CN113174416A - Method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus - Google Patents
Method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus Download PDFInfo
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Abstract
The invention belongs to the technical field of kitchen waste resource utilization. The invention aims to realize the efficient directional conversion of the bacterial cellulose from the kitchen garbage by taking the black tea fungus as a bacterial cellulose production strain, thereby achieving the resource utilization of the kitchen garbage and reducing the production cost of the bacterial cellulose. The invention provides a method for producing bacterial cellulose by fermenting kitchen garbage with black tea fungus, which comprises the following steps: 1) pretreatment of kitchen garbage: 2) efficient biodegradation of kitchen waste: 3) domestication culture of black tea fungus in kitchen garbage; 4) preparing bacterial cellulose by fermenting black tea fungus of the kitchen waste enzymolysis liquid; 5) and (4) collecting and purifying the bacterial cellulose. By adopting the technology provided by the invention, a high-concentration sugar platform can be provided for the directional conversion of the kitchen waste products, and a foundation is laid for realizing the resource utilization and high-valued conversion of the kitchen waste; meanwhile, the method develops cheap and efficient production raw materials for the production of the bacterial cellulose.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of kitchen waste resource utilization, and particularly relates to a method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus.
[ background of the invention ]
Bacterial cellulose is a porous reticular nano-scale biopolymer material produced by microbial fermentation and is named after being synthesized by bacteria. As for chemical components, the cellulose has no obvious difference from plant cellulose in chemical composition and structure, and is a linear high molecular compound formed by connecting beta-D-glucose through beta-1, 4-glycosidic bonds, and the main difference of the two is that the former does not contain hemicellulose, lignin, pectin and other cell wall components. But compared with plant cellulose, the bacterial cellulose has the obvious advantages of high purity (more than or equal to 99 percent), high crystallinity, good water binding capacity, high tensile strength, good biocompatibility and degradability, controllability during biosynthesis and the like. Therefore, the bacterial cellulose has wide application prospect in the industries of medical dressing, tissue engineering, textile, food industry, conductive material, environmental protection and the like. However, in China, the production cost of bacterial cellulose (particularly the culture medium cost is high) and the yield is low, and the problems still restrict the industrialization and the widening of the application range to a certain extent. In fact, the cost of bacterial cellulose fermentation depends on the culture medium, especially the carbon source, so that the search for cheap and efficient production raw materials is one of the development directions for reducing the production cost of bacterial cellulose.
Residues of industrial and agricultural production, food processing wastes, agricultural and sideline products such as fruits and vegetables with high sugar content and starch content are widely used as raw materials for bacterial cellulose production. As one of the important components of organic garbage in municipal solid waste, the discharge amount of kitchen waste is increasing with the increasing population and the increasing living standard in recent years. The kitchen waste contains starch, cellulose, protein, lipid, inorganic salt and the like, and can provide good carbon source and energy for the growth and the propagation of microorganisms. If the kitchen waste can be further recycled, not only can the pollution to the environment be avoided, but also the utilization degree of renewable resources can be improved, a series of biological products can be obtained, the recycling, energy regeneration and the like can be realized, and the development direction of sustainable development and circular economy can be met. However, at present, the treatment of kitchen waste is mainly incineration, landfill, or composting. The kitchen garbage can be degraded through composting, natural fermentation and other modes, so that the reduction of the kitchen garbage is realized, but the recycling is insufficient. And the kitchen waste is directionally converted into a product with a high added value, so that an important way of recycling the kitchen waste is realized.
