TWI683664B - Actinidia chinensis planch. fermented extracts and the use thereof in a composition for increasing the antioxidant activity, increasing the anti-glycation activity, increasing the activity of proteolytic enzymes, improving the gastrointestinal dyspepsia, and increasing the gastrointestinal flora diversity - Google Patents

Abstract

The present invention provides an Actinidia chinensis Planch. fermented extracts and the use thereof in a composition for increasing the antioxidant activity, increasing the anti-glycation activity, increasing the activity of proteolytic enzymes, improving the gastrointestinal dyspepsia, and increasing the gastrointestinal flora diversity. The Actinidia chinensis Planch. fermented extracts can increase the normal function of the gastrointestinal tract, maintain the normal function of the immune system, delay the progression of chronic diseases and aging, and increase the gastrointestinal digestive function to achieve the maintaining of the normal function of the intestinal tract. The Actinidia chinensis Planch. fermented extracts is prepared by fermenting the Actinidia chinensis Planch. extracts to yeast, lactic acid bacteria and acetic acid bacteria for three-stage fermentation.

Description

Kiwi fruit ferment and its use for preparing pharmaceutical composition for increasing antioxidant activity, increasing anti-glycation activity, increasing proteolytic enzyme activity, improving gastrointestinal dyspepsia, and increasing gastrointestinal flora diversity

The present invention relates to a kiwifruit ferment and its use, especially a kiwifruit ferment for the preparation of increased antioxidant activity, increased anti-glycation activity, increased proteolytic enzyme activity, improved gastrointestinal dyspepsia, and increased gastrointestinal tract Use of the medicinal composition of flora diversity.

The main function of the human gastrointestinal tract is to decompose food and absorb nutrients. However, the pressure of modern life and the changes in eating habits have caused many people to suffer from functional dyspepsia. The so-called functional dyspepsia refers to upper abdominal pain and upper abdominal discomfort. Bloating, loss of appetite, nausea, vomiting, etc., symptoms of discomfort in the upper abdomen, but after examination, there are no organic lesions (such as peptic ulcer, gastric cancer, etc.), and the symptoms will continue or recur repeatedly, and currently still have no effect Drugs or methods for the treatment of functional dyspepsia can only recommend patients to improve their eating habits (such as avoiding overeating and eating indigestible foods, etc.), as well as maintaining proper exercise and normal work schedule to improve.

In addition, the gastrointestinal tract is also an important immune organ of the human body. It is an important shield against pathogens entering the body. Therefore, the health of the gastrointestinal tract is closely related to the health of a body. In addition, the health of the gastrointestinal tract is not only affected by the ecology of the flora in the gastrointestinal tract, but the antioxidant capacity of the gastrointestinal tract cells itself is also a key factor. Studies have shown that excessive environmental stress factors (such as pathogens) in the gastrointestinal tract can cause oxidative stress (Oxidative stress), and can cause damage to the integrity of the gastrointestinal tract, inflammation, and even changes in the flora. The gastrointestinal tract cells with good antioxidant strength are tightly connected to each other, which can reduce the movement of pathogens and toxins between intestinal cells on the first line, and strong cells are less affected by inflammation; therefore, once The antioxidant capacity of gastrointestinal cells becomes poor, and immunity will decline accordingly. The digestive and metabolic functions of the intestine will also be affected. Serious or even trigger a chronic inflammatory response or change the intestinal bacterial phase, and cause many other diseases. Therefore, enhancing the antioxidant power of the gastrointestinal tract is also a key point for maintaining the health of the gastrointestinal tract and maintaining its normal function.

Advanced Glycation End Products (AGEs) are a group of highly oxidized compounds, so they are considered to be a glycotoxin (Glycotoxin). Studies have shown that the end products of glycation will combine with the receptors on the cell surface to change their structure and function. It also promotes the increased oxidative stress and inflammation of the cells, and is closely related to the formation of diabetes, arteriosclerosis and chronic kidney diseases. In addition to the production of saccharification end products in the body’s normal metabolic process, many processed foods also contain saccharification end products. Studies have also pointed out that avoiding the intake of saccharification end products from food can help delay the progress of chronic diseases and aging.

