CN111454993B

CN111454993B - Semen cassiae fermentation product and application thereof

Info

Publication number: CN111454993B
Application number: CN202010293704.8A
Authority: CN
Inventors: 邹敏亮; 刘翔; 张泽文; 菅保森
Original assignee: Wuhan Senlan Biotechnology Co ltd
Current assignee: Wuhan Senlan Biotechnology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2021-01-15
Anticipated expiration: 2040-04-15
Also published as: CN111454993A

Abstract

The invention discloses a cassia seed fermentation product and application thereof, and relates to the technical field of microbial fermentation. The method comprises the following steps: (1) crushing 20-60 parts of semen cassiae, 10-30 parts of bean pulp and 10-30 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use; (2) inoculating the mixture A into a bacterial liquid for fermentation treatment to obtain a fermentation product; (3) stirring and extracting the fermentation product with 50% ethanol solution, filtering, concentrating to obtain concentrated solution of the fermentation product, and spray drying the concentrated solution to obtain powder of the fermentation product; the interaction between the fermentation raw materials and the strains can be effectively improved by controlling the weight part ratio of the cassia seeds to the soybean meal in the implementation process, and the spore yield in the fermentation process is improved, so that the fermentation effect is improved; the interaction among microorganisms can be effectively improved by controlling the types and the addition amounts of the microorganisms and the additives, and the release amount of active ingredients of the medicament can be effectively improved.

Description

Semen cassiae fermentation product and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a cassia seed fermentation product and application thereof.

Background

The fermentation method is a traditional method for processing Chinese medicines, and has the effects of saving medicines, reducing toxicity and enhancing efficiency. Although the traditional Chinese medicines are processed by fermentation, the conventional common procedures of decoction, steaming and soaking, refining and the like are changed, the curative effect of the medicines is obviously enhanced. For example, the traditional Chinese medicine fermented soybean is prepared by fermenting mulberry leaves or ephedra herb and the like, and the properties of the medicines can be obviously changed by adopting different culture media and treating the medicines by the same microorganisms, so the traditional Chinese medicines with different indications can be prepared by utilizing the characteristic. The modern traditional Chinese medicine fermentation technology is further innovative and developed on the traditional fermentation technology, combines traditional Chinese medicine fermentation with modern biotechnology such as bioengineering, microecology and the like, treats medicinal materials, and has wide prospect.

The fermentation has multiple functions in the processing of the traditional Chinese medicine, (1) the curative effect of the medicine can be enhanced: the traditional Chinese medicine fermentation processing is implemented by jointly fermenting microorganisms and traditional Chinese medicines, and secondary metabolites and medicinal ingredients generated by fermenting the microorganisms in the process are mutually cooperated to enhance the medicinal effect, so that the metabolic pathway is widened while the secondary metabolites and the medicaments act, and even obviously different effective ingredients are generated; (2) the extraction rate of the effective components is improved: the effective components of the Chinese herbal medicine are mostly wrapped in the cell walls of the Chinese herbal medicine, and the dense and hard cell walls are barriers for preventing the effective components of the medicine from being dissolved out of the cells and preventing the effective components from being absorbed in vivo. Therefore, the microorganism plays a certain role in extracting effective components, can secrete various extracellular enzymes aiming at the wall structure formed by different components, so that the formed compact structure is decomposed, the gaps among cells are enlarged, a channel is provided for the diffusion of substances inside and outside the cells, and the extraction rate and the absorption utilization rate of the effective components can be improved; (3) the toxicity of the medicine is reduced: traditional Chinese medicines are not completely harmless, and some medicines have certain toxicity and irritation due to various chemical components contained in the medicines, so that some side effects can occur when people use the medicines. Some medicinal materials are fermented, and the toxicity or irritation is obviously weakened, because the structure of toxic components is changed under the action of microorganisms, and the toxic components become low-toxicity or even non-toxic substances.

