotein. Enzymatic preparation can maintain the natural structure and active function of zymosan, but the product extracted by enzymatic method has high protein content and low purity. It needs to be further purified by methods such as ion exchange column and electrophoresis, and the enzyme is expensive and costly.

Based on the main problem of extracting the active components of yeast polysaccharides, the present invention is based on the myxobacteria β-1,6-glucanase involved in CN 105524934 A to further protect the enzyme in the comprehensive utilization of yeast cells. . β-1,6-glucanase GluM is purified from the extracellular supernatant of Corallococcus sp.EGB to obtain a specific cleavage with glucose as the constituent unit and β-1,6-glycosidic bond connection Dextran. The specific enzyme activity of the β-1,6-glucanase GluM is as high as 24 000 U·mg ^-1 . The β-1,6-glycosidic bond is an important cross-linking structure in the yeast cell wall, which cross-links the long-chain β-1,3-glucan and the outer mannan into a network structure, β-1,6 -The destruction of glycosidic bonds will result in the lysis of the entire cell wall. In addition, fungal-derived β-1,6-glucanase can also play a similar role. In CN1940084A, three biological enzymes that can break the whole structural connection of yeast cell wall are used, and their enzymatic activity and cost are unknown.

The present invention only utilizes β-1,6-glucanase, which can destroy the cross-linked structure, and combines with low-cost, mature papain on the market, which can efficiently and specifically promote mannose protein and yeast glucan The release of sugar, while making full use of yeast cell resources, to obtain three important active components of yeast glucan, yeast mannoprotein and yeast extract.

Summary of the invention

In order to solve the above-mentioned problems in the background art, the present invention provides a biological enzymatic method, that is, a method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6 glucanase.

In order to achieve the above objectives, the technical solutions adopted by the present invention are:

A method for co-producing yeast glucan, mannose protein and yeast extract by using β-1,6-glucanase. Using yeast cells as raw materials, the yeast cells are inactivated at high temperature, centrifuged and the supernatant is dried. Obtain yeast extract; use β-1,6-glucanase to enzymolyze yeast cell walls and centrifuge to obtain the supernatant of enzymatic hydrolysate and enzymatic precipitation. The supernatant of enzymatic hydrolysate is ethanol-precipitated, centrifuged and dried to obtain mannose protein After the ethanol is recovered, the remaining liquid part is concentrated and spray-dried to obtain a yeast extract; the enzymatic hydrolysis precipitate is sequentially degreasing, protease treatment, and spray drying to obtain yeast glucan.

The yeast extract product obtained by the technical scheme of the present invention is a light yellow, powdery solid; the mannose protein product is a white, loose solid; and the yeast glucan product is a gray-brown, powdery solid.

The method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6 glucanase of the present invention preferably includes the following steps:

(1) Using yeast cells as raw materials, perform high temperature inactivation treatment on yeast cells, and collect the precipitate and supernatant by centrifugation: the precipitate is the treated yeast cell wall; the supernatant is concentrated and spray-dried to obtain the yeast extract;

(2) Add β-1,6-glucanase solution to the yeast cell wall obtained in step (1), perform enzymatic hydrolysis, centrifuge, and collect the supernatant;

(3) Add ethanol to the supernatant of the enzymatic hydrolysate obtained in step (2) for alcohol precipitation, centrifuge, and collect the precipitate;

(4) Add deionized water to dissolve the precipitate obtained in step (3), perform membrane dialysis, and collect the retentate;

(5) Spray drying the retentate obtained in step (4) to obtain mannose protein;

(6) Precipitate the enzymolysis solution obtained in step (2), add petroleum ether, heat to reflux, centrifuge, and collect the precipitate;

(7) Add protease to the precipitate obtained in step (6), perform enzymatic hydrolysis reaction, centrifuge, and collect the precipitate;

(8) Spray drying the precipitate obtained in step (7) to obtain yeast glucan.

(9) The liquid part obtained in step (3) is recovered by ethanol, and the remaining liquid after recovery is concentrated and spray-dried to obtain a 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 discarded industrial Saccharomyces cerevisiae cells need to be pre-treated, including: preparing the discarded industrial Saccharomyces cerevisiae cells into a suspension with a mass percentage of 3-15%, passing through a sieve with an aperture of 80-120 mesh, Centrifuge and wash 3-5 times with distilled water, the centrifugal speed is 10 000-15 000 rpm, and the centrifugal time is 10-15 min.