Currently, research on directional conversion of kitchen waste includes enzymatic and microbial conversion processes. According to the characteristics of raw materials of kitchen garbage, enzyme preparation is adopted to degrade the kitchen garbage into fermentable carbon source, and then the fermentable carbon source is converted into a target product by microorganisms. The directional conversion products of kitchen garbage mainly comprise lactic acid (ZL 201310437655.0), rhamnolipid biosurfactant, ethanol (ZL 201310015313.X,201910361741.5), butanol (ZL201210056810.X), hexanoic acid (202010997594.3) and the like. However, no research related to fermentation of bacterial cellulose by kitchen waste is found. The fermenting microorganisms for bacterial cellulose are mainly from Acetobacter, such as Acetobacter (Acetobacter), foal-shaped bacillus (Komagataeibacter), Gluconacetobacter (Gluconacetobacter) and Gluconobacter (Gluconobacter), which not only can synthesize bacterial cellulose in large quantities, but also can form obvious cellulose membranes. In addition, there are microorganisms belonging to the family of non-acetobacteriaceae, such as Enterobacter (Enterobacter), Pseudomonas (Pseudomonas), Bacillus (Bacillus), and the like. A symbiotic community consisting of various acetic acid bacteria, yeasts and lactic acid bacteria exists in the black tea fungus (Kombucha). Therefore, the black tea fungus is also used for producing bacterial cellulose, for example, patent 201510133651.2 discloses a method for producing bacterial nano cellulose by using black tea fungus and raffinose, wherein raffinose extracted from cottonseed meal is used as a raw material, and the black tea fungus can overcome the inhibition of gossypol on acetobacter xylinum and greatly reduce the production raw material cost of the bacterial nano cellulose; patent 201810424077.X discloses a method for preparing bacterial cellulose by using soybean curd yellow serofluid, and also realizes black tea fungus fermentation of bacterial cellulose. And researches show that compared with acetic acid bacteria separated by independent culture, the cellulose production speed and yield of the black tea bacteria are relatively high due to the strong symbiotic relationship and the complex metabolic pathway formed among various microorganisms of the black tea bacteria. Therefore, the black tea fungus has important application value in the aspect of bacterial cellulose production.
Therefore, the invention discloses a method for producing bacterial cellulose by fermenting kitchen garbage with black tea fungus, which aims to take black tea fungus as a bacterial cellulose production strain and kitchen garbage as a raw material for producing the bacterial cellulose.
[ summary of the invention ]
[ OBJECTS OF THE INVENTION ]
The production cost of the bacterial cellulose (especially the culture medium cost is high) and the yield is low, and the problems still restrict the industrialization and the widening of the application range to a certain extent. In fact, the cost of bacterial cellulose fermentation depends on the culture medium, especially the carbon source, so that the search for cheap and efficient production raw materials is one of the development directions for reducing the production cost of bacterial cellulose. The efficient high-value utilization of the kitchen waste has the dual attributes of recycling and pollution prevention, and the kitchen waste is directionally converted into a product with a high added value, so that the method is an important way for recycling the kitchen waste. One of the purposes of the invention is to provide a method for producing bacterial cellulose by using kitchen garbage as a raw material for producing the bacterial cellulose, thereby providing an idea for realizing the directional high-valued production of the kitchen garbage. The invention also aims to provide a method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus by taking the black tea fungus as a production strain of the bacterial cellulose, so that the fermentation efficiency of the bacterial cellulose is improved and the fermentation cost is reduced through the interaction of multiple black tea fungus strains.
[ technical solution ] A
The invention relates to a method for producing bacterial cellulose by fermenting kitchen garbage with black tea fungus, which comprises the following steps:
1) pretreatment of kitchen garbage: firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) efficient biodegradation of kitchen waste: adding water into the kitchen waste raw material obtained in the step 1) to adjust to a certain solid-to-liquid ratio, and adding high-temperature amylase, pectinase, cellulase and glucoamylase to carry out enzymolysis;
3) domestication and culture of black tea fungus in kitchen garbage: performing gradient dilution on the hydrolysate, performing high-temperature sterilization and cooling, and inoculating various seed solutions of degrading bacteria step by step from high dilution to low dilution for acclimatization to obtain black tea fungus with high fermentation performance;
4) preparing bacterial cellulose by fermenting black tea fungus of the kitchen waste enzymolysis liquid: directly sterilizing the kitchen waste enzymatic hydrolysate obtained in the step 2) at 115 ℃ for 30min or after supplementing nutrient substances, cooling, inoculating domesticated black tea fungus seed liquid, and then standing and culturing in a constant-temperature incubator;
5) collecting and purifying bacterial cellulose: and 4) taking out the bacterial cellulose membrane after the fermentation is finished, and performing degerming, impurity removal and purification to obtain the white transparent bacterial cellulose membrane.
The solid-liquid ratio of the kitchen waste obtained in the step 2) is adjusted to 1: 5-1: 15(w/v, measured by the wet weight of the kitchen waste).
The parameters of the enzymolysis of various enzymes added into the raw materials of the kitchen waste in the step 2) of the invention are that high-temperature amylase is added to treat the kitchen waste for 30-60min at 70-90 ℃, the temperature is cooled to 50 ℃, simultaneously glucoamylase, pectinase and cellulase are added, the temperature is kept at 50 ℃ for enzymolysis for 2-6h, and after the enzymolysis is finished, the filtrate is collected by filtration.