Traditional Chinese medicines are valuable wealth in China, but with the increasing development and unreasonable development and utilization of industrialization, medicinal plant resources are gradually deficient, and wild traditional Chinese medicinal materials are endangered, so that the improvement of the utilization rate of medicinal resources is one of effective ways for developing and utilizing traditional Chinese medicines. The fermentation method which is one of the research hotspots in the field of traditional Chinese medicine processing has the advantages of changing the property of the medicine, improving the curative effect, reducing the toxic and side effect and the like, and widens the research range for further research and development of abundant medicinal resources in China and even development of the traditional Chinese medicine industry.

Semen Cassiae is dried mature seed of Cassia tora or Cassia tora L of Leguminosae, and has effects of clearing heat, improving eyesight, and loosening bowel to relieve constipation. Pharmacological experiments prove that the cassia seed not only has better effects of reducing blood fat, resisting atherosclerosis and the like, but also has the effects of reducing blood pressure, enhancing the immunity of the organism, resisting aging and the like. The cassia seed contains a plurality of chemical components such as anthraquinone, naphthopyrone, polysaccharide and the like, wherein the anthraquinone compound mainly comprises emodin, chrysophanol, physcion and the like, and has the effects of resisting inflammation and bacteria, resisting oxidation, resisting tumors, reducing blood fat, protecting nerves, protecting liver and eyes, relaxing bowels and promoting urination, killing larval activity, and inhibiting acetylcholinesterase and beta-secretase; naphthopyrones mainly include rubrofusarin, nor-rubrofusarin, cassia seed lactone, iso-cassia seed lactone, cassia anthranone, rhein-9-anthracenone, etc., and have antidiabetic, antibacterial, and antiestrogenic effects; the polysaccharide compound mainly comprises pectic polysaccharide, hemicellulose, arabinoglucan, galactomannan, glucuronoxylan, neutral galactomannan and the like, has the functions of resisting oxidation, immunity and tumors, treating diabetes and complications, reducing blood pressure and blood fat, regulating gastrointestinal tracts and protecting livers and eyes, and the cassia seed is one of the first medicines which are both edible and medicinal and published by the Ministry of health of China.

Chinese patent application 201811439573.9 discloses a semen Cassiae composition with blood lipid lowering effect, which is prepared by mixing and fermenting semen Cassiae, fructus crataegi, semen glycines and rice twice, and its preparation process is: firstly inoculating the semen cassiae, the hawthorn and the soybeans into bacillus natto according to a formula ratio for fermentation, adding rice and monascus into the prepared fermentation product for secondary fermentation, wherein the active components in the obtained semen cassiae composition have an obvious synergistic effect, and play a role in actively reducing blood fat in multiple aspects such as reduction of endogenous lipid synthesis, promotion of lipid transportation and excretion, regulation of lipid metabolism and the like, and the semen cassiae composition is safe, non-toxic and harmless.

Chinese patent application 201410692371.0 discloses a composition for improving, treating or preventing constipation containing a lactic acid bacteria fermentation product of cassia seed as an active ingredient, which is highly safe for long-term use and has a low possibility of causing side effects as a crude drug, and which can be used as a pharmaceutical composition for treating or preventing constipation or a health functional food for improving or preventing constipation, and a method for producing the same.

According to the above description of the prior art, the main purpose is to achieve the synergistic effect of the medicinal components in the composition, but there is no description on how to improve the release rate of the effective components in the medicinal components, and the content of various effective components in the fermented cassia seed product obtained by adopting various microbial fermentation methods in the implementation process of the invention is obviously improved.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the cassia seed fermented product and the application thereof.

In one aspect, the invention provides a cassia seed leavening, which comprises the following steps:

(1) crushing the fermentation raw materials, uniformly mixing and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) into a bacterial liquid for fermentation treatment to obtain a fermentation product;

(3) and (3) stirring and extracting the fermentation product obtained in the step (2) by using a 50% ethanol solution, filtering and concentrating to obtain a fermentation product extraction concentrated solution, and then performing spray drying on the concentrated solution to obtain fermentation product extraction powder.