In the step (1), preferably, the yeast cells are formulated into a suspension with a mass percentage concentration of 6-15%, and treated at a high temperature under the conditions of a temperature of 110-130° C., a time of 20-40 min, and a pressure of 0.1Mpa.

In the step (2), the centrifuge speed of the collected enzyme solution is 10 000-15 000 rpm, and the centrifugal time is 10-15 min.

In the step (2), the β-1,6-glucanase is preferably the β-1,6-glucanase shown in GenBank No. MH747076, GenBank No. NP596461 or GenBank No. XP024773174.

In the step (2), the β-1,6-glucanase is preferably derived from Corallococcus sp.EGB with the deposit number CCTCC NO: M2012528 (the accession number of β-1,6-glucanase is GenBank No .MH747076), or from Schizosaccharomyces pombe (GenBank No.NP596461 of β-1,6-glucanase) or Trichoderma harzianum (β-1,6-glucanase) The fermentation supernatant of GenBank No. XP024773174) is further preferably derived from Corallococcus sp. EGB with the deposit number CCTCC NO: M2012528 (the accession number of β-1,6-glucanase is GenBank No. MH747076).

In the step (2), the β-1,6-glucanase is derived from Corallococcus sp.EGB with the deposit number CCTCC NO: M2012528, and the Corallococcus sp.EGB is inoculated in the VY/4 liquid enzyme production medium , Fermentation culture, centrifugation to collect the supernatant to obtain the β-1,6-glucanase described in step (2).

The formula of Corallococcus sp.EGB in VY/4 liquid medium: 0.40% yeast cell wall, 0.1% CaCl ₂ , 0.1% yeast extract (w/v), pH 7.0, rotation speed 150-250rpm, fermentation temperature 20-40 ℃, culture time is 2-5 days, centrifugation to remove bacteria, the supernatant obtained is β-1,6-glucanase enzyme solution, which is used for subsequent yeast cell treatment.

The enzymolysis conditions for enzymatic hydrolysis of yeast cell wall with β-1,6-glucanase derived from Corallococcus sp.EGB with the deposit number of CCTCC NO: M2012528 are: the enzyme dosage is 2-10 U/g yeast, and the pH of the enzymatic hydrolysis system is 6-8, time is 12-24h, temperature is 30-50℃, stirring speed is 150-250rpm.

In the step (3), preferably, the amount of ethanol used is two to four times the volume of the supernatant obtained, the temperature is 0-4°C, overnight; the precipitate is collected by centrifugation, and the centrifugal speed is 6 000-10 000 rpm. The time is 5-10min, or the precipitation can be obtained through plate and frame filter press.

Preferably, in the step (4), the precipitate obtained in step (3) is dissolved in deionized water to prepare a solution to be dialyzed with a mass concentration of 2-8%. The molecular weight cut-off of the dialysis membrane is 5-10kDa, preferably 7kDa, and the temperature is 0-4°C.

In the step (6), preferably, the amount of petroleum ether is 5-40 mL/g, the reflux temperature is 60-95° C., and the reflux time is 2-4 h.

The enzymolysis conditions in the step (7) are preferably: papain is selected, the enzyme dosage is 5000-60000 U/g, the time is 5-15h, the temperature is 50-70°C, and the stirring rate is 150-250rpm.

In an embodiment of the present invention, the yeast extract product obtained in the step (1) is a pale yellow, powdery solid, and the yield is 56.5% (based on the dry weight of yeast cells, the same below).

In an embodiment of the present invention, the mannose protein product obtained in the step (6) is a white, loose solid, with a yield of 11.1% and a purity of 90.66%.

In an embodiment of the present invention, the yeast glucan product obtained in the step (9) is gray-brown, powdery solid, with a yield of 17.4% and a purity of 85.36%.

The β-1,6-glucanase in the fermentation broth of Corallococcus sp. EGB of the present invention has the ability to lyse yeast cell walls. The present invention simultaneously selects β-1,6-glucanase from different microbial sources and β-1,6-glucanase from different preparation methods, such as Schizosaccharomyces pombe (S.pombe(

-Santero E et al., 2010)), Trichoderma harzianum (Manuel Montero et al., 2005) heterologous expression or direct culture and purification of its β-1,6-glucanase, observe β-1 , 6-glucanase on the lysis of yeast cell wall, it is found that β-1,6-glucanase of different sources and different preparation methods can lyse yeast cell wall, and realize the preparation of yeast mannoprotein and yeast glucan. Therefore, the practical application of β-1,6-glucanase from different sources and different preparations also belongs to the protection scope of the present invention.

