阅读说明：本技术 一种蒲公英橡胶的发酵提取方法 (Fermentation extraction method of dandelion rubber ) 是由 张继川 马喆 颉翔 马勇 聂秋海 王峰 张立群 于 2019-04-11 设计创作，主要内容包括：本发明公开了一种蒲公英橡胶的发酵提取方法。包括：(1)蒲公英橡胶草干根洗净、干燥、磨成粉末；(2)将步骤(1)得到的蒲公英橡胶草干根粉末放入反应釜中,加入稀硫酸进行预处理；然后水洗至中性后抽滤,将滤渣放入烘箱中进行干燥；(3)滤渣在纤维素酶的作用下进行水解；(4)步骤(3)得到的酶解液在微生物酿酒酵母的作用下进行发酵；(5)橡胶与发酵液的分离。本发明方法解决了溶剂法提取橡胶过程中有机溶剂的使用,降低了毒害,并且充分利用了蒲公英橡胶草中的非胶组分,使最终发酵后的橡胶纯度达到95.24％,乙醇浓度达到10.29g/L,而且发酵过后的残渣可以作为肥料,从而可以降低蒲公英橡胶提取的工艺成本。(The invention discloses a fermentation extraction method of dandelion rubber. The method comprises the following steps: (1) cleaning dried roots of taraxacum kok-saghyz, drying and grinding into powder; (2) putting the dandelion herb and rubber plant dry root powder obtained in the step (1) into a reaction kettle, and adding dilute sulfuric acid for pretreatment; then washing with water to neutrality, filtering, and drying the filter residue in a drying oven; (3) hydrolyzing the filter residue under the action of cellulase; (4) fermenting the enzymolysis liquid obtained in the step (3) under the action of microbial saccharomyces cerevisiae; (5) and (4) separating the rubber from the fermentation liquor. The method solves the problem of using organic solvent in the process of extracting rubber by a solvent method, reduces toxicity, fully utilizes non-rubber components in the taraxacum kok-saghyz, ensures that the purity of the finally fermented rubber reaches 95.24 percent, the concentration of ethanol reaches 10.29g/L, and the fermented residue can be used as fertilizer, thereby reducing the process cost of extracting the taraxacum kok-saghyz.)

1. A fermentation extraction method of dandelion rubber is characterized by comprising the following steps:

sample preparation, sample pretreatment, hydrolysis, fermentation and separation.

2. The fermentation extraction method of dandelion rubber according to claim 1, characterized in that said method comprises:

(1) cleaning dried roots of taraxacum kok-saghyz, drying and grinding into powder;

(2) putting the dandelion herb and rubber plant dry root powder obtained in the step (1) into a reaction kettle, and adding dilute sulfuric acid for pretreatment; then washing with water to neutrality, filtering, and drying the filter residue in a drying oven;

(3) hydrolyzing the filter residue under the action of cellulase;

(4) fermenting the enzymolysis liquid obtained in the step (3) under the action of microbial saccharomyces cerevisiae;

(5) and (4) separating the rubber from the fermentation liquor.

3. The fermentation extraction method of dandelion rubber according to claim 2, characterized in that:

in the step (1), the fineness of the powder is between 80 meshes and 200 meshes.

4. The fermentation extraction method of dandelion rubber according to claim 2, characterized in that:

in the step (2), the pretreatment temperature is 120-160 ℃; the pretreatment time is 20 min-60 min.

5. The fermentation extraction method of dandelion rubber as defined in claim 4, characterized in that:

the pretreatment temperature is 120-140 ℃; the pretreatment time is 30-45 min.

6. The fermentation extraction method of dandelion rubber according to claim 2, characterized in that:

in the step (2), the solid-to-liquid ratio is 1: 5-1: 15; the concentration of the dilute sulfuric acid is 1 to 6 percent.