In summary, in response to the gastrointestinal tract or overall health problems faced by modern people due to changes in life and eating habits, and based on the improvement of modern people's living standards and the improvement of health care concepts, we have developed a method that can effectively improve gastrointestinal dyspepsia symptoms and increase resistance A pharmaceutical composition composed of active ingredients that oxidize activity and increase anti-glycation activity is really necessary.

Therefore, one object of the present invention is to provide a kiwifruit fermented product, in which a kiwifruit water extract is fermented in order by yeast ( Saccharomyces cerevisiae ), Lactobacillus plantarum , and Acetobacter aceti obtain.

Another object of the present invention is to provide a use of the fermented kiwi fruit as described above for preparing a composition for improving gastrointestinal discomfort symptoms.

Another object of the present invention is to provide a use of the fermented kiwifruit as described in item 1 of the patent application scope for the preparation of a composition for enhancing antioxidant and anti-glycation activity.

Another object of the present invention is to provide a kiwi fermented product as described in item 1 of the patent application scope for preparing a pharmaceutical combination that increases proteolytic enzyme activity, improves gastrointestinal dyspepsia, and increases gastrointestinal flora diversity The purpose of things.

Another object of the present invention is to provide a method for manufacturing a kiwifruit fermented product, which comprises: sequentially carrying out a kiwifruit water extract through the yeast, lactobacilli, and acetobacter described in item 2 of the patent application scope Obtained by fermentation.

In one embodiment of the present invention, wherein the yeast strain is a strain of BCRC20271; the lactobacillus strain is a strain of BCRC910805; the acetate strain is a strain of BCRC11688. And the addition amount of the yeast is 0.01-0.5% (v/v); the addition amount of the Lactobacillus is 0.01-0.25% (v/v), and the addition amount of the Acetobacter is 3-10% (v/v) ). The ratio of the cultivation time of the yeast stage, the lactobacillus stage and the acetate bacilli stage is 1-2.5:1-3:3-10.

In yet another embodiment of the present invention, wherein the improvement of gastrointestinal discomfort symptoms is to increase proteolytic enzyme activity.

In another embodiment of the present invention, wherein the enhanced antioxidant activity is to increase the activity of superoxide dismutase (SOD) and/or increase the total polyphenol content.

At the same time, the invention is used to prepare medicines that increase antioxidant activity, increase anti-glycation activity, increase proteolytic enzyme activity, improve gastrointestinal dyspepsia, and increase gastrointestinal flora diversity The pharmaceutical composition may also contain an effective amount of fermented kiwi fruit and a pharmaceutically acceptable carrier. The composition is in the form of powder, granule, liquid, gel or paste.

The fermented kiwifruit of the present invention can be effectively used to prepare pharmaceutical compositions that increase antioxidant activity, increase anti-glycation activity, increase proteolytic enzyme activity, improve gastrointestinal dyspepsia, and increase gastrointestinal flora diversity, and It can effectively increase the function of gastrointestinal tract to maintain the normal operation of gastrointestinal immune function, and also help to delay the progress of chronic diseases and aging, as well as increase the digestive function of gastrointestinal tract, and improve the stomach and intestines, and maintain the normal intestines. The effect of function.

The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are used to clarify the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with this art without departing from the spirit and spirit of the present invention Within the scope, some changes and retouching can be done, so the protection scope of the present invention shall be subject to the scope defined in the appended patent application.

FIG. 1 is a bar graph of the kiwifruit fermented product for enhancing SOD protein activity according to an embodiment of the present invention.

FIG. 2 is a bar graph of the kiwifruit fermented product to increase the total polyphenol content according to an embodiment of the present invention.

FIG. 3 is a bar graph of the anti-glycation activity of the kiwifruit fermented material according to an embodiment of the present invention.

FIG. 4 is a photograph of a kiwifruit fermented product enhancing proteolytic enzyme activity according to an embodiment of the present invention.

FIG. 5 is a bar graph of kiwifruit fermented products in improving overall gastrointestinal discomfort symptoms of the human body according to an embodiment of the present invention. ***p<0.001.