Compared with the prior art, the present invention has the following advantages and effects:

1. β-1,6-glucan is an important cross-linking structure in the yeast cell wall. The long-chain β-1,3-glucan and the outer mannan are cross-linked into a network structure, and β- The destruction of 1,6-glucan will result in the lysis of the entire cell wall. Therefore, the use of β-1,6-glucanase with high specific enzyme activity to act on the β-1,6-glycosidic bond in the yeast cell wall can efficiently and specifically enzymatically hydrolyze the yeast cell wall and promote the yeast glucose on the cell wall. The release of glycan and mannoprotein improves the yield of yeast glucan and mannoprotein.

2. The present invention adopts β-1,6-glucanase to treat yeast cells to extract yeast mannoprotein, which can avoid the damage of yeast mannoprotein by protease treatment in high temperature water for a long time, and the method of water extraction and alcohol precipitation can be simple Quickly obtain yeast mannoprotein, avoid the equipment and operation complexity brought by technologies such as nanofiltration membrane, and improve the purity of yeast mannoprotein.

3. The present invention utilizes the β-1,6-glucanase secreted by Corallococcus sp.EGB to realize full enzymolysis of yeast cell walls without the need for subsequent complicated purification steps, which greatly reduces the cost of large-scale production.

4. The present invention uses the combination of high temperature inactivation, biological enzymatic hydrolysis and spray drying technology, and the equipment used can realize the needs of industrial production, does not involve reagents such as strong acids and alkalis, does not pollute the environment, and is easy to industrial scale production .

5. The present invention uses yeast cells as raw materials to co-produce three important yeast active components of yeast glucan, mannose protein and yeast extract, and give full play to the added value of yeast cells.

In summary, the present invention uses β-1,6-glucanase for the first time to effectively promote the release of yeast glucan and mannoprotein in the yeast cell wall, and efficiently prepare high-purity and high-yield mannoprotein and yeast glucan. sugar. At the same time, the enzymatic preparation process is simple, avoids complicated process flow, saves costs, avoids the use of strong acids and alkalis, and reduces environmental pollution. This improves the added value of yeast cell products, drives the development of the wine industry, and increases scale. The economic benefits of the enzyme application industry play an important role.

Description of the drawings

Figure 1 is a process flow diagram of the present invention;

Figure 2 is a high performance liquid chromatogram after acid hydrolysis of yeast glucan;

Figure 3 is a high performance liquid chromatogram of the product obtained in the present invention after acid hydrolysis of mannose protein;

Figure 4 is an infrared spectrogram of the yeast glucan obtained in the present invention;

Figure 5 is an infrared spectrogram of the product mannoprotein obtained in the present invention;

Figure 6 is a scanning electron microscope picture of the yeast glucan produced by the present invention.

Figure 7 is a scanning electron microscope picture of the yeast glucan produced by the present invention.

detailed description

The present invention will be further clarified below in conjunction with specific examples. The specific embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these methods are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1 A method for co-producing yeast glucan, mannoprotein and yeast extract using β-1,6 glucanase, using baker's yeast as a raw material, including the following steps (as shown in Figure 1):

(1) Using baker's yeast as the raw material, the yeast cells are formulated into a suspension with a mass percentage of 9%. The suspension is subjected to high temperature inactivation treatment at a temperature of 110°C, a time of 20 minutes, and a pressure of 0.1Mpa. Centrifuge the liquid at 8000 rpm for 10 min, and collect the precipitate and supernatant: the precipitate is the treated yeast cell wall; the supernatant is concentrated and spray-dried to obtain the yeast extract;

(2) Corallococcus sp. EGB was inoculated into VY/4 liquid enzyme production medium (0.40% yeast cell wall, 0.1% CaCl ₂ , 0.1% yeast extract (w/v), pH 7.0), fermented for 2 days, and collected by centrifugation Enzyme solution, the centrifugal speed is 12 000 rpm, and the centrifugation time is 15 minutes, to obtain β-1,6-glucanase enzyme solution;

(3) Add the yeast cell wall obtained in step (1) to the β-1,6 glucanase solution obtained in step (2). The enzyme dosage is 8U/g yeast, the enzymatic hydrolysis system pH7, time 24h, temperature 37℃ , Enzymatic hydrolysis was carried out under the condition of stirring rate of 220 rpm. After the enzymolysis is completed, centrifuge to collect the supernatant, the centrifuge speed is 8 000 rpm, and the centrifuge time is 10 minutes;