7. The fermentation extraction method of dandelion rubber as claimed in claim 6, characterized in that:

the solid-liquid ratio is 1: 8-1: 10; the acid concentration is 1.5-3.5%.

8. The fermentation extraction method of dandelion rubber according to claim 2, characterized in that:

in the step (3), the step (c),

the dosage ratio of the filter residue to the cellulase is as follows: 1g: (0.1-0.3 ml);

the hydrolysis temperature is 45-55 ℃.

9. The fermentation extraction method of dandelion rubber according to claim 2, characterized in that:

step (4) of carrying out a treatment,

the volume of the microbial saccharomyces cerevisiae is 0.5 to 3 percent of the volume of the enzymolysis liquid;

the fermentation temperature is 20-30 ℃, and the fermentation time is 36-48 hours.

Technical Field

The invention relates to the technical field of rubber, in particular to a fermentation extraction method of dandelion rubber.

Background

With the continuous innovation and revolution of industrial technology, the number of natural rubber products on the market is increasing, and thus the demand for the yield of natural rubber is increasing. Natural rubber, petroleum, steel and coal are called as four industrial raw materials in the world, the natural rubber which is one of the four industrial raw materials is favored in various industries, the natural rubber has better elasticity at normal temperature, when the external force applied to the natural rubber is relieved, the natural rubber can be restored to the original state, and the natural rubber has excellent mechanical property, just because of the fact that the natural rubber can be widely applied to daily life, in the era of the rapid economic development today, the wide utilization of the natural rubber causes market crisis that the yield of the natural rubber in China is not in demand, the natural rubber in China is mainly distributed in low-latitude provinces such as Yunnan, Guangdong, Guangxi, Fujian and Hainan, and 1800 ten thousand mu of land suitable for planting the natural rubber in China has 1800 ten thousand mu, but more than 1700 mu has been developed, and the space for further growth is limited, the import of natural rubber in China is more than 80%, so that the search for the second natural rubber has great significance to the rubber industry in China, and the number of plants capable of producing the natural rubber reaches more than two thousand at present, but most of the plants have low rubber content, and the taraxacum kok-saghyz can effectively solve the critical problem of insufficient yield of the natural rubber.

Taraxacum kok-saghyz (TKS) originates from Tianshan region in the border of China and Kazakhstan, is named as Russian taraxacum in early days, belongs to the genus Taraxacum, and further belongs to the family of Compositae, rubber exists in latex tube and vascular bundle at the root of taraxacum kok-saghyz in the form of latex particles in fresh Taraxacum root, and rubber is intertwined with plant tissues in the form of filaments in dry Taraxacum kok-saghyz root, and has obvious drawing phenomenon after being broken off, the chemical structure of Taraxacum kok-saghyz is cis-1, 4-polyisoprene, and has the same structure as that of Hevea trilobata, and the difference between the cis-1, 4-polyisoprene and the Hevea trilobata is only reflected in the chemical components and the content of the non-rubber components, so that the Taraxacum kov.

In addition, the roots of the taraxacum kok-saghyz contain rubber and abundant polysaccharide components, the taraxacum kok-saghyz can be used for preparing ethanol through fermentation and used as a biological energy source, plant residues after gum extraction can be used for preparing organic fertilizers, leaves of the taraxacum kok-saghyz can be used as feeds or feed additives, the production cost of the taraxacum kok-saghyz can be completely reduced due to the benefit of the byproducts, the taraxacum kok-saghyz can be greatly developed, and the most remarkable problem at present is how.

At present, the dandelion rubber is extracted by a solvent method, and the solvent method is to extract the rubber from plant tissues by utilizing good solvents of the rubber by utilizing the principle of similar compatibility. Good solvents commonly used for the extraction of natural rubber are toluene, petroleum ether and chloroform, and toluene and petroleum ether are more commonly used solvents because chloroform corrodes metal containers. However, the organic solvents used in the solvent method have certain toxicity, which not only has certain harm to the health of operators, but also has great pollution to the environment, although the solvent method can extract rubber with high purity, the non-rubber components in the rubber grass are not fully utilized, and the cost of the used organic solvents is high, which causes the cost of rubber extraction to be high.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a fermentation extraction method of dandelion rubber. The method of the invention is not only non-toxic and harmless, but also can fully utilize the non-rubber components in the dandelion herb of Hevea brasiliensis, and can obtain high-purity rubber at the same time.