FIG. 6 is a bar graph of the kiwifruit fermented product in the improvement of the human body's Nipine Defect Index according to an embodiment of the present invention. ***p<0.001.

The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

Example 1 Preparation method of kiwi fermented material of the present invention

Kiwi fruit ( Actinidia chinensis Planch.) is Actinidiaceae ( Actinidiaceae ) Actinidia genus ( Actinidia ) plant, Chinese kiwi fruit, kiwi fruit, carambola, sheep fruit, vine pear or rope pear, native to China, growing at an altitude of 400-2,000 In the mountainous area of Chichi, the stems are elongated, with most branches, the leaves are round or wide oval, the flowers are solitary or several clusters, they are white at the beginning, and then turn to pale yellow, and the fruits are berries, which are oblong Shaped, oval or round, with dense brown fur, seeds are dark brown. At present, the stems of kiwifruit are known to have the effects of clearing heat, diuresis, activating blood circulation, reducing swelling, and anti-cancer.

In an embodiment of the present invention, the kiwi fruit is thoroughly washed, the washed kiwi fruit is mixed with water at a solid-liquid ratio of 1:5-15, and extracted at 50-100°C for 0.5-2.5 hours to obtain kiwi Fruit water extract. The kiwi fruit water extract is cooled to room temperature for subsequent three-stage fermentation. It is first planted with 0.01-0.5% yeast ( Saccharomyces cerevisiae) , purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC20271) Fermentation in this kiwifruit water extract for 1-2.5 days, followed by direct addition of 0.01-0.25% Lactobacillus plantarum TCI028, patent deposited at the Biological Resources Conservation and Research Center, Taiwan, No. BCRC910805) Fermentation 1 -3 days, then add 3-10% Acetobacter aceti (purchased from the Biological Resources Conservation and Research Center, Taiwan, number BCRC11688) and ferment for 3-10 days; of which, Lactobacillus TCI028 is a patent application number in the Republic of China No. 106145146 completed the patent deposit, the fermentation of these three bacteria in order: yeast, lactobacilli, acetobacter, and can not be adjusted back and forth, and finally without removing these three bacteria, use the set sugar content range 2-10 °, pH2-4, alcohol <3% and other specifications, if the test meets the specifications, it is judged that the fermentation is completed and the fermentation broth is obtained. Next, the fermentation broth is concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh screen, then 1-3% citric acid and 40-70% isomalt oligosaccharide are added to adjust the specifications, and then sterilized to obtain The fermented kiwifruit of the present invention is made by microbial fermentation to release a large amount of kiwifruit effective substance.

Example 2 The superoxide dismutase (SOD) activity of the kiwi fermentation product of the present invention is increased

Superoxide dismutase (SOD) is an enzyme that can catalyze the superoxide dismutation reaction and convert it into oxygen and hydrogen peroxide. It is widely found in various animals, plants, and microorganisms. An important antioxidant in the body that protects cells exposed to oxygen. In this example, in order to compare whether the SOD activity contained in the fermented kiwifruit of the present invention is higher than before fermentation, the SOD activity is quantified in order to inhibit the efficiency of pyrogallol (Pyrogallol) auto-oxidation reaction. First, configure solution A and solution B for measurement. Solution A consists of 1.2114g tris(hydroxymethyl)aminomethane (Tris) and 37.2mg ethylenediaminetetraacetic acid (EDTA-2 Na ) Dissolve in 62.4mL 0.1mole/L hydrochloric acid solution, and after dissolution, quantify to 100mL with pure water, solution B is to dissolve 56.7mg pyrogallol (Pyrogallol) in a small amount of 10mmole/L hydrochloric acid solution , And after dissolving, quantify to 100mL with pure water. Next, take 2.35 mL of the A solution in a centrifuge tube, add 1.8 mL of pure water each and 0.2 mL of the kiwi fermentation product (experimental group) and kiwifruit water extract (control group) of the present invention, each tube is shaken After mixing well, add 0.15mL of B solution, shake gently and evenly, then pour into the colorimetric tube, and then measure the initial time at 325nm wavelength and the absorbance value after 1 minute, and add 0.2.mL pure The water group is the blank measurement group, and then the SOD activity is calculated by the following formula.