(4) Add three times the volume of ethanol to the supernatant of the enzymatic hydrolysate obtained in step (3) for alcohol precipitation, at a temperature of 4°C, overnight, and collect the precipitate by centrifugation at a speed of 8 000 rpm and a centrifugal time of 10 min;

(5) Add deionized water to the precipitate obtained in step (4) to prepare a solution with a mass fraction of 5%, and perform membrane dialysis with a membrane with a molecular weight cutoff of 7kDa at a temperature of 4°C, and collect the retentate after 2 days;

(6) Spray drying the retentate obtained in step (5) to obtain yeast mannoprotein.

(7) Precipitate the enzymatic hydrolysate obtained in step (3), add petroleum ether 20mL/g, heat to reflux for 3 hours, at a temperature of 60°C, and collect the precipitate by centrifugation at a speed of 8000 rpm for 10 minutes;

(8) Add papain 40,000 U/g to the precipitate obtained in step (7), the enzymolysis time is 10 hours, the temperature is 65°C, the stirring rate is 180 rpm, the precipitate is collected by centrifugation, the rotation speed is 8 000 rpm, and the time is 10 minutes;

(9) Spray drying the precipitate obtained in step (8) to obtain yeast β-glucan.

(10) The liquid part obtained in step (4) is recovered by ethanol, and the remaining liquid after recovery is concentrated and spray-dried to obtain a yeast extract.

The yeast extract obtained in this example is a pale yellow, powdery solid with a yield of 56.5%; the mannose protein is a white, loose solid, with a yield of 11.1% and a purity of 90.66%. ; Yeast glucan is a gray-brown powdery solid with a yield of 17.4% and a purity of 85.36%.

Example 2

Weigh about 20 mg of yeast glucan and mannoprotein obtained in Example 1, add 10 mL of trifluoroacetic acid (4M), and hydrolyze at 100°C for 4 hours. The hydrolyzed solution was rotary evaporated at 80°C and adjusted to neutral with 4M NaOH. Pipette 150μL of the hydrolysis solution, add 150μL of 0.6M NaOH, and then add 200μL of 0.5M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution, and react in a water bath at 70°C for 90 minutes. After the reaction is over, cool to room temperature, add 0.3M HCl to the reaction solution to adjust to neutrality, and make up to 1 mL with deionized water. Add 1 mL of chloroform to extract PMP, centrifuge at 12,000 rpm for 1 min, and repeat the extraction three times. The upper aqueous phase solution is passed through a 0.22μm water phase filter membrane, and 20μL is taken on the machine. The derivatized sample adopts C18 chromatographic column, UV detector OD ₂₃₀ , mobile phase is a mixed solution of PBS and acetonitrile (PBS is 0.02M, pH6.7, PBS:acetonitrile volume ratio is 83:17), and the flow rate is 1.0ml/min , Column temperature 30℃.

It can be seen from Figure 2 that the high performance liquid chromatogram of the standard product shows that the retention time of the mannose standard product is 25.5 minutes, the retention time of the glucose standard product is 41.3 minutes, and the chromatographic peak with a retention time of 18.3 minutes represents a monosaccharide derivative Solvent PMP. The high performance liquid chromatogram of yeast glucan shows that there are only two monosaccharide peaks. The chromatographic peak with a retention time of 25.5 min is the mannose produced by the hydrolysis of the yeast glucan product, and the peak with a retention time of 41.1 min is yeast The glucose produced by acid hydrolysis of dextran 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 of the present invention has higher purity. It can be seen from FIG. 3 that the high performance liquid chromatogram of the sample shows a chromatographic peak of only one monosaccharide, and the chromatographic peak with a retention time of 25.5 min is the peak of mannose produced after acid hydrolysis of the mannose protein product in Example 1. Therefore, the mannose protein product prepared by the method of the present invention has high purity and no other miscellaneous sugars.

Example 3

The content of each component of yeast glucan and mannoprotein obtained in Example 1 was determined. The crude protein and ash were determined by the AOAC method (1990); the total sugar was determined by the anthrone-sulfuric acid method; the yeast glucan and mannoprotein were determined by referring to Example 2, and the external standard method was used for quantification.

