阅读说明：本技术 一种絮凝酵母生物粘合剂及其制备和使用方法 (Flocculation yeast biological adhesive and preparation and use method thereof ) 是由 刘晨光 冯颀 张家威 于 2020-07-22 设计创作，主要内容包括：本发明公开了一种絮凝酵母生物粘合剂及其制备和使用方法,涉及生物粘合剂领域,包括蛋白质、多糖和絮凝酵母活细胞,其中,蛋白质含有贻贝蛋白多巴(Dopa),絮凝酿酒酵母包括酿酒酵母S.cerevisiae SPSC01、S.cerevisiae 6525Flol和S.cerevisiae 4126Flol中的一种或多种；其制备方法包括活化、培养、收集和混合；液态适合原位使用；固态适合长期保存和运输。Dopa赋予该粘合剂较强的粘合效能,絮凝酵母用于调整粘合强度,同时活性细胞使该粘合剂可以在粘合后的一段时间继续进行生化反应。该生物粘合剂生物粘合性能好,且具有防水、低刺激及抗菌的性能,能减少甲醛释放。(The invention discloses a flocculation yeast biological adhesive and a preparation and use method thereof, relating to the field of biological adhesives, comprising protein, polysaccharide and flocculation yeast living cells, wherein the protein contains mussel protein Dopa (Dopa), and the flocculation saccharomyces cerevisiae comprises one or more of saccharomyces cerevisiae S.cerevisiae SPSC01, S.cerevisiae 6525Flol and S.cerevisiae 4126 Flol; the preparation method comprises activating, culturing, collecting and mixing; the liquid state is suitable for in-situ use; the solid state is suitable for long-term storage and transportation. The Dopa imparts a strong binding efficiency to the binder, and the flocculating yeast serves to adjust the binding strength while the living cells allow the binder to continue biochemical reactions for a period of time after binding. The biological adhesive has good biological adhesion performance, waterproof, low-irritation and antibacterial performances, and can reduce formaldehyde release.)

1. A flocculating yeast bioadhesive, wherein the bioadhesive comprises proteins, polysaccharides and flocculating yeast living cells; wherein the protein comprises mussel protein Dopa (Dopa), and the flocculating yeast comprises one or more of saccharomyces cerevisiae SPSC01, saccharomyces cerevisiae s. cerevisiae 6525 flor, and saccharomyces cerevisiae s. cerevisiae 4126flor.

2. A flocculating yeast bioadhesive according to claim 1, wherein said mussel protein Dopa (Dopa) content of said protein is more than 30%.

3. A flocculating yeast bioadhesive according to claim 1, wherein said saccharomyces cerevisiae s.cerevisiae 6525 flor is obtained by introducing FLO-spsc gene into flocculating yeast s.cerevisiae 6525(ATCC No. 4006525) by genetic engineering means; the saccharomyces cerevisiae s.cerevisiae4126 flor was obtained by introducing FLO-spsc gene into flocculation yeast s.cerevisiae4126 (ATCC No. 4004126) by means of genetic engineering.

4. A flocculating yeast bioadhesive according to claim 1, wherein said bioadhesive comprises a liquid bioadhesive and a solid bioadhesive.

5. A method of preparing a flocculating yeast bioadhesive according to claims 1 to 4, comprising the steps of:

step 1) activation: inoculating the frozen stock solution of flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) into a seed culture solution for activation to obtain activated flocculation Saccharomyces cerevisiae;

step 2) culturing: when the cell growth of the activated flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 1) reaches the middle and later stages, transferring the activated flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) into an improved fermentation culture solution, and culturing in a fermentation tank to obtain a cultured flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae);

step 3) collecting: when the cells of the cultured flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 2) reach the middle growth stage, collecting the flocculation Saccharomyces cerevisiae cells at the lower layer of the fermentation tank to obtain fermented flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae);

step 4), mixing: uniformly mixing the fermented and flocculated Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 3) with a solution of protein and polysaccharide with rich Dopa content to prepare a liquid biological adhesive; freeze-drying the fermented flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 3) to obtain cell dry powder, and uniformly mixing the cell dry powder with the mixed dry powder of protein and polysaccharide with rich Dopa content to prepare a solid biological adhesive; wherein the mass ratio of the flocculent cell dry powder to the mixed dry powder of the protein and the polysaccharide rich in Dopa is 1: 20 to 1: 50, the components of the mixed dry powder are yeast extract, peptone, glucose, mannose and the protein with rich Dopa content.

