阅读说明：本技术 一种菊叶薯蓣低酸预处理提高皂素得率的方法 (Method for improving saponin yield through low-acid pretreatment of dioscorea composita ) 是由 谢君 叶广英 毕桂灿 于 2019-11-20 设计创作，主要内容包括：本发明公开了一种菊叶薯蓣低酸预处理提高皂素得率的方法,先使用低浓度的硫酸预处理菊叶薯蓣,固液分离,固相部分再进行薯蓣皂素的水解。本发明避免了自然发酵的预处理周期长、具有产生杂质的潜在风险的问题；同时本发明避免了薯蓣皂素直接水解的过程中,由于淀粉和木质纤维素随着水解并进一步脱水,带来水解后废酸水中有机物含量的急剧增加,引起严重的环境污染问题；另外本发明提高了薯蓣皂素的水解得率明显提高。本方法具有显著优势,有很好的推广应用价值。(The invention discloses a method for improving saponin yield by low-acid pretreatment of Dioscorea composita. The method avoids the problems of long pretreatment period of natural fermentation and potential risk of generating impurities; meanwhile, the invention avoids the serious environmental pollution problem caused by the rapid increase of the organic matter content in the waste acid water after hydrolysis because starch and lignocellulose are hydrolyzed and further dehydrated in the process of direct hydrolysis of diosgenin; in addition, the invention improves the hydrolysis yield of diosgenin obviously. The method has obvious advantages and good popularization and application values.)

1. The method for improving the saponin yield by the low-acid pretreatment of the dioscorea composita is characterized by comprising the following steps of:

s1, using 0.1-0.2 mol/L sulfuric acid, and pretreating Dioscorea composita and sulfuric acid at the temperature of 110-120 ℃ for 2-6 hours according to the material-liquid ratio of 1: 4-6;

s2, solid-liquid separation, namely mixing solids with deionized water to obtain liquid: 4-6 ml solid: 1g of a compound;

s3, performing solid-liquid separation, mixing the solid with 1.0-1.2 mol/L sulfuric acid, and performing acid hydrolysis on the diosgenin at the solid-liquid ratio of 1: 4-6, wherein the hydrolysis temperature is 115-125 ℃, and the hydrolysis time is 4-6 hours;

s4, cooling to room temperature, then neutralizing to be neutral, filtering by using filter paper, and drying to obtain a hydrolysate;

s5, extracting the diosgenin from the hydrolysate by using petroleum ether Soxhlet.

2. The preparation method according to claim 1, wherein in step S1, 0.1mol/L sulfuric acid is used, and Dioscorea composita and sulfuric acid are pretreated at 105 ℃ for 6h according to a feed-liquid ratio of 1: 6.

3. The method according to claim 1, wherein in step S2, the ratio of liquid: solid 6 ml: 1g of the total weight of the composition.

4. The method according to claim 1, wherein in step S3, the concentration of sulfuric acid is 1.0mol/L, the solid-to-liquid ratio is 1:6, the hydrolysis temperature is 120 ℃, and the hydrolysis time is 4 h.

5. The method according to claim 1, wherein the Dioscorea composita is lyophilized powder of Dioscorea composita.

Technical Field

The invention relates to the technical field of diosgenin extraction, in particular to a method for improving saponin yield through low-acid pretreatment of Dioscorea composita.

Background

Dioscorea composita is a plant of the genus Dioscorea of the family Dioscoreaceae. The method is native to Mexico, and the seeds are successfully tested in Yunnan province in China in the 70 th century of 20 th century, so that the method is widely planted in China at present. The dioscorea composita has high saponin content, which is about twice as high as that of dioscorea zingiberensis, and is an important raw material for extracting saponin and synthesizing other corticoids.

The steroid of diosgenin has simple structure and easy chemical modification, is a main raw material for synthesizing steroid hormone medicines which are second only to antibiotics, is called medical gold, and has high market value. China is a world major producing country of diosgenin in years, and with the development of the saponin industry and the increase of the demand of the international market for diosgenin, the search for a diosgenin preparation method with high diosgenin content and high yield becomes one of the key for solving the current development of the diosgenin industry.

At present, the problems of low hydrolysis yield and serious pollution of the current dioscorea composita saponin hydrolysis production are insufficient, so that the production of the dioscorea composita saponin is limited.

