AU2020102037A4 - A method of efficiently increasing the alpha-glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation - Google Patents

Abstract

This research lies in the field of ecological agriculture and medical science. It is a method of improving the inhibitory activity of a-glucosidase from mulberry leaves by biological fermentation. This method consists of the following steps. Firstly, we choose fresh mulberry leaves and pretreat them. Secondly, the inoculum of Lactobacillus plantarum, Trichoderma, Pleurotus eryngii and so on, are prepared separately and one or more of them are inoculated on fresh mulberry leaf to conduct the fermentation; Thirdly, the active ingredients in the fermented mulberry leaves will be extracted. Finally, The inhibition rate of a-glucosidase in vitro of mulberry leaf extract will be determined. 1

Description

TITLE

A method of efficiently increasing the a-glucosidase inhibitor content in fresh

mulberry leaves by the solid-state fermentation

FIELD OF THE INVENTION

This research lies in the field of ecological agriculture and medical science. It is a

method of improving the inhibitory activity of c-glucosidase from mulberry leaves

by biological fermentation.

BACKGROUND OF THE INVENTION

Among all kinds of plant species that may regarded as edible by general public,

there is a group of plants that functions as medicine, known as food-medicine

homologous plants, or medicinal plants. Some examples of food-medicine

homologous plants are red dates, hawthorn, and perilla leaf. Chinese herbal

medicines have long believed that plants can be used as medicines by its different

characteristics of functions. Therefore, we have started the development of plant

resources.

One key highlight of plant resource development was the discovery of active

ingredient within the plants. We have found that the these active ingredient have

positive physiological effects on human body and may become the potential

treatment of chronic diseases, like diabetes. As the country become more developed,

the rate of chronic diseases have drastically increased, which the rate of diabetes increased about 5 times in China. Modern treatment of diabetes is mainly rely on the injection of insulin. However, many evidence have shown the later dependency of the medicine, indicating that insulin is only alleviating the symptom and stop it from worsening rather than a reliable treatment. Hence, the discovery of the active ingredient's function on diabetes was innovative and astonishing.

Active ingredients that we found in plants are divided into 3 main categories

plant polysaccharide, flavonoids, and alkaloids. The one we are focusing at is one of

the alkaloids, called alpha-glucosidase inhibitor. Alpha-glucosidase inhibitor could

slow down the motility rate of intestines and reduce the absorption of carbohydrates,

as well as slowing down the breakdown of disaccharide. The highlights of treatment

of alpha-glucosidase inhibitor demonstrate us a method to reduce blood glucose

level that involved multi-targeting and more safety - less-dependent. In fact, the

extractions of alpha-glucosidase of current techniques is still low, making the

process less efficient.

Popular techniques of extraction can fulfill some of the desired properties of a

ideal extraction but with apparent disadvantages in regarding to environmental,

economical and safety issues. For instance, enzymolysis extraction is good at

purifying, while the enzyme is costly. On the other hand, current techniques always

involves extracting active ingredients from dried plant materials, like dried folium

mori leaf, due to the fact of economic constraints and the period of transportation.

We are wondering if we can extract active ingredients from fresh plants, since they

are more abundant in plants that is recently alive.

In our study, we are trying to find whether the fermentation of fresh folium mori

leaf would increase the production of alpha-glucosidase inhibitor.

SUMMARY OF THE INVENTION

In order to improve the inhibitory activity of glucosidase in mulberry leaves. We

used microbial fermentation.

Fermentation process of Chinese medicine food homologous plant multiple

substances into the same substance; Microorganisms can change the material

structure of natural active components in medicinal plants and improve the

dissolution rate of active components. This method can solve effectively solve the

problems such as loss of nutrition,reduction of efficiency and pollution of

fermentation products.

In order to maintain the activity of microorganisms and make the experiment run

smoothly, the temperature range of the experiment should be guaranteed to be 25-30

degrees. And this experiment consists of seven steps.

[Step1] Pretreatment of fresh mulberry leaves. Choosing fresh mulberry leaves with

to 80 percent water content and washing them.