The black tea fungus and various enzyme preparations can be purchased from commercial products.
Diluting the kitchen waste hydrolysate with water, sterilizing at high temperature, cooling, inoculating the black tea fungus seed solution into the kitchen waste hydrolysate with the highest dilution according to the inoculation amount of 20%, and standing and culturing at 28-30 ℃ for 7 d; after the bacteria grow well, the obtained black tea fungus fermentation liquid is inoculated into kitchen waste hydrolysate with the next dilution degree step by step according to the dilution times from high to low until the black tea fungus which can grow well in the high-concentration kitchen waste hydrolysate is obtained.
The nutrient substances in the step 4) comprise peptone, yeast extract and corn steep liquor, and the addition amount is 0.5-2%.
The black tea fungus inoculation amount in the step 4) of the invention is 5-30% (v/v), the fermentation culture temperature of the black tea fungus is 25-32 ℃, and the culture time is 7-14 days.
The method for purifying the bacterial cellulose membrane in the step 5) comprises the following steps: treating with 0.1mol/L NaOH solution in 100 deg.C water bath for 30min to obtain white transparent bacterial cellulose membrane.
[ PROBLEMS ] the present invention
The kitchen waste is rich in organic matters, has double properties of resource utilization and pollution prevention and control in high-efficiency utilization, and has important research significance and application value. However, at present, the treatment mode of the kitchen waste is mainly incineration, landfill or composting, so that the added value is low and resources are wasted. Therefore, a technology for directionally converting the kitchen garbage into high value-added products is urgently needed. The bacterial cellulose is a polymer material with wide application prospect in the industries of medicine, textile, food, materials, environmental protection and the like, and the industrialization and the application popularization of the bacterial cellulose are restricted by the problems of high production cost (particularly high culture medium cost), low yield and the like. Therefore, the method for directionally converting the kitchen garbage into the bacterial cellulose by adopting the microbial fermentation method has important significance.
The invention discloses a method for producing bacterial cellulose by fermenting kitchen garbage with black tea fungus, which takes black tea fungus as a bacterial cellulose production strain and kitchen garbage as a bacterial cellulose production raw material. Has the following beneficial effects:
1) the kitchen garbage is efficiently degraded by the synergistic biological depolymerization of multiple enzymes, so that high-concentration fermentable sugar is obtained;
2) the kitchen waste hydrolysate can be directly used for producing the bacterial cellulose or used for compensating a small amount of nitrogen source, so that the production cost of the bacterial cellulose can be obviously reduced;
3) the black tea fungus with excellent fermentation performance is obtained by gradually increasing the concentration of the hydrolysate of the kitchen waste and performing domestication culture;
4) by utilizing the strong symbiotic relationship and the complex metabolic pathway among various microorganisms in the black tea fungus, the speed and the yield of fermenting the bacterial cellulose by the kitchen waste are obviously improved;
in a word, the technology provided by the invention can provide a high-concentration sugar platform for the directional conversion of the subsequent kitchen waste products, and lays a foundation for realizing the recycling and high-valued conversion of the kitchen waste; meanwhile, the method develops cheap and efficient production raw materials for the production of the bacterial cellulose.
[ detailed description ] embodiments
The technical solution of the present invention is further illustrated by the following examples. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into the crushed kitchen waste raw material to adjust the solid-to-liquid ratio to be 1:5(w/v, based on the dry weight of the kitchen waste), uniformly stirring, adding high-temperature amylase to treat at 70 ℃ for 60min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, performing heat preservation and enzymolysis at 50 ℃ for 1h, filtering after the enzymolysis is finished, collecting filtrate, namely kitchen waste hydrolysate, and refrigerating for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 0.5% peptone (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed solution according to the inoculation amount of 20% (v/v), and standing and culturing in a constant-temperature incubator at 28 ℃ for 7 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 6.542g/L (kitchen waste hydrolysate).
Example 2:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into pulverized kitchen waste raw materials to adjust the solid-to-liquid ratio to 1:8(w/v, based on the dry weight of the kitchen waste), stirring uniformly, adding high-temperature amylase, treating at 90 ℃ for 45min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, performing heat preservation and enzymolysis at 50 ℃ for 2h, filtering after the enzymolysis is finished, and collecting filtrate for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 1% of peptone and 0.5% of corn steep liquor (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed solution according to the inoculation amount of 10% (v/v), and standing and culturing in a constant-temperature incubator at 25 ℃ for 9 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 4.65g/L (kitchen waste hydrolysate).