The dandelion taraxacum kok-saghyz contains rich lignocellulose, the non-gum components are polysaccharide, the polysaccharide components in the dandelion taraxacum kok-saghyz are not effectively utilized while gum is extracted in the prior art, although the polysaccharide components in the taraxacum kok-saghyz are difficult to digest and utilize by a human body, the polysaccharide components can be changed into monosaccharide through hydrolysis, the monosaccharide is used as a carbon source in the life activity of microorganisms, the monosaccharide can be metabolized through the generation and metabolism of the microorganisms, the monosaccharide obtained after hydrolysis is used as a nutrient substance to absorb, ethanol is metabolized and used as a biofuel, the environment is not polluted after combustion, and in addition, fermented plant residues can be used as fertilizers, so the whole treatment process is environment-friendly, pollution-free, residue-free and waste-free.

The invention aims to provide a fermentation extraction method of dandelion rubber.

The method comprises the following steps:

sample preparation, sample pretreatment, hydrolysis, fermentation and separation.

The method comprises the following steps:

(1) cleaning and drying the dandelion and taraxacum rubber grass dry roots, and grinding into powder;

(2) putting the dandelion herb and rubber plant dry root powder obtained in the step (1) into a reaction kettle, and adding dilute sulfuric acid for pretreatment; then washing with water to neutrality, filtering, and drying the filter residue in a drying oven;

(3) hydrolyzing the filter residue under the action of cellulase;

(4) fermenting the enzymolysis liquid obtained in the step (3) under the action of microbial saccharomyces cerevisiae;

(5) and (4) separating the rubber from the fermentation liquor.

Among them, it is preferable that,

in the step (1), the fineness of the powder is between 80 meshes and 200 meshes.

In the step (2), the pretreatment temperature is 120-160 ℃; more preferably 120 to 140 ℃;

the pretreatment time is 20min to 60 min; more preferably 30 to 45min.

In the step (2), the solid-to-liquid ratio is 1: 5-1: 15; more preferably 1:8 to 1: 10;

the concentration of the dilute sulfuric acid is 1 to 6 percent; more preferably 1.5% to 3.5%.

In the step (3), the step (c),

the dosage ratio of the filter residue to the cellulase is as follows: 1g: (0.1-0.3) ml;

the hydrolysis temperature is 45-55 ℃.

The higher the activity of the cellulase is, the better the hydrolysis effect of the cellulase is, the activity of the cellulase changes with the temperature to a certain extent, for example, between 45 ℃ and 55 ℃, the activity of the cellulase is increased and then decreased, the activity is the highest when the activity is 50 ℃, and then the activity is in a descending trend with the temperature rise, and the activity can be selected between 45 ℃ and 55 ℃ in comprehensive consideration.

Step (4) of carrying out a treatment,

the volume of the microbial saccharomyces cerevisiae is 0.5 to 3 percent of the volume of the enzymolysis liquid;

the fermentation temperature is 20-30 ℃, and the fermentation time is 36-48 hours.

The fermentation temperature is the growth temperature of the saccharomyces cerevisiae in the fermentation process, the saccharomyces cerevisiae has a temperature system which is most suitable for the saccharomyces cerevisiae per se, generally between 20 ℃ and 30 ℃, and for the fermentation time, the length of the fermentation time has a certain influence on the yield of alcohol, after the saccharomyces cerevisiae is fermented for 36 hours, the alcohol content is slowly increased, since most of sugar is consumed, the saccharomyces cerevisiae lacks certain nutrient substances, and after the saccharomyces cerevisiae is fermented for 48 hours, the alcohol content possibly has a certain tendency of descending, the reason is that when the saccharomyces cerevisiae lacks the nutrient substances, the produced ethanol is used as a carbon source for supplementing the nutrient substances, and if the fermentation time is too short, the sugar in the hydrolysate cannot be fully utilized, so the temperature can be selected from 36 hours to 48 hours.