6. A method of making a flocculating yeast bioadhesive according to claim 5, wherein the flocculating yeast (Saccharomyces cerevisiae) comprises one or more of Saccharomyces cerevisiae SPSC01, Saccharomyces cerevisiae 6525Flol and Saccharomyces cerevisiae 4126 Flol.

7. The method of claim 5, wherein the seed culture solution in step 2) is YPD seed culture solution, and the formulation comprises 1% by weight of yeast extract, 2% by weight of peptone and 2% by weight of glucose; the improved fermentation culture solution formula in the step 3) is characterized in that proteins rich in Dopa and mannose are additionally added on the basis of the YPD seed culture solution, wherein the mass fraction of the Dopa proteins is 0.1-20%, and the mass fraction of the mannose is 0.1-10%.

8. A process for the preparation of a flocculating yeast bioadhesive according to claim 5, wherein said Dopa mass content of said Dopa-rich protein in step 4) is more than 30%.

9. The use method of the flocculation yeast biological adhesive according to claim 1-4, wherein the liquid biological adhesive is suitable for in-situ production and use, and the use method of the liquid biological adhesive is direct adhesion; the solid biological adhesive is suitable for long-term storage and long-distance transportation, and the use method of the solid biological adhesive comprises the following steps: mixing the solid biological adhesive and the improved fermentation liquor in a mass ratio of 1: 20-50, stirring uniformly, placing and culturing at 20-30 ℃ for 1-4 h to obtain a mixture, and collecting the mixture for use, wherein the components of the mixture are flocculating yeast cells, proteins and polysaccharides with abundant Dopa content.

10. The method of claim 9, wherein the modified fermentation broth is prepared by adding 0.1-20 wt% of the protein rich in Dopa and 0.1-10 wt% of mannose to YPD seed broth, wherein the YPD seed broth is prepared from 1 wt% yeast extract, 2 wt% peptone and 2 wt% glucose.

Technical Field

The invention relates to the field of biological adhesives, in particular to a flocculation yeast biological adhesive and a preparation method and a use method thereof.

Background

The condensation of formaldehyde and urea can form urea-formaldehyde resins, so-called resin glues. Because the raw materials are low in price, the application range of the glue is quite common, and the glue is mainly applied to the manufacturing of wood board type materials. Urea-formaldehyde resins contain unreacted formaldehyde in various parts of the panel, and deep formaldehyde release is a slow, continuous process that causes formaldehyde contamination in interior building materials and office supplies. People living indoors are easy to contact formaldehyde for a long time, and are not good for human health. Thus, from an environmental point of view, there appears to be a biological binder that does not release formaldehyde in partial replacement for urea formaldehyde resins.

Mussels living in the sea can quickly and firmly adhere to the reefs by secreting mussel protein, and are effectively prevented from being washed away by sea waves. From the chemical composition, good anti-wet tack in mussel adhesive proteins comes from the characteristic Dopa in proteins (Dopa). Dopa is a post-transcriptional modified amino acid produced by a tyrosine under the catalytic action of a tyrosine hydroxylase, and a catechol group in a side group of the Dopa has chemical versatility and affinity diversity. In alkaline seawater, Dopa is oxidized into dopaquinone, and both Dopa and dopaquinone can form bond crosslinking and non-bond crosslinking on organic, inorganic and metal surfaces, so that the Dopa is the key of interfacial adhesion. The mussel adhesive protein is a natural protein, has excellent waterproof adhesive performance and good biocompatibility, and does not cause human immune reaction. Therefore, the adhesive has wide application prospect in the aspects of ship waterproof sealing, ship bottom protective paint, medical adhesive for tissue rapid suturing in the biomedical field, household adhesive in the building decoration field and the like. However, the use of bioaxtraction to prepare mussel adhesive protein is expensive and limits the use of bioadhesives. On one hand, the mussel adhesive protein is high in extraction cost, for example, a method for extracting adhesive protein from perna viridis and using a waterproof biological adhesive with good biocompatibility is used for directly extracting and preparing the adhesive protein from organisms, so that the cost is reduced, but the prepared adhesive protein is relatively expensive as a raw material of the adhesive. On the other hand, if no mussel adhesive protein is used to produce the bioadhesive, the use of a combination of biological and chemical materials to produce the bioadhesive will introduce additional contamination while reducing costs. For example, the biological adhesive prepared by carrying out Michael addition reaction on structural proteins (collagen, gelatin and the like) and tannic acid, carrying out subsequent treatment and then crosslinking through Transglutaminase (TG) has certain pollution to the environment and certain stimulation to a human body due to introduction of non-biological chemical substances.

