TW202137889A - Plant fermented product, method of manufacturing the same, composition of plant fermented product and use thereof - Google Patents

Abstract

The present invention relates to a plant fermented product, method of manufacturing the same, composition of the plant fermented product and use thereof. A plant-based substrate is fermented by adding a bacterial liquid including multiple strains of Lactobacillus therein, so as to obtain a plant fermented product. A composition including the plant fermented product is administered to a subject, thereby alleviating and/or preventing hyperglycemia and complications thereof.

Description

Plant ferment and its manufacturing method, composition containing it and its use

The present invention relates to a plant ferment and its manufacturing method, a composition containing it and its use, in particular to a plant ferment using multi-strain Lactobacillus ferment and its manufacturing method, and a composition containing it Cum its use.

Hyperglycemia refers to the inability of the body to effectively use or store the carbohydrates it has taken, making the glucose concentration in the blood higher than the standard value. When a high concentration of glucose exists in the blood for a long time, it may cause diabetes, cardiovascular disease, brain atrophy, chronic kidney disease, eye disease, neuropathy, dementia and other diseases, and the wound is not easy to heal, which may easily lead to infection and worsening .

Currently commonly used drugs for lowering blood sugar include Biguanides, Sulfonylurea, Meglitinides, Thiazolidinedione (TZD), Glucosidase inhibitors (α- glucosidase inhibitor) class, Dipeptidyl peptidase-4 inhibitor (Dipeptidyl peptidase-4 inhibitor, DPP-4 inhibitor) and sodium glucose co-transporters 2 inhibitor (SGLT2 inhibitor). However, the aforementioned types of drugs often have side effects such as gastrointestinal upset, decreased appetite, fatigue, weight loss/increase, hypoglycemia (dizziness, palpitations, cold sweats, fainting), edema, flatulence, diarrhea, abdominal pain, headache, muscles Pain, joint pain, urinary tract infection, diuresis, dehydration, hypotension, dyslipidemia and/or hypoglycemia.

Secondly, although probiotics and/or probiotic ferments are currently used to regulate blood sugar, the effect of probiotics in regulating blood sugar varies greatly depending on the type of probiotic used. In the past, researches on multi-strain probiotics and/or probiotic ferment in regulating blood sugar mostly focused on the combination of Lactobacillus acidophilus and other probiotics. Studies have shown that the combination of Lactobacillus acidophilus and Lactobacillus casei (Lactobacillus casei) can reduce the blood glucose concentration of rats. Other studies have shown that lactobacillus acidophilus and Bifidobacterium sp. (Bifidobacterium sp.) dairy fermented products can improve fasting blood glucose levels in diabetic patients. However, the effect of using specific multi-strain Lactobacillus plant ferment in regulating blood sugar has been seldom studied.

In view of this, it is urgent to provide a plant ferment and its manufacturing method, a composition containing it, and its use to solve the above-mentioned problems.

Therefore, one aspect of the present invention is to provide a method for manufacturing plant ferment, which involves adding a multi-strain Lactobacillus strain into a plant matrix, To carry out the fermentation step.

Another aspect of the present invention is to provide a plant ferment, which is prepared by the above method, and the plant ferment contains a specific total number of multi-strain lactobacilli.

Another aspect of the present invention is to provide a composition of a plant ferment, which contains the above-mentioned plant ferment as an effective ingredient.

Another aspect of the present invention is to provide a use of a plant ferment for preparing a composition for regulating blood sugar. The composition includes a plant ferment, which can slow down and/or prevent hyperglycemia and/or its complications.

According to the above aspects of the present invention, a method for producing plant ferment is proposed. First, a cyanolysis step is performed on the plant slurry to obtain a plant substrate, wherein the cyanolysis step is performed at 70° C. to 100° C. for 30 minutes to 60 minutes. Then, a multi-strain Lactobacillus is provided, wherein the multi-strain Lactobacillus includes Lactobacillus plantarum , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus brevis and murine Lactobacillus rhamnosus (Lactobacillus rhamnosus).

The above-mentioned multi-strain Lactobacillus is deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan. The deposit number of Lactobacillus embryos is BCRC 11697, and the deposit number of Lactobacillus casei is BCRC 17942. The deposit number of Lactobacillus deer is BCRC 16091, the deposit number of Lactobacillus brevis is BCRC 11196, and the deposit number of Lactobacillus rhamnosus is BCRC 80663. The aforementioned multi-strain Lactobacillus has been in a divisible state as of the application date. Total number of viable cells of Lactobacillus strains multiple forming units (colony-forming unit, CFU) / milliliter (mL) of at least ¹⁰⁷ colonies.