The results are shown in Table 1. Yeast cells are mainly composed of polysaccharides and proteins, with a total sugar content of 34.32% (wherein, glucan accounts for 20.56% of the dry cell weight and mannan accounts for 13.32%), and protein content is 59.31%. The yield of mannoprotein prepared by the method of the present invention is 11.1% and the purity is 90.66%; the yield of yeast glucan is 17.44% and the purity is 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, 5 mg of anhydrous sample was placed under the probe, and the BIO-RAD Win-IR was used to scan in the range of 500-4500 cm ^-1 .

As illustrated in Figure 4, 1076cm ^-1, absorption peaks 1427 / 1460cm ^-1, the characteristic peaks for the polysaccharide; characteristic peak at around 890cm ^-1 proved characterized deformation vibration β-D- absorbent pyranose CH Peaks, indicating that the component contains β-D-glucopyranose rings, and the molecules are connected by β-glycosidic bonds; the characteristic peaks of 1255/1252cm ^-1 , 1375/1372cm ^-1 , and 2923cm ^-1 indicate that it has β-(1 -3) Bonded dextran. As shown in Figure 5, the absorption peak at 3400/3435 cm ^-1 is the formation of intermolecular and intra-molecular hydrogen bonds by -OH on polysaccharides; the absorption peak at 2900/2930 cm ^-1 is CH stretching vibration, which is a characteristic peak of sugars; 1645 / 1652cm ^-1 amide group absorption peaks, the sample can be inferred protein is a sugar binding protein may be; absorption peak of 810 / 820cm ^-1 is α- mannose pyran configuration characteristic peaks. The above results show that the tested sample has a characteristic absorption peak of mannose protein. The above results show that the tested sample has the characteristic absorption peak of yeast glucan.

Example 5

The yeast extract obtained in Example 1 is a yellow, powdery solid. The determination of various physical and chemical indexes of the yeast extract obtained in the present invention is based on the method described in the national standard GB/T23530-2009.

Table 2 Physical and Chemical Index of Yeast Extraction

Example 6

Take a small amount of dried yeast glucan and mannan samples to adhere to the sample table, place a conductive film (gold) in the vacuum sprayer, and observe using a scanning electron microscope.

As shown in Figure 6 and Figure 7, the yeast cells are inactivated at high temperature, β-1,6-glucanase is enzymatically hydrolyzed, and the supernatant of the enzymatic hydrolysis is subjected to water extraction and alcohol precipitation, membrane dialysis and spray drying to obtain the mannoprotein; The scanning electron microscope image of the yeast glucan obtained by degreasing, protease treatment, and spray drying shows that it presents the characteristic gel-linked and porous state of polysaccharide.

Example 7

A method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6-glucanase, using waste industrial Saccharomyces cerevisiae as raw material, including the following steps:

(1) Using waste industrial Saccharomyces cerevisiae cells as raw materials, the yeast cells are formulated into a suspension with a mass percentage concentration of 5%. The suspension is passed through a sieve with an aperture of 100 mesh, and washed with distilled water for 5 times at a speed of 10,000 rpm and a centrifugal time of 10min.

(2) The yeast cells washed to remove impurities are prepared into a suspension with a mass percentage concentration of 9%, and the suspension is inactivated at a high temperature under the conditions of a temperature of 120°C, a time of 30 minutes, and a pressure of 0.1Mpa. Centrifuge for 15 minutes at 8 000 rpm, and collect the precipitate and supernatant: the precipitate is the yeast cell wall after treatment; the supernatant is concentrated and spray-dried to obtain the yeast extract;

(3) Corallococcus sp.EGB was inoculated into VY/4 liquid enzyme production medium, fermented for 3 days, centrifuged to collect the enzyme solution, centrifuged at 10,000 rpm, and centrifuged for 10 minutes to obtain β-1,6-glucanase solution ；

(4) Add the yeast cell wall obtained in step (1) to the β-1,6 glucanase obtained in step (2). The enzyme dosage is 10U/g yeast, the enzymatic hydrolysis system pH is 7.5, the time is 24h, and the temperature Enzymatic hydrolysis was carried out at 40°C and a stirring rate of 250 rpm. After the enzymolysis is completed, centrifuge to collect the supernatant, the centrifuge speed is 9,000 rpm, and the centrifuge time is 15 minutes;

(5) Add three times the volume of ethanol to the supernatant of the enzymatic hydrolysate obtained in step (3) for alcohol precipitation, at a temperature of 4°C, overnight, and collect the precipitate by centrifugation at a speed of 10,000 rpm and a time of 15 min;

(6) The precipitate obtained in step (4) is added to deionized water to prepare a solution with a mass fraction of 8%, a membrane with a molecular weight cutoff of 7kDa is used for membrane separation, and the temperature is 4°C, and the retentate is collected after 1 day;

(7) Spray drying the retentate obtained in step (5) to obtain mannose protein.