[Step2] Crushing fresh mulberry leaves and control the proportion of adding water.

Solid-to-liquid ratio: 1:2-1:5. Stiring evenly and use for microbial fermentation after sterilization;

[Step3] Preparing the microbial fermented seed liquid. Lactobacillus plantarum,

Saccharomyces cerevisiae, Ganoderma lucidum, Pleurotus eryngii, Bacillus subtilis

and Trichoderma reesei were selected to prepare their seed liquid respectively; One

or more microorganisms were taken for fresh mulberry leaf fermentation.

[Step4] Conducting solid fermentation. Microbial fermentation of fresh mulberry

leaves: the prepared seed liquid was inoculated into the prepared mulberry leaf

fermentation medium, and incubated at 28-30 C for 5-7 days at a constant

temperature;

[Step5] Extracting the active components from fermented mulberry leaves. It

includes the production of Polysaccharides and extraction of total flavonoids and

alkaloids.

[Step6] Determination of -glucosidase inhibitory rate of Mulberry leaf extract in

vitro.

[Step7] Outputting the qualified products.

DESCRIPTION OF THE DRAWINGS

Figure 1:This flowchart describes the process of enrichment, extraction and

purification of the active components in mulberry leaves by means of microbial

fermentation.

DESCRIPTION OF PREFERRED EMBODIMENT

At present, the main problems restricting the application of fresh medicines are as

follows. The previous studies on the medicinal ingredients of mulberry leaves were

mainly carried out after drying, and fermentation research using fresh mulberry

leaves as a substrate has not been seen. The mulberry leaves fermenting

microorganisms have a single type, and there is a lack of in-depth research on the

types and mechanism of microorganisms. There is no research on the changes of

active ingredients and efficacy of mulberry leaves before and after fermentation.

In this experiment. This can maximize the retention of active ingredients in mulberry

leaves and improve the experimental yield. Therefore, different from the Previous

treatment, this project intends to use fresh mulberry leaves as raw materials, through

the study of microbial fermentation of fresh mulberry leaves hypoglycemic active

ingredients (flavonoids, alkaloids and polysaccharides) changes and its

hypoglycemic effect, Establish a new method of fermentation and processing of

mulberry leaf fresh medicine, so as to achieve the purpose of enriching anti-glycemic

active ingredients through biotransformation and improving its efficacy.

In this experiment, we chose the solid fermentation method to extract the effective

substances. Solid fermentation is a biological reaction process that uses one or more

microorganisms in an underwater soluble solid substrate with a certain humidity in

an environment where there is no or almost no free water.

Microorganisms grow on moist and water-insoluble substrates for fermentation, and do not contain any free water during the solid fermentation process. As the free water produced by microorganisms increases, the scope of solid fermentation extends to viscous fermentation and solid particle suspension fermentation. In order to improve the cultivation efficiency, the method of increasing the surface area is adopted. The laboratory generally uses test tube slopes, petri dishes, Erlenmeyer flasks, Kirschner flasks, etc., and most factories use curved plates, curtains, and ventilated koji tanks.

Especially in the cultivation of molds, the solid culture method is still used to

produce koji. Due to the selection of agricultural and sideline products such as bran

as raw materials, the price is low, the surface area of the particles is large, and the

raw materials are easy to obtain in large quantities.

Therefore, they are widely used in the wine industry. However, because there are

still many difficulties in large-scale surface culture technology, in fermentation

production, most of the liquid surface culture can be replaced by liquid deep culture

method.

Specific experimental steps:

Step 1: Pretreatment of fresh mulberry leaves.

Wash the fresh mulberry leaves and drain the water, and crush them with a crusher.

Step 2: Preparation of fermentation medium for fresh mulberry leaves.

In step 1, the pulverized fresh mulberry leaves are combined with auxiliary materials

to obtain a mulberry leaf fermentation substrate, and water is added to adjust the ratio of the mixed mulberry leaf fermentation substrate to water (substrate wet weight: moisture (w/v)) will be 2:1 to 1:5, and stir evenly, after sterilization, it is used for microbial fermentation.