Then, the bacterial liquid of the multi-strain Lactobacillus is added to the plant substrate to perform the fermentation step, thereby obtaining the plant ferment, wherein the fermentation step is performed at 35°C to 40°C for 36 hours to 60 hours.

According to an embodiment of the present invention, the above-mentioned plant slurry is obtained by homogenizing plant materials and water.

According to an embodiment of the present invention, the cell number ratio of any two of the above-mentioned Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordella, Lactobacillus brevis, and Lactobacillus rhamnosus is 3:1 to 1:3.

According to an embodiment of the present invention, the acid-base value of the above-mentioned plant ferment is less than pH 4.

According to an embodiment of the present invention, the above-mentioned plant ferment contains at least 10 ⁷ colony-forming units (CFU)/ml (mL) of total viable bacteria, among which Lactobacillus embryo, Lactobacillus casei, and Loder's milk The content ratio of any two of bacillus, Lactobacillus brevis and Lactobacillus rhamnosus is 5:3 to 3:5.

According to another aspect of the present invention, a plant is proposed a starter Po, Po fermentation method for producing a plant which was obtained by the system described above, wherein the plant comprises a starter Po total number of viable cells of at least ¹⁰⁷ CFU (colony-forming unit , CFU)/ml (mL) of multi-strain Lactobacillus.

According to an embodiment of the present invention, the above-mentioned plant ferment includes plant ferment liquid and/or plant ferment freeze-dried powder.

According to another aspect of the present invention, a composition containing a plant ferment is provided, which contains the above-mentioned plant ferment as an active ingredient.

According to another aspect of the present invention, a use of a plant ferment for preparing a composition for regulating blood sugar is proposed, wherein the composition comprises the plant ferment as described above.

According to an embodiment of the present invention, when the composition is administered to rats, the effective dose of the plant ferment in the composition is 0.1 g to 2.5 g per kilogram of body weight per day.

According to an embodiment of the present invention, when the composition is administered to an adult, the effective dose of the plant ferment in the composition is 0.015 g to 0.5 g per kilogram of body weight per day.

By applying the plant fermented product, its manufacturing method, the composition containing it, and its application, the multi-strain Lactobacillus bacteria liquid can be added to the plant substrate for fermenting, and the plant fermented product can be obtained. In addition, after administering a composition containing plant ferment to a subject, it can slow down and/or prevent hyperglycemia and/or its complications.

In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the detailed description of the attached drawings is as follows:

[Figure 1] is a line graph showing the plasma glucose concentration according to an embodiment of the present invention.

[Figure 2] is a bar graph showing the area under the broken line of the plasma glucose concentration according to Figure 1.

[Figure 3] is a bar graph showing the degree of insulin resistance according to an embodiment of the present invention.

Based on the above, the present invention provides a method for manufacturing plant ferment. First, a cyanolysis step is performed on the plant slurry to obtain a plant substrate, wherein the cyanolysis step is performed at 70° C. to 100° C. for 30 minutes to 60 minutes. If the temperature of the cyanine-killing step is lower than 70°C or the time is less than 30 minutes, the tissues in the plant slurry cannot be effectively softened, some microorganisms are eliminated, and the air in the plant slurry is eliminated. If the temperature of the cyanine-killing step is higher than 100°C or the time is more than 60 minutes, the nutrients in the plant slurry will be destroyed, resulting in the failure to provide sufficient nutrients during the subsequent fermentation step.

In one embodiment, the above-mentioned plant slurry is obtained by homogenizing plant materials and water. The types and parts of plant materials are not limited, such as but not limited to roots, stems, leaves of at least one of pumpkin, bitter gourd, cucumber, carrot, spinach, apple, guava, papaya, blueberry and/or turmeric, Flowers and/or fruits.

In this embodiment, a bacterial liquid of Lactobacillus sp. is provided for use in the subsequent fermentation step. The multi-strain Lactobacillus includes Lactobacillus plantarum , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus brevis , and Lactobacillus rhamnosus . The aforementioned multi-strain Lactobacillus is deposited at the Biological Resource Center (BCRC), Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit number of Lactobacillus embryo is BCRC 11697, and the deposit number of Lactobacillus casei is BCRC 17942. The deposit number of the aforementioned Lactobacillus lordella is BCRC 16091, the deposit number of the aforementioned Lactobacillus brevis is BCRC 11196, and the deposit number of the aforementioned Lactobacillus rhamnosus is BCRC 80663.

In one embodiment, the total number of viable cells of Lactobacillus strains plurality of at least ¹⁰⁷ CFU (colony-forming unit, CFU) / milliliter (mL).

In one embodiment, the cell number ratio of any two of Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordella, Lactobacillus brevis, and Lactobacillus rhamnosus is 3:1 to 1:3. If the cell number ratio of the above-mentioned multi-strain Lactobacillus falls outside the aforementioned range, the subsequent fermentation step will cause the Lactobacillus to fail to achieve better growth.

Then, the bacterial liquid of the multi-strain Lactobacillus is added to the plant substrate to carry out the fermentation step, thereby obtaining the plant fermentation product. The fermentation step is carried out at 35°C to 40°C for 36 hours to 60 hours. If the temperature of the fermentation step falls outside the aforementioned range, the growth of Lactobacillus is poor. If the time for the fermentation step is less than 36 hours, sufficient fermentation product cannot be obtained. If the fermentation step takes more than 60 hours, it will affect the taste and texture of the plant fermented product.

The present invention further provides a plant Po fermentation thereof, the method for producing a plant Po fermentation product which system described above, the obtained, wherein the plant Po yeast containing the total number of viable cells forming units (colony-forming unit, CFU) of at least ¹⁰⁷ cfu / ml (mL) of the multi-strain Lactobacillus, in which the content ratio of any two of Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordrea, Lactobacillus brevis and Lactobacillus rhamnosus is 5:3 to 3:5.

The acid-base value of the obtained plant ferment is less than pH 4. If the acid-base value is at least pH 4, the total viable cell count in the plant ferment will not be maintained at at least 10 ⁷ CFU/mL.

In one embodiment, the plant ferment can be selected as plant ferment liquid and/or plant ferment freeze-dried powder. Plant fermented liquid refers to the liquid plant fermented product obtained after the fermenting step. The freeze-dried plant fermented powder refers to the anhydrous solid plant fermented freeze-dried powder obtained after freeze-drying the plant fermented liquid.

The present invention also provides a composition of plant ferment, which contains plant ferment as an effective ingredient. The composition may be an oral composition, and the composition of the present invention is administered orally.

The composition can also be a food composition or a pharmaceutical composition. For example, the food composition can be, for example, cereal products, fruit products, vegetable products, meat products, fish products, egg products, milk products, Drinks, health foods, health foods, functional foods, nutritional supplements or special nutritional foods.

The pharmaceutical composition may optionally include a mixture of pharmaceutically acceptable ingredients, and may include, but is not limited to, solvents, emulsifiers, suspending agents, decomposing agents, binding agents, excipients, stabilizers, chelating agents, diluents, Gelling agents, preservatives, lubricants, absorption delaying agents and/or liposomes.

In one embodiment, the dosage form of the above-mentioned food composition and medical composition is not limited to a specific form, and may be liquid and/or solid, and may include, but is not limited to, tablets, granules, powders, capsules, solutions, suspensions, and / Or dispersion Wait.

The present invention further provides a use of a plant ferment for preparing a composition for regulating blood sugar, wherein the composition comprises the plant ferment as described above.

The regulation of blood glucose in the present invention refers to the measurement of fasting blood glucose, serum insulin, glucose tolerance, insulin resistance, and glycosylated Hb. , Plasma fat (blood lipid) and lipoprotein (lipoprotein) and other indicators to determine the metabolic status of blood sugar. When blood sugar cannot be metabolized normally, it may cause hyperglycemia and/or its complications.

When applying the above composition, the amount of use, the type of use, and the number of uses can be adjusted flexibly according to demand. In one embodiment, when the plant ferment contains 10 ⁷ CFU/mL of total viable lactobacillus, the effective dose of the plant ferment of the present invention in the composition that can be administered to rats is 0.1 g per kilogram of body weight per day. To 2.5g, or the effective dose that can be administered to adults of the plant ferment of the present invention in the composition is 0.015g to 0.5g per kilogram of body weight per day (According to the 2005 U.S. Food and Drug Administration announced the early experimental estimation method (Estimating the The maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers) is calculated based on 6.2 times the recommended daily intake per kilogram of body weight (g/kg bw/d) of the human body as 1 times the dose of rats]. It can be used once a day or several times a day.

Several embodiments are used below to illustrate the application of the present invention, but they are not used to limit the present invention. Those with ordinary knowledge in the technical field of the present invention, Various changes and modifications can be made without departing from the spirit and scope of the present invention.

1. Preparation of plant ferment

(1) Preparation of plant fermented liquid

Mix the pre-prepared plant substrate and the multi-strain Lactobacillus liquid, and anaerobic culture at 37°C for 48 hours to obtain the plant fermented liquid. The acid-base value of the plant fermented liquid is pH 3.57±0.07. In addition, the standard plate count method was used to determine that the plant fermented liquid contained (1.8±0.3)×10 ⁸ CFU/mL Lactobacillus. The standard plate counting method is well-known to those with ordinary knowledge in the technical field of the present invention, and will not be repeated here.

The above-mentioned plant substrate is to cut pumpkin, bitter gourd, cucumber, carrot, spinach, apple, guava, papaya, blueberry, and turmeric, then mix it with water in a volume ratio of 1:1, and then kill at 95℃ Jing obtained in 40 minutes.

The above-mentioned multi-species Lactobacillus bacterium liquid contains 1% Lactobacillus embryo (BCRC 11697), 1% Lactobacillus casei (BCRC 17942), 3% Lactobacillus lordrea (BCRC 16091), 1% Lactobacillus brevis ( BCRC 11196) and 1% Lactobacillus rhamnosus (BCRC 80663), which can be obtained from BCRC distribution.

(2) Determine the proportion of Lactobacillus strains in the plant fermented liquid

Use random amplification of polymorphic nucleic acid-polymerization chain reaction (Random Amplified Polymorphic DNA-Polymerase chain reaction, RAPD-PCR) was used to determine the content ratio of various lactobacilli in plant ferment. Use Random primers to perform PCR. After amplifying the fragments of Lactobacillus DNA, the PCR products are analyzed by electrophoresis, and the number of fragments, position differences, length differences, and signal strengths presented by the PCR products are used to distinguish Lactobacilli Strains and relative bacterial count percentage.

The present invention uses primers (such as SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or any combination of the foregoing ] Amplify the partial sequence of the multi-strain Lactobacillus in the plant fermented liquid. The methods for DNA extraction, PCR, and electrophoresis are well known to those with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here.

The results show that the plant ferment contains 25±3% of Lactobacillus embryo, 25±2% of Lactobacillus casei, 15±1% of Lactobacillus lordella, 17±3% of Lactobacillus brevis and 18±2% of Lactobacillus brevis Lactobacillus rhamnosus.

(3) Preparation of freeze-dried plant fermented powder

After the plant fermented liquid is freeze-dried in a vacuum at -40°C and 4pa for 24 hours, the plant fermented freeze-dried powder can be obtained. The moisture content of the freeze-dried plant fermented powder measured by a commercially available infrared moisture meter was 8.83%±0.10%. In addition, a standard plate counting method was used to determine that the plant fermented lyophilized powder contained (2.6±0.6)×10 ⁸ CFU/g of Lactobacillus.

2. The anti-oxidant effect of plant fermented liquid

(1) Determination of total phenolic contents (TPC)

Add 1 mL of 2% sodium carbonate (Na ₂ CO ₃ ) solution to 25 μL of plant fermentation solution, mix well and let stand for 2 minutes in the dark, then add 250 μL of 50% Folin-Ciocalteu's phenol reagent After uniformly mixing, let it stand for 30 minutes in the dark, and measure the absorbance at 750 nm with a commercially available spectrophotometer. Make a calibration curve against the standard galic acid (Galic acid), convert the gallic acid equivalents (GAE) contained in each milliliter of plant fermented liquid, to calculate the total phenolic content of the plant fermented liquid, Among them, the results are expressed using mean±SD (n=3).

The results showed that the total phenol content of the plant fermented liquid was equivalent to the gallic acid equivalent (mg GAE/mL) of 17.47±0.29 mg/mL. (2) DPPH free radical scavenging activity measurement

The DPPH radical is blue-violet in methanol, and has a maximum absorbance at a wavelength of 517nm. When the free radical scavenger is present, the color of DPPH free radical changes from blue-violet to light yellow, and the light absorption value also decreases.

Dilute 50μL of plant fermentation solution with distilled water to 400μL, add 1mL 0.2mM 2,2-Diphenyl-1-picryl-hydrazyl (2,2-Diphenyl-1-picryl-hydrazyl, DPPH) solution (weigh 3.9 mg DPPH is dissolved in 50 mL methanol), mix well and stand for 50 minutes in the dark, and measure the absorbance at 517 nm with a commercially available spectrophotometer. The lower the light absorption value, the stronger the scavenging ability of DPPH free radicals and the anti-oxidation The better the ability.

Comparing L-Ascorbic acid (Vitamin C) standard products to make a standard curve, convert the DPPH free radical scavenging capacity of L-ascorbic acid equivalents (Vitamin C equivalents) per milliliter of plant ferment. The results show that the DPPH free radical scavenging ability of the plant fermented liquid is equivalent to the L-ascorbic acid equivalent of 437.02±14.47μg/mL.

In addition, distilled water was used to replace the plant fermented liquid as a blank group, and the following formula (I) was used to calculate the scavenging rate of DPPH free radicals. The results show that the plant fermented liquid has a scavenging rate of DPPH free radicals of more than 90%.

DPPH free radical scavenging rate (%)=(1-A517nm _sample /A517nm _blank )×100% (I)

(3) Determination of reducing power

By reducing substances, potassium ferricyanide [potassium ferricyanide, K ₃ Fe(CN) ₆ ] can be reduced to potassium ferrocyanide [potassium ferrocyanide, K ₄ Fe(CN) ₆ ], and potassium ferricyanide is then combined with Ferric ions (Fe3 ⁺ ) react to produce ferric ferrocyanide {ferric ferrocyanide, Fe4〔Fe(CN) ₆ 〕 ₃ }, which has a maximum absorbance at a wavelength of 700nm. When the reducing ability of the reducing substance is stronger, the more ferrous ferrocyanide is produced, and the light absorption value also increases.

Dilute 50μL of plant fermentation solution with distilled water to 500μL, add 500μL of 0.2M sodium phosphate buffer solution (pH 6.6) and 500μL of 1% K ₃ Fe(CN) ₆ solution, mix well, and incubate in a water bath at 50°C for 20 minutes. Move to ice and quickly cool for 5 minutes, add 500 μL of 10% TCA solution, mix well, and centrifuge at 3000 rpm for 10 minutes. Take 500μL of supernatant and add 500μL of deionized water and 100μL of 0.1% FeCl ₃ . After mixing the 6H ₂ O solution, let it stand for 10 minutes in the dark. Use a commercially available spectrophotometer to measure the absorbance at 700 nm. The higher the absorbance, the stronger the reducing power of the plant fermented liquid. Comparing L-ascorbic acid standard products to make a standard curve, convert the reducing power of vitamin C equivalents per milliliter of plant ferment liquid, where the result is the mean ± standard deviation (mean ± SD) (n= 3) Representation.

The results showed that the reducing power of the plant fermented liquid was equivalent to 13.76±0.01 mg/mL of L-ascorbic acid equivalent.

(4) Determination of the ability to chelate ferrous ions

Ferrous ion (Fe ²⁺ ) and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4”-disulfonic acid sodium salt [3-( 2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4”-disulfonic acid sodium salt, FerroZine (FerroZine)) complex, the complex has a maximum absorbance at a wavelength of 562nm If the antioxidant is chelated with ferrous ions, the ferrous ions cannot form a complex with FerroZine, and the light absorption value will decrease.

Take 400μL plant Po fermentation broth, and methanol was added 40μL 2mM 1480μL ferrous chloride tetrahydrate (FeCl ₂ .4H ₂ O) solution was mixed uniformly for 30 seconds. Then add 80μL of 5mM FerroZine (FerroZine) solution, mix evenly and let stand for 10 minutes in the dark. Use a commercially available spectrophotometer to measure the absorbance at 562nm. The lower the absorbance, it means that the plant fermented liquid chelates ferrous iron. The stronger the ion capacity.

Comparing ethylene diamine tetraacetic acid (EDTA) standard products to make a standard curve, convert the ferrous ion chelating capacity equivalent to EDTA equivalent per milliliter of plant fermented liquid, and the result is the average value ± standard deviation ( mean±SD)(n=3) means.

The results show that the chelating capacity of ferrous ferrous ions is equivalent to EDTA equivalent of 199.07±16.15μg/mL.

In addition, distilled water was used to replace the plant fermented liquid as a blank group, and the chelating rate of ferrous ions was calculated by the following formula (II). The results show that the plant fermented liquid has a ferrous ion chelating rate equivalent to 30%.

Ferrous ion chelation rate (%)=(1-A562nm _sample /A562nm _blank )×100% (II)

3. Establish an animal model for diabetic rats

(1) Induction of diabetic rats

6-week-old Sprague-Dawley (SD) male rats were raised in a constant temperature of 22±2°C, 12 hours of light and 12 hours of darkness in a circulating light environment, and they were given adequate feed and drinking water. After 1 week of domestication and adaptation, the rats started the experiment after 7 weeks of age. The experiment was conducted in accordance with the guidelines for the care and use of laboratory animals of the Agriculture Committee and the relevant regulations of the Taiwan Animal Protection Law.

The rats were randomly divided into 5 groups, each with 10 rats, and the healthy group (control group, which was fed with physiological saline every day), Example 1 to Example 3 and Comparative example 1. Example 1 to Example 3 and Comparative Example 1 induced diabetes in rats according to the current "Evaluation Method for Blood Glucose Regulation Function of Healthy Food" announced by the Ministry of Health and Welfare of Taiwan, which was administered to rats by intraperitoneal injection of a single dose of 230 mg/kg nicotine Nicotinamide (NA), 15 minutes later, rats were intraperitoneally injected with a single dose of streptozotocin (STZ). The streptozotocin medicament is dissolved in physiological saline, containing 65 mg/kg streptozotocin and 10 mM sodium citrate, pH 4.5. One week after the injection of streptozotocin, blood is collected to measure the blood glucose level on an overnight fasting (17-24 hours after eating). If the blood glucose level reaches or exceeds 13mM (230+10mg%), it is considered to have diabetes. Then, feed 0.1g/kg bw/d plant ferment freeze-dried powder (Example 1), 0.5g/kg bw/d plant ferment freeze-dried powder (Example 2), 2.5g/kg bw per day /d of plant fermented freeze-dried powder (Example 3) or physiological saline (Diabetes control group, Comparative Example 1).

After 4 weeks, the test rats were _{stunned with CO 2} and blood samples were collected by blood sampling from the hepatic portal vein for the following evaluations.

(2) The basic physiological parameters of experimental rats

During the experiment, the feed, drinking water and body weight of the rats were recorded twice a week to observe the changes in the basic physiological parameters of the rats.

Table 1 shows the changes in the amount of water the rats drink during the test period, in milliliter (mL). The data in Table 1 are based on Tukey's multiple analysis method. When the sample is 10 rats per group (n=10), the results are expressed as "mean ± standard deviation" (standard deviation). deviation, SD)" said. In addition, the values of different superscript letters represent statistically significant differences between groups (P<0.05). The same applies to Tables 2 to 7 listed below.

Table 1 shows that the water consumption of the rats of Example 3 decreased significantly from the second week, and the rats of Examples 1 and 2 also decreased significantly from the third week.

Table 1

Table 2 shows the changes in food intake of rats during the test period, in grams (g). Table 2 shows that the food intake of the rats in Example 3 decreased significantly from the second week.

Table 2

Table 3 shows the changes in the body weight of rats during the test period, in grams (g). Table 3 shows that the body weight of the rats in Example 3 increased significantly from the third week.

table 3

4. The effect of plant ferment freeze-dried powder in regulating blood sugar

(1) The effect of plant fermented freeze-dried powder in regulating fasting blood glucose (Fasting blood glucose)

The blood glucose concentration of rats was measured according to the hexokinase assay in the above-mentioned "Methods for the Evaluation of Blood Glucose Regulation Function of Health Foods". Using a commercially available blood glucose machine, glucose is catalyzed by hexokinase to generate glucose-6-phosphate (G-6-P), and G-6-P ^{reacts with NADP+} to generate NADPH. Then by measuring the maximum absorbance of NADPH at a wavelength of 340nm, the blood glucose concentration can be quantified.

Table 4 shows the changes in fasting blood glucose levels of rats during the test period, in milligrams per deciliter (mg/dL). Table 4 shows that compared with Comparative Example 1, the fasting blood glucose values of the rats of Examples 1 to 3 have a significant decrease starting from the 4th week.

Table 4

(2) The effect of plant fermented freeze-dried powder on regulating serum insulin (Serum insulin)

A commercially available rat insulin enzyme immunosorbent assay reagent set (Rat Insulin ELISA, Mercodia, Sweden) was used for insulin content determination. Pipette 10 μL of serum and standard solution into a 96-well micro multi-well plate respectively, and then add 100 μL of 1 times the enzyme conjugate solution and place on a rocking shaker at room temperature for 2 hours. Wash with 1 times wash buffer solution (wash buffer solution) 5 times, and remove residual liquid. Next, add 200μL of tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and react for 15 minutes at room temperature. Add 50μL of Stop Solution and place in a rocking shaker for a few seconds. , So that the reagents in the micro porous plate react uniformly. Next, use an enzyme immunoassay to measure the absorbance at 450nm, and calculate the serum insulin content based on the standard curve.

Table 5 shows the content of insulin in the serum of rats during the 4th week of feeding the freeze-dried plant fermented powder. After the rats in Examples 1 to 3 were fed the freeze-dried plant fermented powder, the insulin content was higher than that in Comparative Example 1. Therefore, the plant fermented freeze-dried powder can promote the increase of insulin secretion. It shows that the freeze-dried plant fermented powder may be beneficial to repair damaged pancreatic β cells.

table 5

(3) The effect of plant ferment freeze-dried powder on the adjustment of oral glucose tolerance test (OGTT)

According to the Glucose Tolerance Test in the above-mentioned "Methods for the Evaluation of Blood Glucose Regulation Function of Healthy Foods", the glucose tolerance of rats after eating was tested. Glucose tolerance was measured when the rat plant fermented freeze-dried powder was continuously fed to the 4th week. After the rats were fasted for 10 hours to 14 hours, the rats were given glucose (1g/kg bw) by tube feeding, and then at 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes and 180 minutes after the sugar administration. Blood was collected to determine blood glucose level.

Please refer to FIG. 1 and FIG. 2 at the same time. FIG. 1 is a line graph of plasma glucose concentration according to an embodiment of the present invention. X axis system time, the unit is minutes. The Y-axis is the plasma glucose concentration in milligrams per deciliter (mg/dL). In Figure 1, the figure number ○ represents the control group, the figure number △ represents the embodiment 1, the figure number ◇ represents the embodiment 2, the figure number □ represents the embodiment 3, and the figure number ▲ represents the comparative example 1. Fig. 2 is a bar graph showing the area under the broken line of the plasma glucose concentration according to Fig. 1. The X axis from left to right is the control group, Examples 1 to 3 and Comparative Example 1. The Y-axis is the area under curve (AUG), the unit is plasma glucose concentration × minutes [(mg/dL) × min].

The results show that, compared with Comparative Example 1, the rats of Examples 1 to 3 can significantly reduce the plasma glucose concentration after being fed the freeze-dried plant fermented powder.

(4) The effect of freeze-dried plant fermented powder in regulating insulin resistance

Insulin Resistance (IR) refers to the decreased sensitivity of cells (especially liver, muscle, and fat cells) to insulin. Blood glucose cannot enter the cells smoothly. In order to compensate for this response, the body makes the pancreas secrete more Insulin causes a high concentration of insulin in the blood.

Use the Homeostasis model assessment method-insulin resistance index (HOMA-IR index) to measure the fasting insulin and fasting blood glucose of rats when the rats were continuously fed the freeze-dried plant fermented powder to the 4th week. Value, and the following formula (III) to calculate the degree of insulin resistance. The higher the value, the higher the degree of insulin resistance.

Insulin resistance level = (fasting insulin (μU/mL) × fasting blood glucose value (mmol/L))/22.5...(III)

Please refer to FIG. 3, which shows a bar graph of insulin resistance according to an embodiment of the present invention. The X axis from left to right is the control group, Examples 1 to 3 and Comparative Example 1. The Y axis is the degree of insulin resistance. In Figure 3, the data uses Tukey's multiple analysis method. When the sample is 10 rats per group (n=10), the results are expressed as mean±standard deviation (SD) . In addition, the values of different superscript letters represent statistically significant differences between groups (P<0.05).

The results showed that the insulin resistance of the rats of Examples 1 to 3 was lower than that of the rats of Comparative Example 1. Therefore, after feeding the plant fermented freeze-dried powder, it can indeed reduce the degree of insulin resistance, and it is possible to restore the cell to the islet Susceptibility.

(5) The effect of plant fermented freeze-dried powder in regulating Glycosylated Hb

After the glucose in the blood enters the red blood cells, it will combine with hemoglobin to form glycosylated hemoglobin, and it is not easy to separate. The longer the condition of high concentration of glucose in the blood lasts, the higher the percentage of glycated hemoglobin.

When the rat plant ferment freeze-dried powder was continuously fed to the 4th week, the rats were detected by using the commercially available Premier Hb9210 Glycated hemoglobin assay (Trinity Biotech, USA) with the Premier Hb 9210 system The percentage of glycosylated hemoglobin.

The results are shown in Table 6. The percentage of glycated hemoglobin in the rats of Examples 1 to 3 is lower than that of Comparative Example 1. It shows that after feeding plant fermented freeze-dried powder, the glycosylated hemoglobin decreases, which means that the glucose concentration in the blood decreases.

Table 6

(6) The effect of plant ferment freeze-dried powder in regulating plasma fat and lipoprotein

Studies have shown that many diabetic patients have symptoms of abnormal plasma fat. Plasma fat is combined with special proteins to form lipoproteins, which can be dissolved in plasma and enter the blood circulation. The aforementioned plasma fats include triglycerides (Triglyceride, TG) and total cholesterol (Total cholesterol, TC). The aforementioned plasma lipoproteins include very low density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), among which VLDL and LDL are easy to Causes arteriosclerosis, which belongs to low-quality cholesterol. HDL can remove cholesterol deposited on blood vessel walls and prevent the progress of arteriosclerosis. It is a high-quality cholesterol.

In this example, when the rat plant fermented freeze-dried powder was continuously fed to the 4th week, the medical institution was commissioned to test triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein. The results are shown in Table 7. Show (unit: mg/dL).

Table 7 shows that after feeding the freeze-dried plant fermented powder, compared with Comparative Example 1, the triglyceride and low-density lipoprotein concentrations of Example 3 are significantly reduced, and the results of Examples 1 to 3 There was a significant reduction in total cholesterol. In addition, compared with Comparative Example 1, the high-density lipoproteins of Examples 1 to 3 have no significant difference.

Therefore, the freeze-dried plant fermented powder can indeed reduce triglycerides, total cholesterol and low-density lipoprotein, which can be beneficial to regulate plasma fat and lipoprotein.

Table 7

In summary, it is demonstrated by the above several examples that the plant ferment has the effect of regulating blood sugar, so it can be applied to the preparation of blood sugar regulating composition, which can slow down and/or prevent hyperglycemia and/or its complications.

Secondly, it can be seen from the above-mentioned embodiments that the plant fermented product of the present invention, its manufacturing method, the composition containing it, and its use have the advantage of adding the multi-strain Lactobacillus broth to the plant matrix for fermenting, and it can be obtained Plant ferment. By administering a composition containing plant ferment, it can slow down and/or prevent hyperglycemia and/or its complications.

It should be added that although the present invention uses a specific process, a specific analytical method or a specific instrument as an example to illustrate the plant ferment and its manufacturing method of the present invention, anyone with ordinary knowledge in the technical field of the present invention can know that the present invention The invention is not limited to this. Without departing from the spirit and scope of the invention, the plant ferment and its manufacturing method of the invention can also be carried out using other processes, other analytical methods or other instruments.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.

<110> Chen Junhong

<120> Plant ferment and its manufacturing method, composition containing it and its use

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Claims (11)

A method for manufacturing plant ferment, including: Performing a cyanine-killing step on a plant slurry to obtain a plant substrate, wherein the cyanine-killing step is performed at 70°C to 100°C for 30 minutes to 60 minutes; A multi-strain Lactobacillus is provided, wherein the multi-strain Lactobacillus includes Lactobacillus plantarum , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus brevis and murine Lactobacillus rhamnosus (Lactobacillus rhamnosus), this multi-strain Lactobacillus is deposited at the Bioresource Center (BCRC), Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan. The deposit number of Lactobacillus rhamnosus is BCRC 11697. The cheese The deposit number of Lactobacillus is BCRC 17942, the deposit number of Lactobacillus lordella is BCRC 16091, the deposit number of Lactobacillus brevis is BCRC 11196, the deposit number of Lactobacillus rhamnosus is BCRC 80663, and the multi-strain a total viable cell count of Lactobacillus forming units (colony-forming unit, CFU) / milliliter (mL) of at least ¹⁰⁷ colonies; and One of the multi-strain Lactobacillus strains is added to the plant substrate to perform a fermentation step, thereby obtaining a plant ferment, wherein the fermentation step is performed at 35° C. to 40° C. for 36 hours to 60 hours. The method for manufacturing a plant fermented product according to claim 1, wherein the plant slurry is made from a plant material and water after a homogenization step Income. The method for producing a plant ferment according to claim 1, wherein a number of cells of any two of the Lactobacillus embryo, the Lactobacillus casei, the Lactobacillus lordrea, the Lactobacillus brevis and the Lactobacillus rhamnosus The ratio is 3:1 to 1:3. The method for manufacturing a plant ferment as described in claim 1, wherein one of the plant ferment has a pH value of less than pH 4. The method of producing a plant Po fermentation was the sum a requested item, wherein the plant Po yeast comprises a total number of viable cells forming units (colony-forming unit, CFU) / milliliter (mL) of at least ¹⁰⁷ colonies, wherein the Lactobacillus A content ratio of any two of the bacterium, the Lactobacillus casei, the Lactobacillus lordella, the Lactobacillus brevis, and the Lactobacillus rhamnosus is 5:3 to 3:5. A plant Po fermentation thereof, lines such as the requested item 1 to a method for producing a plant of the of a Po fermentation product of any 5 is obtained, wherein the plant Po fermentation containing a total number of viable cells of at least ¹⁰⁷ CFU (colony-forming unit , CFU)/ml (mL) of the multi-strain Lactobacillus. The plant fermented product according to claim 6, wherein the plant fermented product comprises a plant fermented liquid and/or a plant fermented freeze-dried powder. A composition containing a plant ferment, which contains the plant ferment as described in any one of claims 6 to 7 as an active ingredient. A use of a plant ferment for preparing a composition for regulating blood sugar, wherein the composition comprises the plant ferment as described in any one of claims 6 to 7. The use of the plant fermented substance in claim 9 for preparing a composition for regulating blood sugar, wherein when the composition is administered to a rat, an effective dose of the plant fermented substance in the composition is given per kilogram of body weight per day 0.1g to 2.5g. The cosmetic for external use according to any one of claim 8 or 9, wherein a dosage form of the cosmetic for external use includes a solution, an emulsion, a suspension, a powder, a gel, a lozenge, a shower ball, One foaming tablet and/or one bath salt. The use of the plant fermented substance in claim 10 for preparing a composition for regulating blood sugar, wherein when the composition is administered to an adult, an effective dose of the plant fermented substance in the composition is 0.015 per kilogram of body weight per day g to 0.5g.

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