Therefore, a low-cost, environment-friendly, nontoxic and effective bioadhesive is urgently needed.

Therefore, those skilled in the art have been devoted to develop a low-cost, effective bioadhesive, which partially replaces urea-formaldehyde resin, and has no pollution to the environment and no irritation to the human body.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is to develop a bioadhesive that is low cost, water-resistant, has good bioadhesive properties, is low-irritating and is environmentally friendly. In order to achieve the above object, the present invention provides a bioadhesive containing flocculation yeast, which not only has good bioadhesive performance, water resistance, low irritation and antibacterial effect, but also can be mixed with a traditional adhesive taking urea resin as a main component according to a certain proportion, so as to reduce the usage amount of urea resin, and thus reduce the release of formaldehyde. More importantly, because the adhesive contains active flocculation yeast cells, the formaldehyde remaining in the adhesive can be absorbed and decomposed into safe and environment-friendly carbon dioxide and water in a short time (several days) through cell metabolism, so that the problem of slow release of the formaldehyde can be solved fundamentally.

A flocculating yeast bioadhesive comprising proteins, polysaccharides and flocculating yeast living cells, wherein the proteins comprise mussel protein Dopa (Dopa) and the flocculating yeast comprises one or more of saccharomyces cerevisiae s. Saccharomyces cerevisiae SPSC01 is a fusion strain of Schizosaccharomyces pombe (Schizosaccharomyces pombe number Lindner 2.1461) and Saccharomyces cerevisiae (Saccharomyces cerevisiae 2.607), and is deposited in China general microbiological culture Collection center (CGMCC, China, Beijing) at 5/20 days 2001 with accession number CGMCC 0587.

Further, the content of mussel protein Dopa (Dopa) in the protein exceeds 30%.

Further, saccharomyces cerevisiae s.cerevisiae 6525 flor was obtained by introducing FLO-spsc gene into flocculation yeast s.cerevisiae 6525(ATCC No. 4006525) by means of genetic engineering; saccharomyces cerevisiae4126Flol was obtained by introducing FLO-spsc gene into flocculation yeast S.cerevisiae 4126(ATCC No. 4004126) by genetic engineering means.

Further, the bioadhesive includes liquid bioadhesive and solid bioadhesive.

The invention also provides a preparation method of the biological adhesive containing the flocculating yeast, which comprises the following steps:

step 1) activation: inoculating the frozen stock solution of flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) into a seed culture solution for activation to obtain activated flocculation Saccharomyces cerevisiae;

step 2) culturing: when the cell growth of the activated flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 1) reaches the middle and later stages, transferring to an improved fermentation culture solution, and culturing in a fermentation tank to obtain the cultured flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae);

step 3) collecting: when the cells of the cultured flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 2) reach the middle growth stage, collecting the flocculation Saccharomyces cerevisiae cells at the lower layer of the fermentation tank to obtain the fermented flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae);

step 4), mixing: uniformly mixing the fermented and flocculated Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 3) with a solution of protein and polysaccharide with rich Dopa content to prepare a liquid biological adhesive; freeze-drying the fermented flocculation Saccharomyces cerevisiae (Saccharomyces cerevisiae) obtained in the step 3) to obtain cell dry powder, and uniformly mixing the cell dry powder with the mixed dry powder of protein and polysaccharide with rich Dopa content to prepare a solid biological adhesive; wherein the mass ratio of the flocculent cell dry powder to the mixed dry powder is 1: 20 to 1: 50, the components of the mixed dry powder are yeast extract, peptone, glucose, mannose and protein with abundant Dopa content.

Further, the flocculating Saccharomyces cerevisiae (Saccharomyces cerevisiae) comprises one or more of Saccharomyces cerevisiae SPSC01, Saccharomyces cerevisiae s. cerevisiae 6525 flor and Saccharomyces cerevisiae4126 flor.

Further, the seed culture solution in the step 2) is YPD seed culture solution, and the formula of the seed culture solution comprises 1% of yeast extract, 2% of peptone and 2% of glucose by mass; the formula of the improved fermentation culture solution in the step 3) is that proteins with rich Dopa content and mannose are additionally added on the basis of YPD seed culture solution, wherein the mass fraction of the Dopa proteins is 0.1-20%, and the mass fraction of the mannose is 0.1-10%.