△A ₃₂₅ : pyrogallol auto-oxidation rate

△A ′ ₃₂₅ : Sample inhibits pyrogallol auto-oxidation rate

V: volume of the sample

D: dilution factor of the sample

The result of the increased SOD activity in the kiwi fermentation of the present invention is shown in FIG. 1. After the three-stage fermentation of the present invention, the kiwifruit has a SOD activity that is 3.6 times higher than that of the pure kiwifruit water extract. This result shows that the kiwifruit fermentate of the present invention can be effective after a specific microbial fermentation step To enhance the SOD activity therein, this specific fermentation step can enhance the antioxidant capacity of kiwifruit, and can be used to prepare a composition that enhances the antioxidant capacity of the gastrointestinal tract to increase the gastrointestinal tract function and maintain the normal operation of the gastrointestinal tract immune function.

Example 3 The total polyphenol content of the fermented kiwifruit of the present invention is increased

Polyphenols are an antioxidant. Previous studies have indicated that polyphenols can inhibit the occurrence of neurodegenerative diseases and some cardiovascular diseases. In this example, in order to compare whether the total polyphenol content in the kiwifruit ferment of the present invention is higher than that of the kiwifruit kiwifruit water extract, the total polyphenol content in the kiwifruit kiwifruit colorimetric method is determined by using the Folin-Ciocalteu colorimetric method. Quantitative total polyphenol content of tungsten-molybdenum acid produced a blue compound after its reduction (from Mo ⁶⁺ to Mo ⁵⁺ ), and the depth of the blue compound was positively correlated with the total polyphenol content. First, dilute the kiwifruit fermentation product (experimental group) and kiwifruit water extract (control group) of the present invention with water, take 100 mL into a centrifuge tube, and then add 500 μL of Folin-Ciocalteu phenol reagent to mix and let stand 3 After a minute, add 400 μL of 7.5% sodium carbonate, mix and let stand for 30 minutes, take 200 μL of the reaction solution in a 96-well plate, and measure the absorbance at 750 nm. Among them, using gallic acid as the standard product to prepare a standard curve, dissolve 10g of gallic acid in water, and configure standard 0, 20, 40, 60, 80, and 100μL/mL gallic acid Acid, and take 100 μL of each standard solution of each concentration into a 10 mL centrifuge tube, add 500 μL of Folin-Ciocalteu phenol reagent, mix and stand for 3 minutes, add 400 μL of 7.5% sodium carbonate, mix and stand for 30 minutes, take 200 μL of the reaction solution was measured in 96-well plates, and the absorbance at 750 nm was measured to obtain a standard curve, and then the total polyphenol content in the kiwifruit fermented product and kiwifruit water extract of the present invention was converted via the standard curve.

The result of the increase in the total polyphenol content in the kiwi fermentation of the present invention is shown in FIG. 2. After the three-stage fermentation of the present invention, the total polyphenol content of the kiwi fruit is 5.8 times higher than that of the pure kiwi fruit water extract. This result shows that the kiwi fruit fermented product of the present invention undergoes a specific microbial fermentation step. It can effectively increase the total polyphenol content. This specific fermentation step can enhance the antioxidant capacity of kiwi fruit, and can be used to prepare a composition that improves the antioxidant capacity of the gastrointestinal tract, so that the function of the gastrointestinal tract is increased, and the immune function of the gastrointestinal tract is maintained. Normal operation.

Example 4 The anti-glycation activity (AGEs) of the fermented kiwifruit of the present invention is improved