Example 3:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into the crushed kitchen waste raw material to adjust the solid-to-liquid ratio of the kitchen waste to be 1:12(v/v, based on the dry weight of the kitchen waste), uniformly stirring, adding high-temperature amylase to treat at 90 ℃ for 30min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, carrying out heat preservation and enzymolysis at 50 ℃ for 6h, filtering after the enzymolysis is finished, and collecting filtrate for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 0.5% of yeast extract and 1% of corn steep liquor (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed solution according to the inoculation amount of 30% (v/v), and standing and culturing in a constant-temperature incubator at the temperature of 30 ℃ for 12 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 7.84g/L (kitchen waste hydrolysate).
Example 4:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into the crushed kitchen waste raw material to adjust the solid-to-liquid ratio of the kitchen waste to be 1:15(w/v, based on the dry weight of the kitchen waste), uniformly stirring, adding high-temperature amylase to treat at 90 ℃ for 60min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, performing heat preservation and enzymolysis at 50 ℃ for 4h, filtering after the enzymolysis is finished, and collecting filtrate for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 2% corn steep liquor (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed liquid according to the inoculation amount of 5% (v/v), and standing and culturing in a constant-temperature incubator at 30 ℃ for 14 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 6.31g/L (kitchen waste hydrolysate).
Example 5:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into the crushed kitchen waste raw material to adjust the solid-to-liquid ratio of the kitchen waste to be 1:9(w/v, based on the dry weight of the kitchen waste), uniformly stirring, adding high-temperature amylase to treat at 90 ℃ for 30min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, carrying out heat preservation and enzymolysis at 50 ℃ for 6h, filtering after the enzymolysis is finished, and collecting filtrate for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 2% of yeast extract and 0.5% of corn steep liquor (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed solution according to the inoculation amount of 20% (v/v), and standing and culturing in a constant-temperature incubator at the temperature of 30 ℃ for 9 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 6.74g/L (kitchen waste hydrolysate).
Example 6:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into the crushed kitchen waste raw material to adjust the solid-to-liquid ratio of the kitchen waste to be 1:8(w/v, based on the dry weight of the kitchen waste), uniformly stirring, adding high-temperature amylase to treat at 90 ℃ for 60min, cooling to 50 ℃, simultaneously adding saccharifying enzyme, pectinase and cellulase, performing heat preservation and enzymolysis at 50 ℃ for 4h, filtering after the enzymolysis is finished, and collecting filtrate for later use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 0.5% of corn steep liquor, 1% of peptone and 2% of yeast extract (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed liquid according to the inoculation amount of 10% (v/v), and standing and culturing in a constant-temperature incubator at the temperature of 28 ℃ for 14 d.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 8.32g/L (kitchen waste hydrolysate).
Example 7:
1) firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) adding water into pulverized kitchen waste raw materials to adjust solid-to-liquid ratio to 1:4(w/v, based on the dry weight of the kitchen waste), stirring uniformly, adding high temperature amylase, treating at 90 deg.C for 60min, cooling to 50 deg.C, simultaneously adding diastase, pectinase and cellulase, performing enzymolysis at 50 deg.C for 4h, filtering after enzymolysis, and collecting filtrate for use.
3) Performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization at 115 ℃ for 30min, cooling, performing gradient dilution on the kitchen waste hydrolysate with sterile water, inoculating 20% black tea fungus seed solution to the kitchen waste hydrolysate with the highest dilution, and performing standing culture at 28-30 ℃ for 7 d; after the thallus grows well, inoculating the obtained black tea fungus fermentation liquor into kitchen garbage hydrolysate of the next dilution, and continuously culturing according to the same inoculation amount and culture method until the thallus grows well; then, the acclimatization culture was continued in the order of the dilution from high to low.
4) Adding 0.5% of peptone, 0.5% of corn steep liquor and 0.5% of yeast extract (both w/v) into the kitchen waste hydrolysate obtained in the step 2), sterilizing at the high temperature of 115 ℃ for 30min, cooling, inoculating the domesticated black tea fungus seed solution according to the inoculation amount of 10% (v/v), and standing and culturing at the temperature of 28 ℃ for 9d in a constant-temperature incubator.