Step 3: Preparation of microbial fermentation seed liquid.

Select Lactobacillus plantarum, Saccharomyces cerevisiae, Ganoderma lucidum,

Pleurotus eryngii, Bacillus subtilis, and Trichoderma reesei to prepare their seed

liquid respectively.

Step 4: Fresh mulberry leaf microbial fermentation.

Take one or more of the microorganisms' seed liquid prepared in step 3 inoculate

the fermentation medium for fresh mulberry leaves in step 2. Put the samples in the

temperate box and cultivate it at 28-30 degree for 5-7 days.

Step 5: Extraction of active ingredients from fermented mulberry leaves.

Part a: Preparation of polysaccharides.

Extract with water at a ratio of 1:5 to 1:10 for water and reflux, filter the filtrate with

suction and settle with 95% ethanol until get to a final concentration of 80%. The

precipitation time for alcohol is 24h. Evaporate to dryness in a water bath to obtain

crude polysaccharide extract. The filter residue is used for the extraction of total

flavonoids and total alkaloids.

Part b: Extraction of total flavonoids and alkaloids.

Add ethanol solution with the concentration from 50% to 80% to the polysaccharide extraction residue according to the ratio of material to liquid at 1:15 to 1:20. Reflux extraction at 50-75 degree, and concentrate the extract under reduced pressure until nothing Mellow, concentrated, freeze-dried to obtain mulberry leaf flavonoids and alkaloid extracts.

6.Determination of the inhibition rate of c-glucosidase in mulberry leaf extract in

vitro.

Comments for the experimental steps:

1.The auxiliary materials described in step 2 include bran and glucose, wherein the

ratio of the dry weight of bran to the wet weight of fresh mulberry leaves is 1:5 to

1:15 (w/w). The amount of glucose added to the sample isI 10% Fresh mulberry

leaves (weight ratio based on the wet weight of mulberry leaves).

2.The Lactobacillus plantarum, Saccharomyces cerevisiae, Ganoderma lucidum,

Pleurotus eryngii, and Trichoderma reesei described in step 3 use potato juice

glucose medium. Bacillus subtilis uses LB medium (which prepared with IL

distilled water, 20g glucose, 15g peptone, 5g chloride Sodium and 0.5g beef extract).

3.The amount of seed liquid inoculation in step 4 is 5%-20% (v/w) of the initial wet

weight of the compound mulberry leaf fermentation substrate.

Result

After fermentation and enrichment, the total polysaccharide content, total flavonoid

content, and total alkaloid content were increased by 50-70% compared to the unfermented control group; 125-300% and 85%-150%; 1 mg/ml fermented fresh mulberry leaves the c-glucosidase inhibitory rate of the extract reached more than

%.

Claims (3)

1. Claim 1. A method of efficiently increasing the a -glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation, characterized in that, including: use solid fermentation in microbial fermentation, microorganisms can biotransform or decompose the natural active components in mulberry leaves, change their original biological activities, and produce new components or effects, microorganisms can change the material structure of the natural active components of medicinal plants and improve the dissolution rate of alkaloids, polysaccharides and other active components.
2. 2. According to method of claim 1, wherein production of polysaccharides: with a solid-liquid ratio of 1:5-10, add water reflux extraction, extraction filtrate, with 95% ethanol precipitation to the final concentration of 80%, alcohol precipitation time is 24h, decompressed extraction of precipitation part, water bath steam dry polysaccharide crude extract; The filtrate residue is used for extracting total flavonoids and total alkaloids.
3. 3. According to method of claim 1, wherein extraction of total flavonoids and alkaloids: 50%~80% ethanol solution was added to the polysaccharide extraction residue according to the ratio of 1:15-20, and reflux extraction was performed at -75°Q the extraction solution was decompressed and concentrated to no alcohol taste, and the flavonoid and alkaloid extracts from mulberry leaves were concentrated and freeze-dried.

   Figure 1