This example is to compare whether the anti-saccharification activity of the kiwifruit ferment of the present invention is higher than that of the kiwifruit water extract, so as to inhibit the saccharification of bovine serum albumin (BSA) by D-fructose Efficiency, quantify saccharification activity. First, were taken 0.25mL diluted to 20% (v / v) of kiwifruit water extract (control group) or fermented kiwifruit (experimental group) of the present invention, and comprises the addition of 0.25mL of 60mg 0.06% of NaN ₃ /mLBSA solution (configured with 200mM sodium phosphate buffer, pH7.4), and evenly mixed with 1.5M D-fructose (configured with 200mM sodium phosphate buffer, pH7.4), then take 0.1mL The fluorescence value of the mixed solution was measured under excitation light 360nm and emission light 460nm, which was taken as the zero point before the reaction, and 0.45mL of the mixed solution was incubated at 50°C for 24 hours, and then 0.1mL was taken out to measure the fluorescence value. It is the end of the reaction. The same amount of 3 mM Aminoguanidine (AG) (configured with 200 mM sodium phosphate buffer) was used to dissolve the solvent to an equal volume to serve as a positive control group. Aminoguanidine (AG) is known to have the effect of inhibiting glycation. Finally, the efficiency of the ability to eliminate glycation end products (AGEs) is calculated by the following formula to represent its anti-glycation activity.

The results of the increased anti-glycation activity in the kiwi fermentation of the present invention are shown in FIG. 3. After the three-stage fermentation of the present invention, the ability of kiwifruit to remove the final saccharification product is 1.4 times higher than that of the pure kiwifruit water extract. This result shows that the kiwifruit fermented product of the present invention undergoes a specific microbial fermentation step Afterwards, it can effectively enhance its ability to clear the final saccharification products. This specific fermentation step can enhance the anti-glycation activity of kiwifruit, which helps delay the progression of chronic diseases and aging.

Example 5 The activity of the proteolytic enzyme of the fermented kiwifruit of the present invention is increased

In this example, in order to compare whether the proteolytic enzyme activity in the kiwi fermentation of the present invention is higher than that of the kiwi fruit water extract, the activity of the proteolytic enzyme was quantified using skim milk medium (Skim Milk Agar, purchased from HIMEDIA, India). This medium can be used to test whether the test object has the ability to decompose protein. If it can decompose protein, a transparent circle will be generated around the test object. First, prepare Skim Milk Agar solution (purchased medium powder contains 2.8% skimmed milk powder, 0.5% casein hydrolysate, 0.25% yeast extract, 0.1% glucose, and 1.5% carrageenan, all of which are w/ v) After high temperature and high pressure sterilization, the temperature is reduced to 40-50°C, and then poured into a petri dish to make a flat plate, and then the kiwi fruit kiwi fruit water extract and the kiwi fruit fermented product of the present invention are diluted by 10 times, then taken Two sample dilutions of the same concentration were placed on different Skim Milk Agar and incubated in a 37°C growth chamber for 24 hours. Observe and record the size of the transparent circle produced by each group of samples. Among them, water is used as a blank control group.

The results of increased proteolytic enzyme activity in the kiwi fermentation of the present invention are shown in FIG. 4. After the three-stage fermentation of the present invention, the kiwifruit produced a transparent ring on the skim milk medium that was 3.5 times as high as the pure kiwifruit water extract. This result shows that the kiwifruit fermented product of the present invention is passing through After a specific microbial fermentation step, the activity of proteolytic enzymes in it can be effectively increased to increase the digestive function of the gastrointestinal tract and avoid discomfort symptoms caused by indigestion.

Example 6 The effect of the fermented kiwifruit of the present invention to improve the digestion of human gastrointestinal tract

In order to confirm the effect of the fermented kiwifruit of the present invention on improving digestion of human gastrointestinal tract, 7 adults with functional dyspepsia (FD) were first recruited as subjects, taking 5-20 mL of this product after daily lunch The invented kiwifruit fermented product was filled in the questionnaire of the Nepean Dyspepsia Index (NDI) before taking, after taking 2 weeks, and taking after 8 weeks (see World J Gastroenterol. 2009 Jul 7; 15(25): 3173-7.) Give feedback and analysis. Nipin dyspepsia index was created by Talley et al. The index scale contains a total of 25 items and is divided into stress and sleep, daily life intervention, There are 5 areas including diet, knowledge and self-control, and work or study. The scale is a tool for measuring the health-related quality of life (HRQL) of the digestive system, which provides a measurement standard for dyspepsia symptoms and assesses the HRQL specific to dyspepsia.