The fermentation raw material in the step (1) is one or more of cassia seed, soybean meal and wheat bran;

preferably, the fermentation raw materials in the step (1) are cassia seeds, soybean meal and wheat bran;

the fermentation raw material in the step (1) comprises the following components in parts by weight: 20-60 parts of cassia seed, 10-30 parts of bean pulp and 10-30 parts of wheat bran.

Preferably, the fermentation raw material in the step (1) comprises the following components in parts by weight: 30-50 parts of cassia seed, 15-25 parts of bean pulp and 15-25 parts of wheat bran.

Still preferably, the fermentation raw material in the step (1) comprises, in parts by weight: 40-50 parts of cassia seed, 18-22 parts of bean pulp and 20-25 parts of wheat bran.

In some embodiments, the weight ratio of the cassia seeds to the soybean meal is 1-6: 1; preferably, the weight part ratio of the cassia seeds to the soybean meal is 2-5: 1; preferably, the weight part ratio of the cassia seeds to the soybean meal is 4-5: 1.

The bacterial strain in the bacterial liquid in the step (2) is selected from one or more of aspergillus niger, geotrichum candidum, aspergillus fumigatus, lactic acid such as coccus, penicillium, lactobacillus reuteri, lactobacillus gasseri, lactobacillus salivarius, bifidobacterium adolescentis, pichia pastoris powder and kluyveromyces marxianus;

preferably, the strain is selected from one or more of aspergillus niger, lactic acid such as coccus, lactobacillus salivarius, pichia farinosa and kluyveromyces marxianus;

still more preferably, the species is selected from the group consisting of Aspergillus niger, Lactobacillus salivarius and Kluyveromyces marxianus.

Further preferably, the ratio of the addition amount of the lactobacillus salivarius, the kluyveromyces marxianus and the aspergillus niger is 5-10: 3-5: 1; preferably 8 to 10: 4-5: 1; more preferably 9 to 10: 5: 1; more preferably 10: 5: 1.

the addition amount of the strain is 5-10% of the fermentation raw material, preferably, the addition amount of the strain is 5%, 6%, 7%, 8%, 9% or 10% of the fermentation raw material.

In some preferred embodiments, the bacterial liquid further contains an auxiliary agent, wherein the auxiliary agent is prepared from the following components in a mass ratio of 2-3: 1 sucrose and skim milk.

The addition amount of the auxiliary agent is 2-5% of the fermentation raw material; preferably, the addition amount of the auxiliary agent is 2%, 3%, 4% or 5% of the fermentation raw material.

The fermentation time is 36-48 hours.

In the implementation process, the fact that the survival rate of the strains can be effectively improved by adding a certain content of the auxiliary agent into the bacterial liquid in the fermentation process is unexpectedly found, so that the fermentation effect of microorganisms is improved, and the release rate of the effective components of the medicine is improved.

On the other hand, the invention also provides an application of the cassia seed fermentation product as an effective component in preparing a pharmaceutical composition for resisting inflammation and bacteria, resisting tumors, reducing blood fat and protecting liver and eyes.

The pharmaceutical composition can be prepared into powder, granules, tablets, capsules or syrup and the like.

The pharmaceutical composition also comprises a flavoring agent, a filling agent or an emulsifying agent which is acceptable on health-care food.

The invention provides a cassia seed leavening, which specifically comprises the following steps:

(1) crushing 20-60 parts of semen cassiae, 10-30 parts of bean pulp and 10-30 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) in a mixture with the addition amount ratio of 5-10: 3-5: 1, fermenting in a bacterial liquid of a mixed strain of lactobacillus salivarius, kluyveromyces marxianus and aspergillus niger to obtain a fermentation product;

The number of viable bacteria of Aspergillus niger, Lactobacillus salivarius and Kluyveromyces marxianus is 1 × 10⁴-1×10⁶CFU/mL。

In some preferred embodiments, the bacterial liquid further contains an auxiliary agent, wherein the auxiliary agent is prepared from the following components in a mass ratio of 2-3: 1 sucrose and skim milk; preferably, the mass ratio of the auxiliary agent is 2: 1 or 3: 1 sucrose and skim milk.