Disclosure of Invention

The invention aims to overcome the defects of low hydrolysis yield and serious pollution in the current diosgenin hydrolysis production in the prior art, and provides a method for improving the saponin yield by low-acid pretreatment of dioscorea composita.

The invention aims to provide a method for improving saponin yield by low-acid pretreatment of Dioscorea composita.

In order to achieve the purpose, the invention is realized by the following technical scheme:

in the process of direct hydrolysis of diosgenin, starch and lignocellulose are hydrolyzed and further dehydrated, so that the content of organic matters in waste acid water after hydrolysis is increased rapidly, and the problem of serious environmental pollution is caused. The inventor finds that after low-acid pretreatment, solid-liquid separation is carried out, and the solid phase part is hydrolyzed by diosgenin, so that furostane saponin can be converted into spirostane saponin, the hydrolysis yield of the diosgenin is improved, the content of organic matters in wastewater can be reduced, and the cleanness of production is facilitated.

Therefore, the invention claims a method for improving saponin yield by low-acid pretreatment of Dioscorea composita, which comprises the following steps:

s1, using 0.1-0.2 mol/L sulfuric acid, and pretreating Dioscorea composita and sulfuric acid at the temperature of 110-120 ℃ for 2-6 hours according to the material-liquid ratio of 1: 4-6;

s2, solid-liquid separation, namely mixing solids with deionized water to obtain liquid: 4-6 ml solid: 1g of a compound;

s3, performing solid-liquid separation, mixing the solid with 1.0-1.2 mol/L sulfuric acid, and performing acid hydrolysis on the diosgenin at the solid-liquid ratio of 1: 4-6, wherein the hydrolysis temperature is 115-125 ℃, and the hydrolysis time is 4-6 hours;

s4, cooling to room temperature, then neutralizing to neutrality, filtering by using filter paper, and drying (105 ℃ overnight) to obtain a hydrolysate;

s5, extracting the diosgenin from the hydrolysate by using petroleum ether Soxhlet.

Preferably, in step S1, 0.1mol/L sulfuric acid is used, the Dioscorea composita and the sulfuric acid are pretreated for 6 hours at 105 ℃ according to the feed-liquid ratio of 1: 6;

preferably, in step S2, the liquid: solid 6 ml: 1g of the total weight of the composition.

Preferably, in step S3, the concentration of sulfuric acid is 1.0mol/L, the solid-to-liquid ratio is 1:6, the hydrolysis temperature is 120 ℃, and the hydrolysis time is 4 h.

Preferably, the Dioscorea composita adopts Dioscorea composita freeze-dried powder.

Compared with the prior art, the invention has the following beneficial effects:

the method avoids the problems of long pretreatment period of natural fermentation and potential risk of generating impurities; meanwhile, the invention avoids the serious environmental pollution problem caused by the rapid increase of the organic matter content in the waste acid water after hydrolysis because starch and lignocellulose are hydrolyzed and further dehydrated in the process of direct hydrolysis of diosgenin; in addition, the invention improves the hydrolysis yield of diosgenin obviously. The method has obvious advantages and good popularization and application values.

Drawings

Fig. 1 is a standard curve of diosgenin.

FIG. 2 shows the time variation of diosgenin yield in Dioscorea composita under different concentrations of sulfuric acid.

Fig. 3 shows the variation of the content of the four steroidal saponins during pre-acidification.

FIG. 4 shows the effect of different time pre-acidolysis treatment on diosgenin yield.

FIG. 5 shows the change in glucose content during pre-acidification.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

1. Dioscorea composita

Dioscorea composita is 2 years old, provided by Huake biotechnology limited of Fengyuan city, and identified as Dioscorea composita (Dioscorea composita Hemsl) of Dioscorea of Dioscoreaceae (Dioscorea) by energy plant resources and utilization key laboratories of agriculture department of south China university. Cleaning tuber, naturally drying, slicing, freeze drying, pulverizing, and storing in a dryer.

2. HPLC determination of diosgenin

Conditions for HPLC determination of diosgenin: the chromatographic column was Shimazu-GL intersdustain C18 (30X 150mm, S/N8 AR55067) and the column temperature was 40 ℃; the mobile phase is methanol water (90 percent) and the flow rate is 1 mL/min; the detector is Shimadzu ELSD-10A, (Gain 6; 46 deg.C; atomizing gas N)₂The pressure is 350 MPa).