The results of the kiwifruit fermented product of the present invention in improving the overall gastrointestinal discomfort symptoms of the human body are shown in FIG. 5; the results in improving the human body's Niping dyspepsia index are shown in FIG. 6. After taking the kiwi fermented product of the present invention for 4 weeks, the overall gastrointestinal discomfort symptoms can be improved by up to 42%; and according to the Niping adverse index, the kiwi fruit fermented product of the present invention can improve gastrointestinal discomfort symptoms by up to 49%; And the improvement rate is as high as 100% (the results are not shown). These results show that the kiwi fermented product of the present invention has an excellent effect of improving the overall gastrointestinal dyspepsia. In addition, after taking the kiwi ferment of the present invention for 4 weeks, the diversity of intestinal bacteria can be increased by up to 1.5 times, and the ratio of the number of people can be improved by as much as 60% (the results are not shown). Can reduce the risk of many diseases, including Contains gastrointestinal related diseases. Therefore, these results show that the kiwi fermented product of the present invention has an excellent ability to improve gastrointestinal upset symptoms and increase the diversity of gastrointestinal flora, and can effectively achieve the effects of strengthening the stomach and intestines and helping digestion.

In summary, in the present invention, the kiwifruit fermented product obtained by performing three-stage fermentation of kiwifruit with yeast, lactobacilli, and acetobacter can effectively improve the SOD activity, the total polyphenol content, and the removal thereof The ability to saccharify the final product, enhance the activity of proteolytic enzymes therein, improve the overall gastrointestinal dyspepsia, and increase the diversity of gastrointestinal flora, and has a better effect than kiwi fruit water extract, and can be more effectively used for preparation A pharmaceutical composition that increases antioxidant activity, increases anti-glycation activity, increases proteolytic enzyme activity, improves gastrointestinal dyspepsia, and increases gastrointestinal flora diversity, and can effectively increase gastrointestinal function to maintain gastrointestinal immunity The normal operation of the function can also help to delay the progress of chronic diseases and aging, increase the digestive function of the gastrointestinal tract, and achieve the effect of strengthening the stomach and intestines and maintaining the normal function of the intestines.

Claims (10)

A kiwifruit fermented product is obtained by sequentially fermenting kiwifruit water extract through yeast ( Saccharomyces cerevisiae ), Lactobacillus plantarum , and Acetobacter aceti . The kiwifruit fermentation product as described in item 1 of the patent application scope, wherein the addition amount of the yeast is 0.01-0.5% (v/v); the addition amount of the lactobacilli is 0.01-0.25% (v/v), and The added amount of the Acetobacter is 3-10% (v/v). The kiwifruit fermentation product as described in item 1 of the patent application scope, wherein the yeast strain is a strain of BCRC20271; the lactobacillus strain is a strain of BCRC910805; and the acetate strain is a strain of BCRC11688. A use of the fermented kiwifruit as described in item 1 of the patent application scope for preparing a composition for improving gastrointestinal discomfort symptoms. The use as described in item 4 of the patent application range, wherein the improvement of gastrointestinal discomfort is to increase the activity of proteolytic enzymes. A use of the fermented kiwifruit as described in item 1 of the patent application scope for preparing a composition for enhancing antioxidant and anti-glycation activity; wherein the enhancing antioxidant activity is to increase superoxide dismutase (SOD) )active. A use of a kiwifruit fermented product as described in item 1 of the patent application scope for the preparation of a pharmaceutical composition that increases proteolytic enzyme activity, improves gastrointestinal dyspepsia, and increases gastrointestinal flora diversity. A method for manufacturing a kiwifruit fermented product includes: obtaining a kiwifruit water extract by sequentially fermenting yeast, lactobacilli, and acetobacter as described in item 2 of the patent application scope. The manufacturing method as described in item 8 of the patent application scope, wherein the addition amount of the yeast is 0.01-0.5% (v/v); the addition amount of the Lactobacillus is 0.01-0.25% (v/v), and the acetic acid The amount of Bacillus added is 3-10% (v/v). The manufacturing method as described in item 9 of the patent application range, wherein the culture time ratio of the yeast stage, the lactobacilli stage and the acetobacteria stage is 1-2.5:1-3:3-10.

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