CN103263448B - Fermentation bacteria used for fermentation pretreatment to improve extraction of Ginkgo biloba L. leaf flavone and application - Google Patents

Abstract

The invention discloses a fermentation strain used for fermentation pretreatment to improve extraction of flavonoids from ginkgo leaves and an application method. The fermentation strain is Aspergillus fungus, and the patent strain preservation number is CCTCCM2012515. First, crush the ginkgo leaves to 20-60 mesh; add water to the ginkgo leaves and mix, add water 45%-90%; sterilize the mixture at 115°C for 15 minutes, and put it into a fermenter; inoculate the strains according to the weight of the ginkgo leaves in the mixture, the inoculation amount 0.01%-0.02%; aeration and fermentation at 25-30°C for 2-11 days; according to the final parameters, the solid-to-liquid ratio is 1:20, the ethanol content is 60%, add alcohol and water in an appropriate amount, and continuously stir to extract flavonoids; filter and suspend liquid to obtain an extract; the extract is concentrated in a vacuum to obtain a finished product, or further dried into a solid state. The invention uses a strain of Aspergillus niger to ferment and pretreat the ginkgo leaves, degrades the leaf cells, and then extracts them by a traditional extraction method to improve the extraction rate of the ginkgo flavonoids. Compared with the traditional alcohol solvent extraction method, the amount of flavonoids extracted from ginkgo leaves is increased by 20-60%.

Description

Fermentation strains used for fermentation pretreatment to improve extraction of flavonoids from Ginkgo biloba leaves and their application

technical field

The invention belongs to the technical field of extraction of ginkgo flavonoids, in particular to a fermentation strain for improving the extraction of ginkgo flavonoids through fermentation pretreatment and an application method thereof.

Background technique

Since the 1980s, with the continuous improvement of people's living standards in our country, people's dietary patterns have also undergone great changes. The proportion of refined food and animal products in daily life is increasing day by day. The incidence of cardiovascular disease in the elderly has increased significantly. In China, high blood pressure alone accounts for 25%-50% of all deaths, and the number of people who die from cardiovascular diseases rises to the first or second place every year. In recent years, the incidence of cardiovascular diseases in middle-aged and elderly people is still on the rise. trend. Therefore, a reasonable and healthy diet is particularly important to prevent and reduce the morbidity and mortality of cardiovascular diseases. For middle-aged and elderly patients with severe cardiovascular disease, in addition to active drug therapy, diet therapy is also an important means of disease control; for middle-aged and elderly patients with mild cardiovascular disease, healthy diet therapy should be more focused on controlling and alleviating The disease comes first.

Ginkgo biloba L. is an ancient relic plant native to my country. Its leaves are sweet, bitter, and astringent; flat in nature, beneficial to the heart, restraining the lungs, dispelling dampness and stopping diarrhea. Control chest tightness and heartache, palpitation, cough with asthma, dysentery and leucorrhea. "Pinhui Jingyao" records that ginkgo leaves "make cakes for the powder and noodles, cook them over a simmer, and stop diarrhea and dysentery". "Diet Therapy Materia Medica" records that Ginkgo biloba can be used for palpitations, lung deficiency cough and asthma and other diseases.

Ginkgo biloba contains flavonoids as the main active ingredient, the content is generally 2.5%-3.8%, containing isorhamnetin, kaempferol, kaempferol-3-rhamnoside, quercetin, rutin, quercetin , Ginkgo biflavonoids, isogingko biflavones, ginkgo picrolactone A, catechin, epicatechin, Hanshizi catechin ingredients. Studies have shown that ginkgo leaf flavonoids can protect capillary permeability, dilate coronary arteries, restore arterial blood vessel elasticity, lower serum cholesterol, increase coronary blood flow, improve cardiovascular and cerebrovascular circulation, relieve smooth muscle spasm, relax bronchi and inhibit bacteria, and nourish the brain. Cells and other organs, but also has the unique effect of maintaining blood quality and cholesterol in arteries, peripheral blood vessels, and capillaries at normal levels. It is used in the treatment of coronary heart disease. Preliminary observations have a good effect on angina pectoris, and it also has a certain effect on lowering cholesterol and blood pressure.

At present, ginkgo extract products are all over the market, including various buccal tablets and oral liquids, as well as various functional foods added with extracts. The method of the invention is simple and scientific, the production cost is low, and the yield of flavonoids is significantly increased, so it has broad application prospects.