Taking 1.0mg/mL diosgenin standard solutions, diluting the solutions with methanol and other standard solutions with gradient concentration of 500 ug/mL, 250ug/mL, 125ug/mL, 62.5ug/mL and 31.25ug/mL, measuring peak areas under HPLC, and performing linear fitting with origin2018 to obtain a standard curve (figure 1), wherein the linear regression curve is y-2675 x + 6970; coefficient of correlation R²＝0.9996。

3. Measurement of COD

The COD of the hydrolyzed wastewater is determined by a potassium dichromate method, and the COD is the mass concentration of oxygen corresponding to potassium dichromate consumed by soluble substances and suspended substances when a water sample is subjected to potassium dichromate oxidation treatment under certain conditions. Adding a known amount of potassium dichromate solution into a water sample, taking silver salt as a catalyst under a strong acid medium, boiling and refluxing for 2 hours, then taking resorufin as an indicator, titrating unreduced potassium dichromate in the water sample by using ammonium ferrous sulfate, and converting the amount of the consumed ammonium ferrous sulfate into the mass concentration of consumed oxygen.

Diluting 1mL of water sample to 20.00mL, refluxing in a 250mL ground conical flask, accurately adding 10.00mL of potassium dichromate standard solution, slowly adding 30mL of sulfuric acid-silver sulfate solution, shaking gently, mixing uniformly, and heating and refluxing for 2h (timing from the beginning of boiling). After cooling, 3 drops of the ferroxyl indicator solution are added, the solution is titrated by using an ammonium ferrous sulfate standard solution, the end point is obtained when the color of the solution is reddish brown, and the dosage K of the ammonium ferrous sulfate standard solution is recorded. While measuring the water sample, a blank test was carried out with 20.00mL of double distilled water by the same procedure.

COD＝C(V₂-V₁)×8×1000/V

In the formula: c is the concentration of the standard solution of ferrous ammonium sulfate, mol/L;

v1-consumption of standard solution of ferrous ammonium sulfate in titration blank, mL;

v2-consumption of standard solution of ferrous ammonium sulfate in titrating water sample, mL;

v-water sample volume, mL;

8-molar mass of oxygen (1/20), g/mol.

4. Computing

The yield of the diosgenin is the mass of the diosgenin actually extracted from each gram of dioscorea composita, wherein the mass of the diosgenin actually extracted is the product of the concentration of the diosgenin in the extraction solution and the volume of the solution, and the unit of the yield of the diosgenin is mg/g.

The yam yield is the percentage of the actual measured value to the theoretical maximum value of diosgenin in the substrate-based dioscorea composita dry powder; the theoretical content of diosgenin is calculated as the ratio of the molecular weight of diosgenin 414 to the molecular weight of the four saponins (Dioscin, protodioscin, Asperin and asperioside) 869,1049,1015 and 1195, respectively.

The glucose yield is the mass of glucose actually extracted after each gram of dioscorea composita is hydrolyzed, the mass of glucose actually extracted is the glucose concentration and the hydrolysis reaction volume after hydrolysis, and the unit of the glucose yield is mg/g.

All experiments were performed in triplicate and the results are presented as mean values. Data standard error and error bars are calculated using analysis of variance (ANOVA), representing the range of values; statistical analysis was performed using MS Excel, Origin 8.5 and SPSS (Microsoft corp., USA); data were analyzed using one-way analysis of variance (ANOVA) and Student's t test; when the p-value was <0.05, the reading was considered significant.

5. The components of Dioscorea composita

The composition of dioscorea composita is shown in table 1, wherein the content of total saponins is 104.4mg/g (wherein dioscin is 60.8mg/g, saponin pb37.9mg/g, protodioscin is 4.6mg/g, and protodioscin is pb1.1m/g), and the theoretical content of diosgenin is 46.2mg/g by theoretical calculation, so when dioscorea composita is used as a raw material to produce diosgenin, the yield of diosgenin is calculated on the basis of the theoretical content. Besides the steroid saponin component, the freeze-dried powder of the dioscorea composita contains 348.2mg/g of starch, 90.4mg/g of cellulose and 110.2mg/g of hemicellulose.

Table 1 dioscorea composita components: