JP5845240B2 - Gold wire continuous fermentation product, production method and use thereof - Google Patents

Description

  The present invention relates to a functional fermentation product, and more particularly, to a gold wire continuous fermentation product, a production method thereof, and an application.

  In recent years, due to lifestyle changes, many people have changed from eating habits to eat at home to eating habits where at least two meals are mainly eating out. As a result of eating out for a long time, the intake of vegetables tends to decrease and the intake of meat increases. This not only results in a biased nutritional balance, but many eating outs, such as fried chicken and soft drinks, have a high content of oil, sugar and salt, so obesity and metabolic syndrome due to excessive intake of calories. It will cause problems such as, and will further affect the health of individuals. Not only obesity due to excessive intake of high-calorie foods, but too much fat in the liver tends to become fatty liver because the liver cannot play the role of normal fat metabolism and cannot process excess fat.

  In general, when the fat content in the body is higher than a certain level, it is called obesity. The definition of obesity varies with time and place. The World Health Organization defines body mass index (BMI) as a criterion for obesity, and in a report published in 2012, defines overweight as body mass index 25 or more and obesity as body mass index 30 or more. Since the beginning of obesity changes only the appearance of the body, it is often neglected that obesity is a chronic disease. In fact, the effect of obesity on humans not only looks but leads to an increase in morbidity and mortality of non-infectious diseases, resulting in a decrease in quality of life. Specifically, when the body reaches the standard for obesity or overweight, abnormalities in the body's physiology occur. For example, abnormal insulin secretion can cause abnormal fat metabolism in the body, directly or indirectly, type 2 diabetes, hypertension, hyperlipidemia, nonalcoholic fatty liver, cancer, gout, cholecystitis, degenerative joints In particular, type 2 diabetes and obesity are most closely related. A report published in 2012 by the World Health Organization clearly pointed out that 12% of the world's population suffers from obesity and that overweight and obesity are the world's fifth risk factors related to mortality risk. .

  Obesity has a major impact on human health and appearance, and therefore how to prevent and treat is currently the subject of medical research. As obesity prevention, maintaining long-term dietary restrictions and good exercise habits are considered effective in preventing or treating obesity. However, it is difficult for obese patients to maintain long-term dietary restrictions and good exercise habits, and it is difficult to control weight by simply eating and exercising. Currently, gastric bypass surgery exists as the most clinically effective method for treating obesity. However, because surgery is an invasive treatment, it poses a very high risk to the patient himself and cannot be widely applied to all obese people. Therefore, besides surgery, there are various commercially available diet drugs. Regardless of the serious effects of illegal drugs on human health, phenylpropanolamine hydrochloride (PPA), approved by the Taiwan Health Ministry, promotes metabolism and increases calorie consumption. Because it is a sympathomimetic drug, taking it as a dose increases the risk of stroke as well as general dry mouth, palpitation, dizziness and other side effects.

  Many studies tend to develop into traditional Chinese medicines to avoid side effects from Western medicine or invasive treatments. Among them, the “Anoectochilus spp.” Called “Yakuo” is currently regarded as the center of gravity for many literature studies. The gold line is a plant of the orchid family, and the whole plant is prized as a material for herbal medicine, and is often used for the treatment of various diseases of mankind. At present, there are many recognized effects in the gold wire ream, such as Taiwan Patent No. I369207, Taiwan Patent No. I379688 and Taiwan Patent Application No. 09810042, in which the gold wire ream extract is used for liver protection, anti-osteoporosis agent, Effects such as inflammation and anti-allergy are disclosed. Japanese Patent Application No. 8-302556 and Japanese Patent Application No. 2001-263528 disclose that extracts of gold wire reams have a hypoglycemic and dietary effect, respectively. From the above, it is known that the gold wire ream surely has many actions on the human body. However, until now, research has obtained an extract of the gold wire series by the extraction method, and then the extract of the gold wire series is applied to the pharmaceutical composition. Have the following disadvantages.

  1. In the extraction method, an organic solvent is often used as an extraction medium, so that the extract contains a toxic component and causes side effects on the human body.

  2. In order to prevent the extract from containing toxic components such as organic solvents, there is currently an extract using water as an extraction medium. However, since an extraction process includes a high-temperature treatment step, toxic components are dissolved in water, so that the extract contains harmful substances.

  3. Since the extract obtained by extraction cannot improve the content of the active ingredient, it is difficult to satisfy industrial applicability.

  The main object of the present invention is to use a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or excipient by use in hypoglycemia, obesity treatment or metabolic syndrome, fatty liver treatment and diabetes treatment. Is prepared and mixed with at least one food composition to provide a food product for continuous fermentation that can be used to form a food for disease prevention and health maintenance.

  When used as a pharmaceutical composition, the dosage form is not limited and can be appropriately changed as necessary. For example, pill, powder, liquid, spray, colloid, tablet, suspension Provided in the form of In addition, seasonings, sweeteners, or colorants can be added to alter the overall taste and appearance.

  Moreover, when using as a foodstuff, the eating method and form are also not limited, For example, it provides with forms, such as a drink, a chewing tablet, an oral tablet, a spray, a suspension.

  Another object of the present invention is to provide a method for producing a gold wire continuous fermentation product that effectively reduces production costs and produces a large amount of gold wire continuous fermentation products.

Specifically, the wire continuous fermentation product disclosed in one preferred embodiment of the present invention includes at least one wire continuous glycoside whose composition is represented by the following formula (I).

  The gold wire continuous fermentation product disclosed in the second preferred embodiment of the present invention is used for the production of a pharmaceutical composition for hypoglycemia, wherein the gold wire continuous fermentation product is at least represented by the above formula (I). ) Including gold-linked glycosides.

  The continuous wire fermentation product disclosed in the third preferred embodiment of the present invention is used for producing a pharmaceutical composition for the treatment of obesity, wherein the continuous wire fermentation product has at least the above formula ( Gold-linked glycosides as shown in I) are included.

  The gold wire continuous fermentation product disclosed in the fourth preferred embodiment of the present invention is used for the production of a pharmaceutical composition for the treatment of fatty liver, of which the gold wire continuous fermentation product has at least the above formula ( Gold-linked glycosides as shown in I) are included.

  A food product disclosed in a fifth preferred embodiment of the present invention includes the gold wire continuous fermentation product and at least one food composition, wherein the gold wire continuous fermentation product is at least represented by the above formula (I). Includes gold-linked glycosides as shown.

  The method for producing a continuous wire fermentation product disclosed in the sixth preferred embodiment of the present invention includes the following steps.

  Step a: Fermentation reaction of lactic acid bacteria and gold wire continuous medium.

  Step b: A gold wire continuous fermentation product was obtained from the fermentation broth obtained in step a.

  The lactic acid bacterium in step a is Lactobacillus casei subsp.

  The conditions for the fermentation reaction performed in step a are a temperature of 25 to 42 ° C., a pH value of 4.0 to 7.0, and a stirring speed of 0 to 100 rpm.

  The fermentation broth in step b is sterilized and centrifuged to obtain the gold wire continuous fermentation product.

It is a figure which shows the reverse phase high performance liquid chromatograph of a gold wire continuous glycoside standard goods. It is a figure which shows the calibration curve of a gold wire continuous glycoside standard goods. It is a figure which shows the reverse phase high performance liquid chromatograph of an unfermented gold wire line. It is a figure which shows the reverse phase high performance liquid chromatograph of LcR gold wire continuous glycoside standard goods. It is a figure which shows the body weight change of the mouse | mouth of each group by a different process. It is a figure which shows the perirenal fat tissue weight of the mouse | mouth of each group. It is a figure which shows the testicular fat tissue weight of the mouse | mouth of each group. It is a figure which shows the fat tissue weight of the mouse | mouth of each group. It is a figure which shows the body weight change of the mouse | mouth of each group by a different process. It is a figure which shows the liver weight of the mouse | mouth of each group. FIGS. 3A to 3C are diagrams showing livers taken out from mice of the first group to the third group. FIGS. FIGS. 4A to 4C are diagrams showing the results of tissue staining of hepatoxylin / eosin on liver tissue sections of mice of Group 1 to Group 3. FIG. FIGS. 4A to 4C are diagrams showing the results of immunohistochemical staining of liver tissue sections of mice of Group 1 to Group 3 with 4-hydroxynonenal. A to E are diagrams showing the results of immunohistochemical staining of insulin receptor in liver tissue sections of mice of Group 1 to Group 5. A to E are diagrams showing the results of immunohistochemical staining of glucose transporters in liver tissue sections of mice of Group 1 to Group 5.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples are only for illustrating the contents of the present invention. The specification and claims of the present invention are not limited by these examples.

Example 1:
Take the gold wire continuous medium and place it in a fermenter, inoculate the gold wire continuous medium with an appropriate amount of Lactobacillus casei, subspecies, and rhamnosus. Fermentation culture is performed at a pH value of 4.0 to 7.0. After completion of the culture, the fermentation broth was taken and sterilized and centrifuged to obtain an LcR gold continuous fermentation product.

  Moreover, based on the said preparation process, each LGG and the Lactobacillus plantarum, and the said gold wire continuous culture medium were made to ferment, and the LrG gold wire continuous fermentation product and the Lp gold wire continuous fermentation product were obtained, respectively. .

  Example 2: Qualitative quantification of gold-linked continuous glycoside standard product.

  A gold-linked glycoside standard product was taken, and pure water / acetonitrile as a mobile phase was 98.5: 1 using a reversed-phase high performance liquid chromatography column (Mightysil ODS, 4.6 × 250 mm, 5 μm, ELSD detector). .5, Analysis is performed with the flow rate of 0.2 ml / min and the column temperature at room temperature. As a result, as shown in FIG. 1, it was found that the residence time of the gold wire continuous glycoside standard product was about 22.895 minutes.

Next, an appropriate amount of each gold-linked glycoside is taken, and a standard solution is blended with water. A standard curve is prepared with the peak area ratio of the vertical axis as the vertical axis and the sample injection amount of the gold-linked continuous glycoside standard product as the horizontal axis. As a result, as shown in Table 1 and FIG. 2, the regression equation: Y = 817.53X-73981, R ² = 0.9967 was obtained.

Table 1 shows the mass concentration and the measurement peak value of the gold-linked continuous glycoside standard product.

  Example 3: Gold wire continuous fermentation product contains a large amount of gold wire continuous glycosides.

  The LcR gold-line continuous fermentation product and the unfermented gold-line string obtained in Example 1 were taken and analyzed using a reversed-phase high performance liquid chromatography column under the conditions in Example 2, respectively. The results are shown in FIG. 3 and FIG. 4, in which FIG. 3 is a diagram showing a reverse-phase high-performance liquid chromatograph of unfermented gold wires, and FIG. 4 is a standard product of LcR gold wires. It is a figure which shows the reverse phase high performance liquid chromatograph.

  The results of the above analysis are substituted into the regression equation in Example 2, and the LcR gold-line continuous fermentation product and the unfermented gold-line continuous glycoside content are shown in Table 2.

Table 2 shows the content of the LcR gold continuous fermentation product and the unfermented gold wire continuous glycoside glycoside.

  According to the analysis results, it was found that the content of the gold-linked glycosides by the LcR gold-wire continuous fermentation product disclosed in the present invention was significantly improved as compared with the unfermented gold-wire continuous.

  Example 4: Gold wire continuous fermentation products can reduce the risk of obesity induced by high fat diet.

  A plurality of C57BL / 6 mice were divided into 3 groups, and after one week had passed to adapt to the environmental conditions, they were raised for 12 weeks under different conditions, and then the changes in body weight of the mice in each group were measured. As shown in FIG. Among them, the first group of mice is fed with normal food as a normal group. The second group of mice, as a control group, is fed a high fat diet to induce obesity. The third group of mice is fed with the high fat diet and simultaneously with the LcR gold wire continuous fermentation product prepared in Example 1, and the dosage of the LcR gold wire continuous fermentation product is 660 mg / kg.

  According to the results of FIG. 5, it was found that the weight of the second group of mice was significantly heavier than the weight of the first group of mice. By feeding a high fat diet, the weight of the mouse can be reliably increased, and an obese model mouse is formed. Furthermore, when the mice of the second group and the third group are compared, the weight of the mice of the third group is significantly lighter than the weight of the mice of the second group. By doing so, obesity induced by a high fat diet could be effectively reduced.

  Example 5: Gold wire continuous fermentation products can reduce body fat.

  FIG. 6 shows the results of sacrificing the perirenal adipose tissue and peri-testicular adipose tissue and measuring the weight of each group of mice in Example 4 sacrificed. 6A is a diagram showing the peri-renal adipose tissue weight of each group of mice, and FIG. 6B is a diagram showing the peri-testicular adipose tissue weight of each group of mice.