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

(1) according to the invention, the ratio of the weight parts of the cassia seeds to the soybean meal in the fermentation raw materials is controlled to be 1-6:1 in the implementation process, so that the interaction between the fermentation raw materials and the strains can be effectively improved, the spore yield in the fermentation process is improved, and the fermentation effect is improved;

(2) the invention uses mixed strains for fermentation, and controls the adding amount ratio of the microorganisms lactobacillus salivarius, kluyveromyces marxianus and aspergillus niger to be 5-10: 3-5: 1, the interaction among microorganisms can be effectively improved by adopting mixed strain fermentation, the fermentation efficiency is improved, and the fermentation time is shortened;

(3) in order to ensure that the fermentation effect is better and the survival time of microorganisms is longer, a certain amount of auxiliary agents are added into the bacterial liquid, namely the mass ratio of the auxiliary agents is 2-3: 1, the cane sugar and the skim milk can obviously improve the survival time of microorganisms, improve the fermentation effect and promote the full release of the effective components of the medicine.

Detailed Description

The present invention is described below by way of specific embodiments, and unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications can be made in the material composition and the amount of the embodiments without departing from the spirit and scope of the invention.

Wherein, Lactobacillus salivarius (Lactobacillus salivarius), the number is: CICCNo.23174, purchased from China center for Industrial culture Collection of microorganisms;

kluyveromyces marxianus (kluyveromyces marxianus), no: CICCNo.1276, which is purchased from China center for culture Collection of Industrial microorganisms;

aspergillus niger (Aspergillus niger), number CICCNo.2108, purchased from China center for Industrial culture Collection of microorganisms.

In addition, the raw materials used in the present invention, i.e., reagents, are commercially available, and the units used in the present invention may be: parts, percentages, grams or kilograms, as desired.

EXAMPLE 1 fermentation product of Cassia seed

The method specifically comprises the following steps:

(1) crushing 20 parts of semen cassiae, 10 parts of bean pulp and 10 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) in a mixture with an addition ratio of 5: 3: 1, adding 3.6 parts of auxiliary agent-containing bacterial liquid of lactobacillus salivarius, kluyveromyces marxianus and aspergillus niger, and performing fermentation treatment to obtain a fermentation product;

(3) and (3) stirring and extracting the fermentation product obtained in the step (2) for 1 hour by using a 50% ethanol solution, filtering the obtained extract, concentrating the filtrate to obtain a fermentation product extract concentrated solution, and then performing spray drying on the concentrated solution to obtain fermentation product extract powder.

The auxiliary agent in the bacterial liquid is 1.2 parts by mass ratio of 2: 1 sucrose and skim milk.

Example 2A fermentation product of Cassia seed

The method specifically comprises the following steps:

(1) crushing 60 parts of semen cassiae, 30 parts of bean pulp and 30 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) in a mixture with an addition ratio of 5: 5:1, adding 10.56 parts of auxiliary agent into the bacterial liquid of the lactobacillus salivarius, the Kluyveromyces marxianus and the Aspergillus niger, and performing fermentation treatment to obtain a fermentation product;

(3) and (3) stirring and extracting the fermentation product obtained in the step (2) for 2 hours by using a 50% ethanol solution, filtering the obtained extract, concentrating the filtrate to obtain a fermentation product extract concentrated solution, and then performing spray drying on the concentrated solution to obtain fermentation product extract powder.

The auxiliary agent in the bacterial liquid is 2.4 parts by mass ratio of 3: 1 sucrose and skim milk.

EXAMPLE 3 fermentation product of Cassia seed

The method specifically comprises the following steps:

(1) crushing 60 parts of semen cassiae, 10 parts of bean pulp and 15 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) in a mixture with an addition ratio of 10: 3: 1, adding 8.33 parts of auxiliary agent-containing bacterial liquid into the lactobacillus salivarius, the kluyveromyces marxianus and the aspergillus niger, and performing fermentation treatment to obtain a fermentation product;

(3) and (3) stirring and extracting the fermentation product obtained in the step (2) for 1.5 hours by using a 50% ethanol solution, filtering the obtained extract, concentrating the filtrate to obtain a fermentation product extract concentrated solution, and then performing spray drying on the concentrated solution to obtain fermentation product extract powder.