(8) Precipitate the enzymatic hydrolysate obtained in step (3), add petroleum ether 40mL/g, heat to reflux for 2h, the temperature is 65°C, centrifuge to collect the precipitate, rotate speed 8000rpm, time 10min;

(9) Add papain 20,000U/g to the precipitate obtained in step (7), enzymolysis time is 15h, temperature is 40°C, stirring rate is 220rpm, centrifuge to collect the precipitate, rotation speed is 8000rpm, time 10min,;

(10) Spray drying the precipitate obtained in step (8) to obtain yeast glucan.

(11) The liquid part obtained in step (5) is recovered by ethanol, and the remaining liquid after recovery is concentrated and spray-dried to obtain a yeast extract.

The yeast extract obtained in this example is a pale yellow, powdery solid with a yield of 58.9%; the mannose protein is a white, loose solid, with a yield of 9.5% and a purity of 89.24%; Yeast glucan is a gray-brown powdery solid with a yield of 16.3% and a purity of 81.32%.

Example 8 A method for co-producing yeast glucan, mannose protein and yeast extract by using β-1,6 glucanase derived from Schizosaccharomyces pombe.

For the heterologous expression, isolation and purification of β-1,6-glucanase (NM 001922380.2) derived from Schizosaccharomyces cerevisiae S. pombe, refer to CN 105524934A for the method. Using the genome of S.pombe as a template, PCR amplification of the full length of β-1,6-glucanase gene was carried out, and the enzyme-linked with pMD19-T Vector was carried out overnight. The enzyme ligation product was transformed into E. coli DH5α competent cells, single clones were selected for culture, and sequenced to verify that the sequence was correct. The recombinant plasmid extracted above and pET-29a(+) were digested with EcoI and HindIII and linked by enzyme. The verified pET-29a(+)-exg was transformed into E. coli BL21(DE3) competent cells, the expression strain was constructed, and the single clone was picked and cultured. The bacterial cells are collected, sonicated to disrupt the bacterial cells, and the supernatant obtained by centrifugation is the crude β-1,6-glucanase enzyme solution. The crude β-1,6-glucanase solution produced is subjected to ammonium sulfate fractionation precipitation, and combined with purification methods such as adsorption and desorption, the recombinant β-1,6-glucanase is purified, and the purified β -1,6-glucanase is subjected to dialysis treatment and stored for later use.

Using baker’s yeast as a raw material, using heterologously expressed β-1,6 glucanase from S.pombe to co-produce mannoprotein, yeast glucan and yeast extract, including the following steps:

(1) Using baker's yeast as the raw material, the yeast cells are formulated into a suspension with a mass percentage of 9%, and the yeast suspension is inactivated at a high temperature under the conditions of a temperature of 115°C, a time of 20 minutes and a pressure of 0.1Mpa. Centrifuge at 8 000 rpm for 15 minutes to collect the precipitate and supernatant: the precipitate is the yeast cell wall after treatment; the supernatant is concentrated and spray-dried to obtain the yeast extract;

(2) Add the yeast cell wall obtained in step (1) to the β-1,6-glucanase derived from S.pombe, at an enzyme dosage of 12U/g yeast, enzymatic hydrolysis system pH7, time 24h, temperature 37℃ , Enzymatic hydrolysis was carried out under the condition of stirring rate of 220 rpm. After the enzymolysis is completed, centrifuge to collect the supernatant, the centrifuge speed is 8 000 rpm, and the centrifugation time is 10 min;

(3) Add three times the volume of ethanol to the supernatant of the enzymatic hydrolysate obtained in step (4) for alcohol precipitation, at a temperature of 4°C, overnight, and centrifuge to collect the precipitate at a centrifuge speed of 8 000 rpm and a centrifugal time of 10 min;

(4) The precipitate obtained in step (5) is added to deionized water to prepare a solution with a mass fraction of 5%, the molecular weight cutoff is 7kDa membrane for membrane separation, the temperature is 4°C, and the retentate is collected after 2 days;

(5) Spray drying the retentate obtained in step (6) to obtain yeast mannoprotein.

(6) Precipitate the enzymatic hydrolysate obtained in step (2), add 30mL/g of petroleum ether, heat to reflux for 3 hours, at a temperature of 60°C, centrifuge to collect the precipitate, rotate at 8 000 rpm, for 10 minutes;

(7) Add papain 40000U/g to the precipitate obtained in step (6), the enzymolysis time is 10h, the temperature is 65°C, the stirring speed is 180rpm, the precipitate is collected by centrifugation, the speed is 8 000rpm, time is 10min,;

(8) Spray drying the precipitate obtained in step (7) to obtain yeast glucan.

The yeast extract obtained in this example is a pale yellow, powdery solid, with a yield of 54.3%; mannoprotein is a white, loose solid, with a yield of 10.9% of yeast mannoprotein; yeast glucan It is a gray-brown powdery solid with a yield of 16.5%.

Example 9 A method for co-producing yeast glucan, mannoprotein and yeast extract using β-1,6 glucanase derived from T. harzianum.

Fermentation culture of β-1,6-glucan crude enzyme solution derived from T. harzianum. T. harzianum was inoculated into PDA liquid medium and fermented at 28°C for 48h. After that, the bacteria were washed with sterile water and transferred to the enzyme production medium (recipe: 0.5g/LMgSO ₄ , 0.01g/LFeSO ₄ , 0.425g/L KCl, 0.115g/L MgCl ₂ , 2.1g/L NH ₄ Cl , 0.92g/L NaH ₂ PO ₄ , 5g/L yeast cell wall), fermented at 28°C for 48h to obtain β-1,6-glucanase enzyme derived from T.harzianum.

Using baker’s yeast as a raw material, using the β-1,6-glucanase enzyme solution derived from T.harzianum to treat the yeast cell wall to produce mannoprotein, yeast glucan and yeast extract, including the following steps :

(1) Using baker's yeast as raw material, formulate yeast cells into a suspension with a mass percentage of 9%, inactivate the yeast at a high temperature under the conditions of a temperature of 115°C, a time of 20 minutes, and a pressure of 0.1Mpa to suspend the yeast Centrifuge the liquid at 8000 rpm for 10 min, and collect the precipitate and supernatant: the precipitate is the treated yeast cell wall; the supernatant is concentrated and spray-dried to obtain the yeast extract;

(2) Add the yeast cell wall obtained in step (1) to the β-1,6-glucanase solution derived from T.harzianum above. The enzyme dosage is 40U/g yeast, the enzymatic hydrolysis system pH7, time 24h, temperature 37 Enzymatic hydrolysis was carried out under the conditions of ℃ and 220 rpm stirring speed. After the enzymolysis is completed, centrifuge to collect the supernatant, the centrifuge speed is 8 000 rpm, and the centrifugation time is 10 min;

(6) Precipitate the enzymatic hydrolysate obtained in step (2), add petroleum ether 20mL/g, heat to reflux for 3h, the temperature is 60℃, the centrifugal rotation speed is 8 000rpm, the time is 10min, and the precipitate is collected;

(7) The precipitate obtained in step (6) is added with 50 000 U/g of papain for degreasing and precipitation, enzymolysis time is 10 hours, temperature is 65°C, stirring speed is 180 rpm, centrifugal speed is 8 000 rpm, time is 10 minutes, and the precipitate is collected;

(8) Spray drying the precipitate obtained in step (7) to obtain yeast glucan.

The yeast extract obtained in this example is a pale yellow, powdery solid with a yield of 52.5%; mannose protein is a white, loose solid, with a yield of 10.5% of yeast mannose protein; yeast β-glucose The glycan is a gray-brown powdery solid with a yield of 21.3%.

It can be seen from the results that β-1,6-glucanases from different sources and different preparation methods can cleave yeast cell walls, realizing the function of mild enzyme preparation of yeast mannoprotein and yeast glucan.