  According to the results of FIG. 6, it can be seen that the weight of the perirenal and peritesticular adipose tissues of the first group of mice is significantly lighter than that of the second group of mice, that is, by feeding a high fat diet, You can definitely increase your weight. Further, when comparing the second group and the third group, the weight of the perirenal adipose tissue and the testicular adipose tissue of the third group of mice compared to the weight of the perirenal adipose tissue and peritesticular adipose tissue of the second group of mice. It has been found that the amount of visceral fat accumulated can be effectively reduced by feeding the LcR gold wire continuous fermentation product disclosed in the present invention.

  Furthermore, as a result of inferring the body fat tissue content from the fat tissue weight of each group of mice as described above, as shown in FIG. 7, the fat tissue content of the second group of mice is significantly higher than that of the first group of mice. Thus, it was found that the second group of mice surely became an obese model mouse and that its body fat increased. In addition, since the body fat tissue content of the third group of mice is significantly lower than that of the second group of mice, when feeding the LcR gold line continuous fermentation product disclosed in the present invention, body fat can be effectively reduced. I understood.

  Example 6: Gold wire continuous fermentation product can reduce obesity induced by high fat diet.

  A plurality of C57BL / 6 mice were divided into 3 groups, and after 1 week to adapt to environmental conditions, they were reared for 8 weeks under different conditions. Thereafter, the weight of each group of mice was measured and the results are shown in FIG. Show. Among them, the first group of mice is fed with normal water as a normal group. As a control group, the second group of mice is fed with 30% sugar water to induce obesity. The third group of mice is fed with 30% concentration of sugar water and simultaneously with the LcR gold wire continuous fermentation product disclosed in the present invention, and the dose of the LcR gold wire continuous fermentation product is 660 mg / kg.

  According to the result of FIG. 8, it was found that the weight of the second group of mice was significantly heavier than that of the first group of mice. It is shown that obesity in mice can be reliably induced by feeding a high concentration of sugar water. In addition, the weight of the mice in the third group is significantly lighter than the weight of the mice in the second group. Obesity induced by high-concentration sugar water by feeding the LcR gold line continuous fermentation product disclosed in the present invention. Was able to descend effectively.

  Example 7: Gold wire continuous fermentation product can lower blood sugar.

  In this example, the blood glucose level measuring device measures the glucose concentration in the tail vein blood of each group of mice of Example 6. Here, first, the fasting blood glucose levels of the mice of each group were measured, and then the mice of each group were fed with 2.0 g / kg of sucrose, and the blood glucose levels of the mice of each group were fed, respectively. Table 3 below shows the results measured again at intervals of minutes, 60 minutes, 90 minutes, and 120 minutes.

Table 3 shows the sucrose tolerance test of each group of mice.

  According to the results of Table 3 above, it was found that the blood glucose level of the third group of mice on an empty stomach was significantly lower than that of the second group of mice. In addition, the blood glucose level of the third group of mice at each time point after feeding sucrose is also lower than the blood glucose level of the second group of mice, that is, the mice of the third group do not cause insulin resistance. By feeding the LcR gold line continuous fermentation product disclosed in the present invention, it was possible to reduce the occurrence of insulin resistance and to regulate blood glucose normally.

  Example 8: Gold wire continuous fermentation product can suppress the formation of fatty liver.

  FIG. 9 and FIG. 10 show the results obtained by sacrificing the mice of each group in Example 6 and, first, taking the liver of each group of mice and taking a photo to measure the weight. 10A is a photograph of the liver of the first group of mice, FIG. 10B is a photograph of the liver of the second group of mice, and FIG. 10C is a photograph of the liver of the third group of mice.

  Thereafter, the livers of the mice of each group were fixed with formalin, and after paraffin-embedded sections, stained with hematoxylin / eosin staining, dehydrated, sealed, and observed with an optical microscope. The photographed result is shown in FIG. 11A shows the result of staining the liver tissue section of the first group of mice, FIG. 11B shows the result of staining the liver tissue section of the second group of mice, and FIG. 11C shows the result of the third group of mice. It is the result of having dye | stained the liver tissue section | slice of a mouse | mouth.

  Moreover, the result of having performed the immunohistochemical dyeing | staining of the liver tissue section | slice of the mouse | mouth of each said group by 4-hydroxynonenal (4-hydroxynonenal) is shown in FIG. 12A is a diagram showing the result of staining the liver tissue section of the first group of mice, and FIG. 12B is a diagram showing the result of staining the liver tissue section of the second group of mice. These are figures which show the result of having dye | stained the liver tissue section | slice of the 3rd group of mice | mouths.