The existing technology for extracting ginkgo leaf flavonoids generally adopts a single alcohol solution extraction method, and the extraction efficiency is low. The main reason is: the particle size of leaf crushing is limited (20-60 mesh), most of the leaf cells are still in a complete state, which hinders the dissolution of intracellular substances, and research shows that too fine particle size is not conducive to the extraction of flavonoids. There are some reports on microwave and ultrasonic assisted extraction techniques, but the equipment is expensive and difficult to popularize.

Contents of the invention

The purpose of the embodiments of the present invention is to provide a method for improving the extraction of ginkgo flavonoids by fermentation pretreatment, aiming at solving the problem that the existing technology for extracting ginkgo leaf flavonoids generally adopts a single alcohol solution extraction method, and the extraction efficiency is low; , Ultrasonic-assisted extraction technology reports, but the equipment is expensive and difficult to popularize.

The embodiment of the present invention is to provide a method for improving the extraction of ginkgo flavonoids by fermentation pretreatment, the method comprising the following steps:

Ginkgo leaves are crushed to 20-60 mesh;

Ginkgo biloba leaves are mixed with water, and the amount of water added is 45%-90%;

The mixture is sterilized at 115°C and loaded into a fermenter;

Inoculate bacterial classification by the weight of ginkgo leaf in the mixture, inoculation amount is 0.01%-0.02%;

Aerated fermentation at 25-30°C for 2-11 days;

According to the final parameter solid-liquid ratio 1:20, ethanol content 60%, add alcohol and water in proper amount, and keep stirring to extract flavonoids;

Filter the suspension to obtain an extract;

The extract is concentrated in a vacuum to obtain the finished product, or further dried into a solid state.

Further, the mixture was sterilized at 115° C. for 15 minutes, and charged into a fermenter.

Further, the condition of vacuum concentration of the extract is: 70° C., 130 mbar.

Further, filter the suspension to obtain an extract; extract once more, and combine the two extracts.

Further, the fermentation pretreatment can adopt solid state or liquid state fermentation.

Further, the raw materials in the solid-state fermentation pretreatment process are only ginkgo leaves and water; the fermentation conditions are: the water content of the mixture is 45%-65%, the fermentation temperature is 20-35°C, and the fermentation time is 2-11 days.

Further, the raw materials in the liquid fermentation pretreatment process are only ginkgo leaves and water, and the fermentation conditions are: the water content of the mixture is 70-95%, the fermentation temperature is 20-35°C, and the fermentation time is 2-11 days.

Further, the fermentation strain is Aspergillus fungus, and the patent strain preservation number is CCTCC M2012515.

Another object of the embodiments of the present invention is to provide the application of the method for improving the extraction of flavonoids from ginkgo biloba leaves by fermentation pretreatment in the pretreatment of extracting flavonoids from plant tissues.

In the present invention, a strain of Aspergillus niger (patented strain preservation number CCTCC M2012515) isolated from ancient ginkgo soil is used to ferment and pretreat ginkgo leaves, degrade leaf cells, and then extract using traditional extraction methods to improve the extraction of ginkgo flavonoids Rate. Compared with the traditional alcohol solvent extraction method, the amount of flavonoids extracted from Ginkgo biloba is increased by 20-60%.

Description of drawings

Fig. 1, the HPLC fingerprint of the flavone provided by the present invention: (A) control (unfermented sample); (B) the flavone fingerprint of the patent strain fermentation product;

Fig. 2, flavone content and enzyme activity changes during the fermentation of the patent strain provided by the present invention;

Fig. 3, the phylogenetic tree of the bacterial strain provided by the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The embodiment of the present invention provides a fermentation strain used for fermentation pretreatment to improve the extraction of ginkgo leaf flavonoids. The fermentation strain is Aspergillus fungus, and the patent strain preservation number is CCTCC M2012515.

The patented strain used in this invention is isolated from the soil at the bottom of the ancient ginkgo tree. After the mycelium is treated with liquid nitrogen, it is ground, DNA extracted, 18SrDNA sequence amplified and sequenced. The determined sequence is obtained using NCBI Blast and RDP Chimera Check programs were analyzed and determined to be a strain of Aspergillus niger (the species of the strains listed in Table 1 should be determined by this molecular method). The present invention uses this Aspergillus niger to ferment Ginkgo biloba leaves is the first report. Compared with other bacterial strains, this Aspergillus niger grows particularly vigorously in Ginkgo biloba leaves. The components of the processed flavone products were consistent with those of the unfermented products (the chromatographic peaks of the fermented and unfermented samples were consistent in Figure 2).