Further, the mass content of the Dopa in the protein rich in the Dopa in the step 4) exceeds 30%.

The invention also provides a using method of the biological adhesive containing the flocculating yeast, the liquid biological adhesive is suitable for in-situ production and use, and the using method of the liquid biological adhesive is direct adhesion; the solid biological adhesive is suitable for long-term storage and long-distance transportation, and the use method of the solid biological adhesive comprises the following steps: mixing a solid biological adhesive and an improved fermentation liquor in a mass ratio of 1: 20-50, stirring uniformly, placing and culturing at 20-30 ℃ for 1-4 h to obtain a mixture, and collecting the mixture for use, wherein the components of the mixture are flocculating yeast cells, proteins and polysaccharides with abundant Dopa content.

Furthermore, the improved fermentation culture solution formula is characterized in that proteins with rich Dopa content and mannose are additionally added on the basis of YPD seed culture solution, wherein the mass fraction of the Dopa proteins is 0.1-20%, and the mass fraction of the mannose is 0.1-10%.

Further, a bioadhesive containing flocculated yeast active cells, which mainly comprises the mussel protein Dopa (Dopa).

Further, the flocculation yeast SPSC01 is a fusion strain of fission yeast Schizosaccharomyces pombe (Schizosaccharomyces pombe pommbelindner 2.1461) and saccharomyces cerevisiae (saccharomyces cerevisiae 2.607), which was deposited at the common microorganism center of the china committee for culture collection and management of microorganisms (CGMCC, china, beijing) 5/20 days in 2001 with the accession number of CGMCC 0587 (published by patent CN 1436851A).

Further, flocculation yeast 6525Flol was obtained by introducing FLO-spsc gene into Saccharomyces cerevisiae S.cerevisiae 6525(ATCC No. 4006525) by genetic engineering means.

Further, flocculation yeast 4126 flor was obtained by introducing FLO-spsc gene into flocculation yeast s.cerevisiae 4126(ATCC No. 4004126) by means of genetic engineering.

Further, the surface of the flocculated Saccharomyces cerevisiae has proteins with adhesion, flocculation factors (Flocculin) and exopolysaccharides assisting adhesion, so that the surface is rougher and the specific surface area is larger than that of the free Saccharomyces cerevisiae.

Further, the extraction method of the protein rich in the Dopa comprises the following steps:

step a: taking live marine mussel feet, pretreating, cutting into pieces, and homogenizing;

step b: centrifuging the treated homogenate to separate a supernatant, adding perchloric acid (PCA), stirring, centrifuging, and recovering the supernatant;

step c: cooling the supernatant in glacial ethanol, adding concentrated sulfuric acid for acidification while stirring, and adding acetone into the acidified mixed solution;

step d: standing for a period of time, centrifuging, removing supernatant, dissolving precipitate in acetic acid, centrifuging, collecting supernatant, and lyophilizing to obtain protein dry powder with abundant Dopa content.

Further, the two forms of liquid bioadhesive and solid bioadhesive are suitable for different applications; the liquid biological adhesive is suitable for in-situ production and use, does not need complex subsequent processing, maintains high cell activity and biological adhesive activity, and has low cost; the solid biological adhesive can be stored for a long time and transported for a long distance due to dehydration treatment, water is needed to be added for activation before use, water for activation is often replaced by improved fermentation liquor for activation for better adhesion effect, and the growth middle stage is reached after mixed culture for about 2 hours, so that the transformation activity of yeast cells can be better exerted.

The Dopa in the flocculent yeast biological adhesive of the invention endows the adhesive with stronger adhesive performance, the flocculent yeast is used for adjusting the adhesive strength, and simultaneously, the active cells enable the adhesive to continue biochemical reaction for a period of time after the adhesion. The adhesive not only has better biological adhesive property, waterproofness, low irritation and antibacterial effect, but also can be mixed with a traditional adhesive taking urea-formaldehyde resin as a main component according to a certain proportion, so that the use amount of the urea-formaldehyde resin is reduced, and the release of formaldehyde is reduced. More importantly, because the adhesive contains active flocculation yeast cells, the formaldehyde remaining in the adhesive can be absorbed and decomposed into safe and environment-friendly carbon dioxide and water in a short time (several days) through cell metabolism, so that the problem of slow release of the formaldehyde can be solved fundamentally.

In example 1, which is a preferred embodiment of the present invention, the method of extracting a protein rich in Dopa will be described in detail.

In another preferred embodiment of the present invention, example 2, the preparation process of the flocculation yeast bio-adhesive is described in detail.