  Comparing the results of FIGS. 9 to 12 together, the liver of the first group of mice is a healthy liver, its color is vermilion, and the color of the tissue section stained with hematoxylin / eosin is relatively bright . Since the second group of mice is a fatty liver model mouse, the liver contains a large amount of fat, the liver weight is significantly higher than the liver weight of the first group of mice, the color becomes whitish, and the adipose tissue empty. It shows that cysts are conspicuous, lipid peroxide precipitation is relatively increased, and high concentration sugar water is fed to form fatty liver, liver weight is increased, and fatty tissue is accumulated in the liver. Compared with mice in group 2, liver weight of mice in group 3 is significantly lower, liver color is significantly closer to group 1, and adipose tissue vacuoles are relatively inconspicuous. Less.

  From the above, feeding the LcR gold-line continuous fermentation product disclosed in the present invention suppresses the formation of adipose tissue and accumulation in the liver, and prevents fatty liver induced by high-concentration sugar water. It can be effectively suppressed.

  Example 9: Gold wire continuous fermentation product can treat diabetes.

  As in the embodiment shown in Example 6, a plurality of C57BL / 6 mice are divided into 5 groups, and after one week has passed to adapt to environmental conditions, they are raised for 8 weeks under different conditions. Among them, the first group of mice is fed with normal water as a normal group. As a control group, the second group of mice is fed with 30% sugar water to induce fatty liver. A third group of mice is fed with 30% strength sugar water and simultaneously with the LcR gold line continuous fermentation product prepared in Example 1. A fourth group of mice is fed with 30% strength sugar water and simultaneously with the LrG gold line continuous fermentation product prepared in Example 1. A fifth group of mice is fed with 30% strength sugar water and at the same time with the Lp gold wire continuous fermentation product prepared in Example 1.

  After breeding for 8 weeks, the mice of each group were sacrificed, the livers of the mice of each group were collected, and each of the groups was embedded with a freezing embedding solution, and then subjected to immunohistochemical staining. Insulin-receptor and glucose transporter (GLUT4) in mouse liver are observed. As shown in FIGS. 13 and 14, FIGS. 13A to 13E are diagrams showing the results of immunohistochemical staining of insulin receptors in the liver of each group of mice. FIG. 14A to FIG. 14E are diagrams showing the results of immunohistochemical staining of glucose transporters in the liver of each group of mice.

  According to the results of FIGS. 13 and 14, the second group of insulin receptors is less than the first group of insulin receptors, and the insulin receptor content of any one of the third group to the fifth group is the first group. The insulin receptor content is approaching. In addition, the glucose transporters observed in the cells in the liver tissue section of the second group were relatively few as compared to the first group, and the glucose transporters observed in the cells of the third group to the fifth group were respectively the second group. Much more than that. Therefore, each said gold wire continuous fermentation product disclosed by this invention has an effect | action of the treatment or improvement of diabetes, respectively.

  As described above, according to the embodiments, the production method disclosed in the present invention is capable of mass production and production of a continuous wire fermentation product by a low cost method using a production system using microorganisms. Based on the inclusion of large amounts of gold-linked glycosides in the product, for diseases such as weight gain, fatty liver, visceral fat accumulation, elevated blood sugar or diabetes induced by high fat or high sugar, respectively By having an inhibitory or mitigating action, it can be prepared as an active ingredient of a pharmaceutical composition or a food for health.

  As described above, the present invention has been described in detail with reference to the embodiments. However, all simple modifications and changes made by those skilled in the art to the embodiments of the present invention are within the scope of the claims of the present invention. Shall be included.

Claims (3)

In the method for producing the gold-line continuous fermentation product,
A step of fermenting a lactic acid bacterium and a gold wire continuous medium;
Including step b of obtaining a continuous fermented gold wire product from the fermentation liquid obtained in step a ,
The lactic acid bacteria in Step a are Lactobacillus casei subsp. Rhamnosus (Lactobacillus casei subsp. Rhamnosus), Lactobacillus rhamnosus g. Selected from the group,
A method for producing gold-line continuous fermentation products. Conditions of the fermentation reaction performed in the step a, the temperature is 25 to 42 ° C., pH value is 4.0 to 7.0, stirring speed is 0 to 100 rpm, according to claim 1 A method for producing gold-line continuous fermentation products. 3. The method for producing a continuous wire fermentation product according to claim 1 or 2 , wherein the fermented gold solution is obtained by sterilizing and centrifuging the fermentation broth in step b.