The invention can adopt the following technical scheme:

the fermentation process of taraxacum kok-saghyz as one kind of lignocellulose material includes four basic steps, sample preparation, sample pre-treatment, hydrolysis and fermentation.

(1) Cleaning the dandelion and rubber grass dry roots, and drying at room temperature;

(2) grinding the dandelion roots dried at room temperature into powder by a stirrer;

(3) putting powdery dandelion roots into a stainless steel reaction kettle for pretreatment;

(4) washing the pretreated dandelion roots with water to be neutral, then carrying out suction filtration, and putting filter residues into an oven for drying;

(5) hydrolyzing the dried filter residue under the action of cellulase;

(6) fermenting the enzymolysis liquid under the action of microbial saccharomyces cerevisiae;

(7) separating the rubber from the fermentation liquor;

the glue extraction principle is as follows: the invention provides a fermentation method gelatin extraction process, which comprises the following four parts: the basic principle of the method is that the fermentation principle is utilized to decompose lignocellulose so as to separate rubber from plant tissues, the rubber is lighter than water and floats on the water surface, plant tissue residues are heavier than water and are settled at the bottom, and fermentation liquor is arranged in the middle, so that the extraction of the rubber and the preparation of byproduct ethanol are realized.

The invention can adopt the following technical scheme:

preparation of samples: adopting a batch of dandelion taraxacum hevea dry roots, continuously washing the roots with clear water until the water after washing is not turbid, then placing the roots at room temperature for drying, and grinding the dried dandelion roots into powder by a stirrer.

The pretreatment of the sample is the most important step in the whole fermentation process, the pretreatment effect influences the subsequent process indexes, and the purpose of the pretreatment is mainly embodied in three aspects, one is to remove the internal structure of the biomass which hinders hydrolysis and fermentation; secondly, the protection of the crushed lignin on the cellulose is realized, and the crystal structure of the cellulose is broken down; thirdly, the biological enzyme is fully contacted, and a good hydrolysis effect is obtained.

And (3) pre-treatment process exploration: the pretreatment of the taraxacum kok-saghyz dry roots is carried out in a stainless steel reaction kettle, firstly, system variables of an experiment are determined, a preliminary single-factor experiment finds that the reaction temperature, the reaction time, the solid-liquid ratio and the acid concentration have great influence on the experiment result, and according to the single-factor experiment result, the reaction temperature is selected to be 120-160 ℃; selecting the reaction time to be between 20min and 60 min; the solid-liquid ratio is selected to be between 1:5 and 1: 15; the acid concentration is selected to be between 1% and 6%; through carrying out hydrolysis experiments on the pretreated sample, the concentration of glucose monosaccharide after the sample is hydrolyzed is measured, the quality of the pretreatment effect of the sample at the early stage can be reflected according to the concentration of glucose, the concentration of glucose determines the concentration of byproduct ethanol after fermentation at the later stage, and meanwhile, the pretreatment effect of the sample has great influence on the purity of the rubber.

