TWI636134B - A lactobacillus plantarum, composition, culturing method and use of decrease blood lipids and/or decrease function index - Google Patents

Abstract

The present invention provides a lactobacillus lactis GKM3 having a specific gene sequence, which has Acid, alkali and / or cell adsorption capacity, and can achieve the elimination of body fat, lowering blood lipids, reducing liver enlargement, lowering liver function index, anti-inflammatory, lowering uric acid, improving allergies and / or lowering blood sugar. Accordingly, the present invention provides a new type of Lactobacillus germium, and further develops the medical application of the Lactobacillus germium as an alternative choice in the field for preventing and / or treating related diseases caused by obesity.

Description

Lactobacillus germ, composition, culture method and blood lipid lowering and / or reducing Use of liver function index

The invention relates to a lactobacillus germ, a composition containing the same, a culture method and use thereof; more specifically, it relates to a bacterium lactobacillus germ that contains a pheS gene, a recN gene, and a plastid, and the bacterium Use of fat, lowering blood lipids, reducing hepatomegaly, lowering liver function index, anti-inflammatory, lowering uric acid, improving allergies and / or lowering blood sugar for the prevention and / or treatment of obesity-related diseases.

obesity

With the change of life style, especially the change of eating habits, the obesity rate has increased, making obesity a serious health problem in modern countries. The World Health Organization (WHO) has listed obesity as the most important topic of public health and preventive medicine in the 21st century. According to the WHO definition of obesity, BMI (Body mass index) is used as the standard. BMI greater than 30 kg / m ² is obese, and BMI between 25 and 29.9 kg / m ² is overweight (WHO, 1998).

The main cause of obesity is that the long-term calorie intake is greater than the consumption, which causes fat to accumulate in the body, thus forming obesity. The formation of obesity can be divided into primary and spontaneous. Spontaneous obesity is caused by environmental factors such as diet and exercise; spontaneity is caused by inheritance or disease (Hong, 1992). Cell biology defines obesity as the increase in the number and size of preadipocytes that differentiate into adipocytes in adipose tissue (Furuyashiki et al., 2004). Adipose tissue can secrete a variety of substances, including hormones, growth factors, enzymes, cytokines, complements and matrix proteins, etc., whose main role is to regulate metabolism, reproduction, immunity, blood pressure and angiogenesis (Youn et al., 2004). According to this, obesity can induce a variety of diseases throughout the body.

Hepatomegaly

High oil and fat diets lead to obesity, which leads to the accumulation of excessive fat particles in liver cells. When the fat accumulation exceeds 5% of liver weight, it is fatty liver, which causes symptoms such as hepatomegaly. Common causes of hepatomegaly include viral hepatitis, cirrhosis, toxic and drug-induced hepatitis, congestion, fatty liver, tumors, or leukemia caused by metabolic abnormalities.

Liver function index

The indexes measured with the GOT and GPT of the liver index are Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT), which are the two most common enzymes produced by liver cells. When the liver is inflamed, liver cells will be necrotic, and AST and ALT will enter the blood, causing the liver index GOT and GPT to increase. Therefore, it is often used medically to evaluate the degree of liver inflammation or damage. The reasons for the high liver index may come from different factors, such as viral-induced hepatitis, drug-induced hepatitis, explosive hepatitis, liver suppuration, liver cancer, lower blood pressure, acute myocardial infarction, hyperthyroidism, and fatty liver caused by obesity. one of the reasons.

Blood lipids

Fat in blood is mainly divided into two types, namely cholesterol and triglycerides, and it is further subdivided into chylomicrons (CM), very low density lipoprotein (VLDL), and low density lipoprotein (LDL) according to their ratio to protein ) And high-density lipoprotein (HDL) If the good cholesterol in the blood is too low or the bad cholesterol is too high, it is called hyperlipidemia. The causes of hyperlipidemia can be divided into primary or secondary. Primary hyperlipidemia is inherited from the family, and secondary causes are endocrine diseases such as diabetes and nail. Hypogonadism, or liver and kidney disease, living habits, drugs and other reasons. Hyperlipidemia can cause cholesterol to accumulate in blood vessels, which can lead to atherosclerotic arteriosclerosis, which can lead to poor blood flow, triggering myocardial infarction, stroke, and peripheral arterial blockage.

Ketone body

The ketone body contains three compounds: acetone, acetoacetic acid, and β -hydroxybutyric acid. When the body is hungry, fasting, or certain pathological conditions (such as diabetes), fat metabolism increases, and a large amount of fatty acids are absorbed and oxidized by liver cells. At the same time, in order to maintain a stable blood glucose concentration, gluconeogenesis in the body will also be activated , Which in turn generates ketone bodies.