In another preferred embodiment example 3 of the present invention, the use of the liquid bioadhesive or the activated solid bioadhesive for preparing the plywood is explained in detail.

In another preferred embodiment of the present invention, example 4, the embodiment of the flocculation yeast biological adhesive for degrading formaldehyde is described in detail.

Compared with the prior art, the biological adhesive contains the flocculating yeast, so that the yeast metabolizes the formaldehyde to generate carbon dioxide and water, and secondary release of the formaldehyde is avoided. Thus, formaldehyde gas can be effectively absorbed and removed by using yeast cells; on the other hand, the flocculation yeast is beneficial to reducing the cost of the product, is mainly applied to ethanol fermentation at present, and can automatically flocculate to form cell masses in the fermentation process and separate from the fermentation product, so that the cost required by product purification is greatly saved; meanwhile, the flocculation property also ensures that extracellular polysaccharide and protein secreted by the yeast cells are greatly higher than those of common free cells. Glycoproteins on the surface of yeast cells form hydrogen bonds with residual glycosyl groups on the surface of adjacent cells, thereby linking the different cells. Flocculation is proved to be regulated and controlled by FLO related genes, and flocculation-breaking of yeast cells can be regulated and controlled by regulating and controlling the expression of FLO1, FLO5 and FLO11 genes. Among them, flocculation of flocculation yeast of flo1 type is mainly based on the ability to form hydrogen bonds between mannose residues and proteins on its surface.

The invention has the following beneficial effects:

1. the saccharomyces cerevisiae SPSC01, 6525Flol and 4126Flol can spontaneously flocculate, and the cell collection process is low in energy consumption and environment-friendly. The three strains are constructed flocculation saccharomyces cerevisiae which can be used for industrial production at present, and the strains have stable flocculation characteristics and can be used for industrial production to obtain a large amount of yeast cells.

2. The surface of the flocculated saccharomyces cerevisiae has adhesive proteins, is rough, and has larger specific surface area compared with the free saccharomyces cerevisiae cells, so that more surface hydroxyl groups are exposed, and the higher surface energy is possessed. Therefore, compared with free yeast, the surface of the flocculated saccharomyces cerevisiae cell can generate higher thermodynamic reaction potential, is more beneficial to the yeast cell to adhere other substances, and increases the viscosity of the adhesive in use.

3. The invention directly uses live yeast cells rather than yeast component extracting solution, reduces the cost of the process of extracting effective substances, and simultaneously keeps the activity of the cells so that the cells can complete various biochemical reactions, such as the function of removing formaldehyde.

4. The cell surface of the flocculation saccharomyces cerevisiae is rich in reducing groups such as hydroxyl, aldehyde group and the like, and can combine and reduce high-valence heavy metal ions on the surface of the material.

5. The biological adhesive of the flocculating yeast is used in the home decoration process, so that gases harmful to human bodies, such as formaldehyde brought by common chemical adhesives, can be reduced from the source, meanwhile, the effective metabolism and absorption of formaldehyde in the air can be realized, the formaldehyde is converted into carbon dioxide, and the process is environment-friendly and efficient.

6. The biological adhesive of the invention has enriched content of living cells, so that the functions of removing formaldehyde toxicity, adsorbing and reducing heavy metal ions, inhibiting infectious microbes and the like of the flocculation saccharomyces cerevisiae are enhanced.

7. The application method of the solid biological culture medium is that the dry cell powder and the improved fermentation culture medium are mixed according to a certain proportion and cultured for about 2 hours to reach the middle growth stage, so that the transformation activity of the yeast cells can be better exerted. And the powder can be kept in a dry powder state at ordinary times, so that the powder can be stored for a longer time.

8. The flocculation yeast after being modified by a genetic engineering means can improve the adhesiveness and the formaldehyde degradation capability of the flocculation yeast.

9. The yeast has beer or bread fragrance, and improves the sensory acceptability of the adhesive. The yeast has high biological safety, no potential microbial risk, easy and safe decomposition after use and no influence on the adhesion effect.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a flow chart of the preparation of a flocculating yeast bioadhesive according to a preferred embodiment 2 of the present invention;

FIG. 2 is a schematic view of the process of formaldehyde degradation by the flocculation yeast biological binder according to a preferred embodiment of the present invention 5;

wherein, 1-fermentation tank; 2-stirring paddle; 3-flocculating yeast; 4-Dopa protein; 5-flocculating yeast active cells; 6-urea formaldehyde resin.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.