Claims

A method for co-producing yeast glucan, mannoprotein and yeast extract by using β-1,6 glucanase, which is characterized in that yeast cells are used as raw materials, and the yeast cells are inactivated by high temperature and centrifuged. , Dry the supernatant to obtain yeast extract; use β-1,6-glucanase to enzymatically hydrolyze the yeast cell wall in the precipitate, centrifuge to obtain the supernatant of the enzymatic hydrolysis solution and the enzymatic hydrolysis precipitate, and the supernatant of the enzymatic hydrolysis solution is precipitated by ethanol and centrifuged Mannoprotein is obtained after separation and drying; after ethanol is recovered, the remaining liquid part is concentrated and spray-dried to obtain yeast extract; the enzymatic precipitation is successively subjected to degreasing, protease treatment, and spray drying to obtain yeast glucan.
The method according to claim 1, characterized by comprising the following steps:

(1) Using yeast cells as raw materials, perform high-temperature wall breaking treatment on yeast cells, and collect the precipitate and supernatant by centrifugation: the precipitate is the treated yeast cell wall; the supernatant is concentrated and spray-dried to obtain yeast extract;

(2) Add β-1,6-glucanase solution to the yeast cell wall obtained in step (1), perform an enzymatic hydrolysis reaction, centrifuge, and collect the supernatant of the enzymatic hydrolysis solution and the enzymatic hydrolysis precipitate;

(3) Add ethanol to the supernatant of the enzymatic hydrolysate obtained in step (2) for alcohol precipitation, centrifuge, and collect the precipitate;

(4) Add deionized water to dissolve the precipitate obtained in step (3), perform 5-10kDa membrane dialysis, and collect the retentate;

(5) Spray drying the retentate obtained in step (4) to obtain mannose protein;

(6) Centrifuge the enzymolysis precipitate obtained in step (2), add petroleum ether, heat to reflux, centrifuge, and collect the precipitate;

(7) Add protease to the precipitate obtained in step (6), perform enzymatic hydrolysis reaction, centrifuge, and collect the precipitate;

(8) Spray drying the precipitate obtained in step (7) to obtain yeast glucan.

(9) The liquid part obtained in step (3) is recovered by ethanol, and the remaining liquid after recovery is concentrated and spray-dried to obtain a yeast extract.
The method according to 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.
The method according to claim 3, characterized in that, in the step (1), the discarded industrial Saccharomyces cerevisiae cells need to be pre-treated, including: formulating the discarded industrial Saccharomyces cerevisiae cells into suspension with a mass concentration of 3-15% The liquid passes through a sieve with an aperture of 80-120 mesh, and is centrifuged with distilled water for 3-5 times. The centrifugal speed is 10 000-15 000 rpm and the centrifugal time is 10-15 min.
The method according to claim 2, characterized in that, in the step (1), the yeast cells are formulated into a suspension with a mass concentration of 6-15%, at a temperature of 110-130°C, and a time of 20-40 min, High temperature wall breaking treatment under the condition of 0.1Mpa pressure.
The method according to claim 2, wherein in the step (2), β-1,6-glucanase is selected from GenBank No. MH747076, GenBank No. NP596461 or GenBank No. XP024773174 β-1,6-glucanase.
The method according to claim 2, wherein in the step (2), the β-1,6-glucanase is derived from Corallococcus sp.EGB with the deposit number CCTCC NO: M2012528, and the enzymatic hydrolysis conditions are : The enzyme dosage is 2-10U/g yeast, the pH of the enzymatic hydrolysis system is 6-8, the time is 12-24h, the temperature is 30-50°C, and the stirring rate is 150-250rpm.
The method according to claim 7, characterized in that the Corallococcus sp. EGB is inoculated into VY/4 liquid enzyme production medium, fermented and cultured, and centrifuged to collect the supernatant to obtain the β-globulin in step (2). 1,6 glucanase.
The preparation method according to claim 2, characterized in that, in the step (3), the amount of ethanol is two to four times the volume of the supernatant obtained, and the temperature is 0-4°C overnight; the precipitate is collected by centrifugation, The said centrifugal speed is 6 000-10 000 rpm, the centrifugal time is 5-10 min or the precipitation is obtained through plate and frame filter press.
The preparation method according to claim 2, wherein in the step (4), the precipitate obtained in step (3) is dissolved in deionized water to prepare a solution to be dialyzed with a mass concentration of 2-8%. The molecular weight cut-off of the membrane is 5-10kDa and the temperature is 0-4°C.
The preparation method according to claim 2, wherein the amount of petroleum ether in the step (6) is 5-40 mL/g, the reflux temperature is 60-95° C., and the reflux time is 2-4 h.
The preparation method according to claim 2, characterized in that the enzymolysis conditions in the step (7) are: papain is selected, the enzyme dosage is 20 000-60 000 U/g, the time is 5-15 h, and the temperature is 30 -70°C, stirring speed is 150-250rpm.