Uric acid

The purines contained in foods are broken down to produce uric acid. Uric acid is excreted through the kidneys or intestines. When there is too much uric acid produced in the body or the kidney function is impaired, uric acid cannot be smoothly excreted from the body, so that the content of uric acid in the blood is too high, which is called hyperuricemia. In addition, uric acid in the blood accumulates in the joints or tissues, which causes swelling and deformation, that is, gout. There are many causes of hyperuricemia, which are divided into two types: primary and secondary. The primary causes include genetics, improper hyperpurine diet, drugs, alcohol, etc., and ketone bodies produced by lipolysis. It will inhibit uric acid excretion, so obese people may develop symptoms of excessive uric acid, and then evolve into related diseases.

Inflammation and allergies

When biological tissues are stimulated by external virus infection, they will induce allergic reactions, release inflammatory substances, and cause inflammatory reactions. Tumor necrosis factor (TNF-α) is mainly produced by macrophages. It is a cytokine involved in systemic inflammation. It is also a member of cytokines that cause acute immune response. Its role is to regulate the function of immune cells. Endogenous pyrogens can promote fever and cause apoptosis in the body to prevent tumorigenesis and virus replication. The imbalance of TNF-α is considered to be related to many human diseases. Recent studies have also pointed out that diet-induced obesity can cause macrophages, which play an important role in allergic reactions, to accumulate in adipose tissue and cause inflammation of adipose tissue.

When the bronchial tube overreacts to a stimulus, it develops into a chronic chronic inflammatory response in the respiratory tract, which is called asthma. When there is an allergic reaction in the bronchi, excessive secretion of inflammatory substances causes increased secretion of mucus, which in turn obstructs the airway, causes narrowing of the airway, and increases respiratory resistance. As a result, symptoms of dyspnea, dyspnea, wheezing, chest tightness, and cough appear.

blood sugar

Blood glucose is the glucose in the blood, which enters the blood from the small intestine after being digested by food. It is constant and usually maintained at 900 mg / L (5 mmol / L). The normal range is 4-6 mmol / L. Blood sugar rises one to two hours after eating, and goes down to a minimum in the morning. Imbalanced blood glucose concentrations, such as persistently high or low blood sugar, can cause a variety of diseases, the most significant of which is diabetes.

Lactobacillus

Lactobacillus spp. Exists in the general environment and can ferment carbohydrates into lactic acid, so it is mostly used in the manufacture of fermented foods. This bacterium has multiple benefits for human health, and has the effect of helping digestion and improving intestinal health. Even if they are of the same genus Lactobacillus, if they are different species, or even the same species but different strains have different characteristics, they will have different effects on the human body. For example, Lactobacillus plantarum has been studied to improve human health. For example, L. plantarum PH04 has the ability to reduce blood cholesterol; L. plantarum 299V can reduce the colon in IL-10 deficient mice. Symptoms of inflammation; L.plantarum 10hk2 can increase pro-inflammatory mediators, such as IL-1β, IL-6 and TNF-α, and anti-inflammatory interleukin-10 (IL-10), to achieve anti-inflammatory effects; L.plantarum K21 has Reduce blood cholesterol, triglyceride content, and have anti-inflammatory effects.

However, because of the symptoms and diseases caused by obesity as mentioned above, it can be known that obesity comprehensively changes the physiological environment, causing the body to face a variety of different aspects of the disease. Therefore, strains with a single efficacy cannot meet the need for the relief of multiple symptoms caused by obesity, and there is still a need to develop strains with complex efficacy in the field of medicine for prevention and / or treatment to more effectively target obesity-related The condition has improved.

One object of the present invention is to provide a Lactobacillus plantarum, which comprises a pheS gene with a sequence of SEQ ID No: 2 and a recN gene with a sequence of SEQ ID No: 3.

Preferably, the Lactobacillus embryonicus further comprises a plastid having a sequence of SEQ ID No: 1.

Preferably, the lactobacillus germ is deposited at the Institute of Food Industry Development, and its deposit number is BCRC 910787.

Preferably, the Lactobacillus germium is resistant to acids, alkalis and / or cells.

Preferably, the Lactobacillus embryonicus has the effects of eliminating body fat, reducing hepatomegaly, and / or anti-inflammatory.

Preferably, the Lactobacillus embryonicus has the effects of lowering uric acid, improving allergies and / or lowering blood sugar.

Preferably, the Lactobacillus embryonicus has the effects of lowering blood lipid, lowering liver function index, lowering uric acid and / or anti-inflammatory.

Another object of the present invention is to provide a composition comprising the Lactobacillus germium as described above, thereby enhancing the application value of the Lactobacillus germium.