The auxiliary agent in the bacterial liquid is 3.4 parts by mass ratio of 3: 1 sucrose and skim milk.

Example 4A fermentation product of Cassia seed

The method specifically comprises the following steps:

(1) crushing 50 parts of semen cassiae, 10 parts of bean pulp and 25 parts of wheat bran, uniformly mixing, and sterilizing to obtain a mixture A for later use;

(2) inoculating the mixture A obtained in the step (1) in a mixture with an addition ratio of 10: 5:1, adding 6.8 parts of auxiliary agent-containing bacterial liquid into the lactobacillus salivarius, the kluyveromyces marxianus and the aspergillus niger, and performing fermentation treatment to obtain a fermentation product;

The auxiliary agent in the bacterial liquid is 4.25 parts by mass ratio of 3: 1 sucrose and skim milk.

Comparative examples 1 to 6A fermented product of Cassia seed

The difference from example 4 is that: the specific data are shown in Table 1 below (unit: parts)

TABLE 1

The preparation methods of comparative examples 1 to 6 were the same as those of example 4 except that the contents of the respective components were different.

Comparative example 7

The difference from example 4 is that: the addition ratio of the lactobacillus salivarius, the kluyveromyces marxianus and the aspergillus niger is 2: 5: 3, namely 1.36 parts of lactobacillus salivarius, 3.4 parts of kluyveromyces marxianus and 2.04 parts of aspergillus niger, and the other operations and steps are the same as the example 4.

Comparative example 8

The difference from example 4 is that: the mass ratio of the sucrose to the skim milk in the auxiliary agent is 0.7: 1, namely 1.75 parts of cane sugar and 2.5 parts of skim milk, and the other operations and steps are the same as those in example 4.

Comparative example 9

The difference from example 4 is that: the auxiliary agent only contains cane sugar, namely 4.25 parts of cane sugar, and other operations and steps are the same as those of the example 4.

Comparative example 10

The difference from example 4 is that: the weight portion ratio of the cassia seed to the bean pulp is 0.5: 1, namely 20 parts of cassia seeds and 40 parts of soybean meal, and the other operations are the same as the steps in the example 4.

Comparative example 11

The difference from example 4 is that: the weight portion ratio of the cassia seed to the soybean meal is 9: 1, namely 54 parts of cassia seeds and 6 parts of bean pulp, and the other operations are the same as the steps in the example 4.

Test example 1 viable cell count detection

And (3) taking fermentation products with the same volume at time nodes of 0 hour, 6 hours, 12 hours, 24 hours and 48 hours of fermentation respectively, and measuring the number of viable bacteria in the fermentation products, wherein specific detection results are shown in the following table 2.

TABLE 2 viable count test

According to the detection data in the table 2, the number of viable bacteria is obviously increased after 48 hours of fermentation in the embodiment of the invention, the interaction between the fermentation raw materials and the strains can be effectively improved by controlling the mass ratio of the components, and the spore yield in the fermentation process is improved, so that the fermentation effect is improved, while the comparative example changes the mass ratio of the components out of the protection range disclosed by the invention, or reduces the number of viable bacteria which can obviously influence the fermentation process by one component, so that the fermentation effect is influenced.