Hydrolysis of the pretreated sample: washing dandelion roots treated in a reaction kettle with water, performing suction filtration after the dandelion roots are washed to be neutral, drying filter residues in a drying oven, taking out a dried sample for next hydrolysis, selecting cellulase to realize hydrolysis of cellulose polysaccharide in the hydrolysis process, wherein the factors influencing the hydrolysis effect mainly comprise the temperature of an enzymolysis environment, the enzymolysis time, the dosage of the cellulase, the pH value of the enzymolysis environment and the proportion of dregs and water, because the cellulase has self-adaptive conditions to the environment temperature and the environment pH value, selecting the enzymolysis temperature of 50 ℃ in turn, the environment pH value of 4.8, exploring the enzymolysis time, the dosage of the cellulase and the proportion of dregs and water further, in a hydrolysis experiment, firstly preparing a citric acid-sodium hydroxide buffer solution with the pH value of 4.8, pouring 50ml of the buffer solution into a 100ml triangular flask, adding 5g of pretreatment sample, covering a sealing film, tying with a rubber band, sterilizing for 25min in a sterilization pot at 115 ℃, adding 0.75ml of cellulase on a clean bench, finally putting into a shaking incubator at 50 ℃ for enzymolysis for 72h, fishing out crude rubber floating above the enzymolysis liquid after the enzymolysis is finished, then carrying out suction filtration on the slag and the enzymolysis liquid, collecting filtrate and the crude rubber for next fermentation, carrying out multiple test exploration comparison on the dosage of the cellulase and the ratio of the slag to water, selecting 1g of sample from the dosage of the cellulase, adding 0.1 to 0.3ml of the cellulase, and selecting 1:5 to 1:20 of the feed-water ratio.

And (3) fermentation of enzymatic hydrolysis sample: for a sample subjected to enzymolysis, a microorganism saccharomyces cerevisiae is selected for anaerobic fermentation, the saccharomyces cerevisiae has good alcohol production capacity, the principle is that sugar in an enzymolysis liquid is taken as a nutrient substance to be absorbed, ethanol is metabolized through life activities, the saccharomyces cerevisiae needs certain growth conditions in the fermentation process, besides the nutrient substance for the growth of the saccharomyces cerevisiae needs to be added in the fermentation process, the environmental temperature and the environmental pH value of the fermentation need to be strictly controlled, generally the temperature which is most suitable for the growth of the saccharomyces cerevisiae is about 30 ℃, the environmental pH value is about 5.0 to 6.0, the influence on the concentration of the target product ethanol also has factors such as fermentation time, inoculation quantity of the yeast and the like besides the factors, in the shaking flask fermentation process, firstly, the enzymolysis liquid and a crude rubber triangular flask are poured into the flask, the pH value is adjusted to be 5.4, and then peptone with the volume of 2 percent of the enzymolysis liquid and yeast with the volume of 1 percent of the enzymolysis liquid are added to provide the nutrient substance for the growth of the saccharomyces, and then, covering a sealing film, tying the sealing film with a rubber band, sterilizing the bottle for 25min in a sterilizing pot at the temperature of 115 ℃, then inoculating saccharomycetes on a superclean workbench, finally putting the shake bottle into a water bath shaking table at the temperature of 30 ℃ for fermenting for 48h, after final fermentation, firstly fishing the rubber floating above the fermentation liquor, then carrying out suction filtration and separation on the fermentation liquor and fermentation residues, finally distilling and collecting ethanol in the fermentation liquor, and selecting the yeast with the volume of 0.5-3% of the volume of the enzymolysis liquid for fermentation according to the inoculation amount of the saccharomycetes.

Effects of the invention

The preparation of the byproduct ethanol is successfully realized while extracting the rubber by the processes of pretreatment, hydrolysis, fermentation and the like of the dry roots of the dandelion. The method solves the problem of using organic solvent in the process of extracting rubber by a solvent method, reduces the toxicity in the experimental process, fully utilizes non-rubber components in the taraxacum kok-saghyz, ensures that the purity of the finally fermented rubber reaches 95.24 percent, the concentration of ethanol reaches 10.29g/L, and the fermented residue can be used as fertilizer, thereby reducing the process cost of extracting the taraxacum kok-saghyz.

Drawings

FIG. 1 is an infrared image of dandelion rubber extracted in the example.

Detailed Description

The present invention will be further described with reference to the following examples.