The embodiment of the present invention also provides a method for improving the extraction of ginkgo flavonoids by fermentation pretreatment, the method comprising the following steps:

Ginkgo leaves are crushed to 20-60 mesh;

Ginkgo biloba leaves are mixed with water, and the amount of water added is 55%-90%;

The mixture is sterilized at 115°C for 15 minutes and loaded into a fermenter;

Inoculate bacterial classification by the weight of ginkgo leaf in the mixture, inoculation amount is 0.01%-0.02%;

Aeration and fermentation at 25-30°C for 2-4 days;

According to the final parameter solid-liquid ratio 1:20, ethanol content 60%, add alcohol and water in proper amount, and keep stirring to extract flavonoids;

Filter the suspension to obtain an extract;

The extract is concentrated in a vacuum to obtain the finished product, or further dried into a solid state.

In the embodiment of the present invention, the mixture was sterilized at 115° C. for 15 minutes, and loaded into a fermenter.

In the embodiment of the present invention, the condition of vacuum concentration of the extract is: 70° C., 130 mbar.

In the embodiment of the present invention, the suspension is filtered to obtain the extract; the extract is extracted again, and the two extracts are combined.

In the embodiment of the present invention, the fermentation pretreatment may adopt solid state or liquid state fermentation.

In the embodiment of the present invention, the raw materials in the solid-state fermentation pretreatment process are only ginkgo leaves and water. The fermentation conditions are as follows: the water content of the mixture is 45-65%, the fermentation temperature is 26-30°C, and the fermentation time is 2-4 days.

In the embodiment of the present invention, the raw materials of the liquid fermentation pretreatment process are only ginkgo leaves and water. The fermentation conditions are as follows: the water content of the mixture is 70-95%, the fermentation temperature is 20-35°C, and the fermentation time is 3-6 days.

The method provided by the embodiment of the present invention can be applied in the pretreatment of extraction of flavonoid substances in other plant tissues.

In the embodiment of the present invention, fungi and bacteria capable of producing lignocellulose such as Penicillium, Mucor, Fusarium and Trichoderma can also be used to achieve the effect of the present invention, but the effect is relatively poor.

The method is a pretreatment method, and after treatment, solvent extraction can be used alone, or combined with ultrasonic wave and microwave to assist solvent extraction.

The following is a comparison table of total flavonoid content and cellulose activity extracted by different fungi after fermentation.

Table 1 The total flavonoid content extracted by different fungi after fermentation

*There is no significant difference in the means marked with the same lowercase letters (α=0.05)

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (1)

1. a method for fermentation pretreatment improving ginkgo flavonoids extraction, is characterized in that, the method comprises the following steps: Ginkgo leaves are crushed to 20-60 mesh; Ginkgo biloba is mixed with water, the amount of water added is 45%-90%; The mixture is sterilized at 115°C and loaded into a fermenter; Inoculate strains according to the weight of ginkgo leaves in the mixture, and the inoculation amount is 0.01%-0.02%; Aerated fermentation at 25-30°C for 2-11 days; According to the final parameters, the solid-to-liquid ratio is 1:20, the ethanol content is 60%, add alcohol and water in an appropriate amount, and continuously stir to extract flavonoids; Filtrating the suspension to obtain an extract; extracting once more, combining the extracts twice; concentrating the extract in a vacuum to obtain a finished product, or further drying to a solid state; Among them: the mixture is sterilized at 115°C for 15 minutes, and put into a fermenter; the extract is concentrated under vacuum conditions: 70°C, 130mbar; Fermentation pretreatment adopts solid state or liquid state fermentation; The raw materials in the solid-state fermentation pretreatment process are only ginkgo leaves and water; the fermentation conditions are: the water content of the mixture is 45%-65%, the fermentation temperature is 20-35°C, and the fermentation time is 2-11 days; The raw materials in the liquid fermentation pretreatment process are only ginkgo leaves and water, and the fermentation conditions are as follows: the water content of the mixture is 70-95%, the fermentation temperature is 20-35 DEG C, and the fermentation time is 2-11 days.