Test example 2 detection of active ingredient of drug

The detection method comprises the following steps: performing ultrasonic extraction for 30 minutes at room temperature by taking methanol as an extraction solvent according to a sample before fermentation of the semen gmelini, fermentation products obtained in examples 1-4 and comparative examples 1-11, wherein the material-liquid ratio is 1:10, filtering an extracting solution by using a filter membrane, and obtaining a filtrate to be tested for later use;

a chromatographic column: c₁₈-ODS reverse phase column (250 mm. times.4.6 mm,5 μm); mobile phase: methanol (A) -0.1% phosphoric acid water solution (B), gradient elution (volume fraction of A is 63% in 0-33min, 63-75% in 33-40min, and 75-95% in 40-60 min); flow rate: 1 mL. min^-1(ii) a Column temperature: 25 ℃; detection wavelength: 280 nm. The separation degree of each peak is more than 1.5, the theoretical plate number is more than or equal to 3000 calculated according to chrysophanol peak, and the detection results are shown in tables 3 and 4.

Percent change%

TABLE 3 measurement results (mg. g) of anthraquinone component content in the cassia seeds before fermentation and the cassia seeds after fermentation in examples 1 to 4 and comparative examples 1 to 6^-1)

According to the detection data in the table 3, the total increase trend of the anthraquinone content after fermentation can be seen, but the increase amplitudes of aloe-emodin, rhein and emodin in the examples 1-4 are relatively small, and are only increased by about 10%, the chrysophanol is increased by about 120%, and the physcion is increased by about 40%, which indicates that the fermentation method provided by the invention is favorable for releasing the active ingredient anthraquinone, but according to the fermentation result, the free anthraquinone content after fermentation is in the increase trend, which indicates that the cassia seed is favorable for increasing the active ingredient anthraquinone aglycone through fermentation; according to the detection data of the comparative examples 1 to 6, the extraction rate of the effective components in the cassia seeds is obviously reduced compared with the examples because the effective components in the cassia seeds are released and obviously influenced by reducing one component in the components.

TABLE 4 measurement results (mg. g) of anthraquinone components in fermented cassia seeds of comparative examples 7 to 11^-1)

According to the detection data in the above table 4, it can be seen that when the mass ratio of two or more ingredients in the fermentation components is not within the protection range of the present invention, the content of the effective ingredients in the obtained fermented product is increased compared with that before fermentation, but the increase is reduced compared with that in the example 4, so that the fermentation effect is best only when the ingredients and contents disclosed by the present invention are used in the fermentation process, the content release rate of the effective ingredients in the obtained product is high, and further the extraction rate is high.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A semen cassiae fermentation product is characterized in that: the preparation method comprises the following steps:

(2) inoculating a fermentation strain into the mixture A obtained in the step (1) for fermentation treatment to obtain a fermentation product;

(3) stirring and extracting the fermentation product obtained in the step (2) by using a 50% ethanol solution, filtering and concentrating to obtain a fermentation product extraction concentrated solution, and then spray-drying the concentrated solution to obtain fermentation product extraction powder;

the fermentation raw materials in the step (1) comprise the following components in parts by weight: 20-60 parts of cassia seed, 10-30 parts of bean pulp and 10-30 parts of wheat bran;

the weight part ratio of the cassia seeds to the soybean meal is 1-6: 1;

the strains are lactobacillus salivarius, kluyveromyces marxianus and aspergillus niger;

the addition ratio of the lactobacillus salivarius, the Kluyveromyces marxianus and the Aspergillus niger is 5-10: 3-5: 1;

the bacterial liquid in the step (2) also contains an auxiliary agent, wherein the auxiliary agent is prepared from the following components in a mass ratio of 2-3: 1 sucrose and skim milk.

2. The fermentation product of cassia seed according to claim 1, characterized in that: the fermentation raw materials in the step (1) comprise the following components in parts by weight: 30-50 parts of cassia seed, 15-25 parts of bean pulp and 15-25 parts of wheat bran.

3. The fermentation product of cassia seed according to claim 1, characterized in that: the addition amount of the strain is 5-10% of the fermentation raw material.

4. The fermentation product of cassia seed according to claim 1, characterized in that: the addition amount of the auxiliary agent is 2-5% of the fermentation raw material.

5. The use of the fermentation product of cassia seed of claim 1 in the preparation of a pharmaceutical composition for anti-inflammatory, anti-bacterial, anti-tumor, blood lipid lowering, liver protection and eye protection.