5) And after the fermentation is finished, taking out the bacterial cellulose membrane, removing thalli and impurities, and treating the bacterial cellulose membrane in a water bath at 100 ℃ for 30min by using 0.1mol/L NaOH solution to obtain the purified white transparent bacterial cellulose membrane.
According to the method, the yield of the bacterial cellulose is 7.93g/L (kitchen garbage hydrolysate).
Claims (8)
1. The invention relates to a method for producing bacterial cellulose by fermenting kitchen garbage with black tea fungus, which is characterized by comprising the following steps of:
1) pretreatment of kitchen garbage: firstly, picking up non-degradable inorganic impurities such as waste paper, disposable tableware, bones and the like from kitchen waste, and then crushing and slurrying the kitchen waste;
2) efficient biodegradation of kitchen waste: adding water into the kitchen waste raw material obtained in the step 1) to adjust to a certain solid-to-liquid ratio, and adding high-temperature amylase, pectinase, cellulase and glucoamylase to carry out enzymolysis;
3) domestication and culture of black tea fungus in kitchen garbage: performing gradient dilution on the kitchen waste hydrolysate, performing high-temperature sterilization and cooling, and inoculating various seed solutions of degrading bacteria step by step from high dilution to low dilution for acclimatization to obtain black tea fungus with high fermentation performance;
4) preparing bacterial cellulose by fermenting black tea fungus of the kitchen waste enzymolysis liquid: directly sterilizing the kitchen waste enzymatic hydrolysate obtained in the step 2) at 115 ℃ for 30min or after supplementing nutrient substances, cooling, inoculating domesticated black tea fungus seed liquid, and then standing and culturing in a constant-temperature incubator;
5) collecting and purifying bacterial cellulose: and 4) taking out the bacterial cellulose membrane after the fermentation is finished, and performing degerming, impurity removal and purification to obtain the white transparent bacterial cellulose membrane.
2. The method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus according to claim 1, wherein the solid-to-liquid ratio of water adjustment during biodegradation of the kitchen waste in step 2) is 1: 1-1: 7(w/v based on dry weight of the kitchen waste).
3. The method for producing bacterial cellulose by black tea fungus fermentation kitchen waste according to claim 1, wherein the enzymolysis parameters of various enzymes in the step 2) are that high-temperature amylase is added to treat the kitchen waste for 30-60min at 70-90 ℃, the kitchen waste is cooled to 50 ℃, simultaneously saccharifying enzyme, pectinase and cellulase are added, the temperature is kept at 50 ℃ for enzymolysis for 2-6h, and after the enzymolysis is finished, filtrate is collected by filtration.
4. The method for producing bacterial cellulose from black tea fungus fermented kitchen waste according to claim 1, wherein said black tea fungus and various enzyme preparations are commercially available.
5. The method for producing bacterial cellulose by black tea fungus fermentation kitchen waste according to claim 1, wherein the domestication culture method of black tea fungus in the step 3) is to dilute kitchen waste hydrolysate at different concentrations, sterilize at high temperature, cool, inoculate black tea fungus seed solution into kitchen waste hydrolysate with highest dilution according to an inoculation amount of 20%, and perform standing culture at 28-30 ℃ for 7 d; after the bacteria grow well, the obtained black tea fungus fermentation liquid is inoculated into kitchen waste hydrolysate with the next dilution degree step by step according to the dilution times from high to low until the black tea fungus which can grow well in the high-concentration kitchen waste hydrolysate is obtained.
6. The method for producing bacterial cellulose by black tea fungus fermentation kitchen waste according to claim 1, wherein the nutrient substances in step 4) comprise one or more of peptone, yeast extract and corn steep liquor, and the addition amount of the nutrient substances is 0.5-2% (w/v).
7. The method for producing bacterial cellulose by black tea fungus fermentation kitchen waste according to claim 1, wherein the black tea fungus inoculation amount in the step 4) is 5-30% (v/v), the black tea fungus fermentation culture temperature is 25-32 ℃, and the culture time is 7-14 d.
8. The method for producing bacterial cellulose by fermenting kitchen waste with black tea fungus according to claim 1, wherein the method for purifying the bacterial cellulose membrane in the step 5) comprises the following steps: treating with 0.1mol/L NaOH solution in 100 deg.C water bath for 30min to obtain white transparent bacterial cellulose membrane.
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| CN111057722A (en) * | 2019-12-24 | 2020-04-24 | 刘洋 | Method for fermenting corn straws by using black tea leavening agent |
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