Preferably, the lactobacillus germ in the composition is deposited in the Food Industry Development Research Institute, and its deposit number is BCRC 910787.

Preferably, the composition comprises an additive selected from the group consisting of an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a combination thereof.

Preferably, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

Preferably, the composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule, liquid spray or suppository.

Preferably, the composition has the effects of eliminating body fat, reducing hepatomegaly, and / or anti-inflammatory.

Preferably, the composition has the effects of lowering uric acid, improving allergies and / or lowering blood sugar.

Preferably, the composition has the effects of lowering blood lipids, lowering liver function index, lowering uric acid and / or anti-inflammatory.

In order to achieve the foregoing object, the present invention provides a method for culturing a lactobacillus germula, which comprises culturing a lactobacillus germium deposited under the number BCRC 910787 in a medium, wherein the medium comprises sucrose, yeast extract, or a combination thereof, and the sucrose Or the ratio of the yeast extract is 1 wt% to 10 wt% relative to the total weight of the medium.

Preferably, the lactobacillus germ line in the method is cultured at a rotation speed of 5 to 50 rpm.

Preferably, the Lactobacillus embryonicus in the method is cultured at a temperature of 32 ° C to 42 ° C.

Another object of the present invention is to develop the medicinal use of the lactobacillus germium to enhance the medicinal value of the lactobacillus germium.

One of the purposes of the present invention is to provide an application of Lactobacillus embryonicus, which is used to prepare a pharmaceutical composition for reducing fat mass, liver enlargement, and / or anti-inflammatory.

Preferably, the reduced fat mass used in this application is reduced visceral fat mass, subcutaneous fat mass or total fat mass.

Preferably, the reduced fat mass used in this application is to reduce liver fat mass.

Preferably, the reduction of fat mass in the use is to increase the amount of fat discharged through the fat.

Preferably, reducing hepatomegaly in this use is reducing liver weight.

Preferably, the anti-inflammatory in this application is to reduce the content of the inflammatory substance TNF-α in the liver.

One of the purposes of the present invention is to provide an application of a lactobacillus germium strain, which is used to prepare a pharmaceutical composition for reducing uric acid, improving allergies and / or lowering blood sugar, wherein the registration number of the lactobacillus germium is BCRC 910787.

Preferably, the uric acid lowering in this application is to reduce the uric acid content in the serum.

Preferably, the improvement of allergy in this application is to reduce the resistance of the respiratory tract.

Preferably, the hypoglycemic effect in this application is to reduce the amount of blood glucose increase caused by nutritional supplements.

One of the purposes of the present invention is the use of a lactobacillus germium strain for preparing a pharmaceutical composition for improving blood lipids, reducing liver function index, lowering uric acid, and / or anti-inflammatory. The registration number of the lactobacillus germium is BCRC 910787 .

Preferably, the blood lipid improvement in this application is reducing triglyceride, reducing total cholesterol, increasing high-density lipoprotein cholesterol, or reducing ketone bodies.

Preferably, the improvement of liver function index in this application is to reduce the content of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in serum.

The first figure shows the evolutionary tree of pheS genes of L. Plantarum GKM3 and other strains.

The second figure shows the evolutionary tree of the recN gene of L. Plantarum GKM3 and other strains.

The third figure shows the acid resistance of L.Plantarum GKM3 and other strains.

The fourth figure shows the bile salt tolerance of L. Plantarum GKM3 and other strains.

The fifth figure shows the intestinal adsorption capacity of L. Plantarum GKM3 with other strains.

The sixth graph shows the initial weight, final weight, and weight change of the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given the Lactobacillus GKM3 group (HFD + GKM3).

The seventh figure shows the liver weight of rats fed the Lactobacillus GKM3 group (HFD + GKM3) in the normal diet control group (ND), high-fat diet group (HFD), and high-fat diet group.

Figure 8 shows the normal diet control group (ND), high-fat diet group (HFD), and high-fat diet group of rats given the Lactobacillus GKM3 group (HFD + GKM3), and their different parts (perirenal, parasacral, mesenteric) (Outer peritoneum and groin) relative to the weight of fat in rats.

The ninth figure shows the rats of the normal diet control group (ND), high-fat diet group (HFD) and high-fat diet group given Lactobacillus GKM3 group (HFD + GKM3), different types of adipose tissue (total fat, visceral fat) Tissue and subcutaneous adipose tissue) relative to the body weight of the rat.

The tenth figure shows the rats in the normal diet control group (ND), high-fat diet group (HFD) and high-fat diet group given lactobacillus GKM3 group (HFD + GKM3). Cholesterol content.

The eleventh figure shows the content of liver TNF-α in the control group (ND), high-fat diet group (HFD), and high-fat diet group of rats administered the Lactobacillus GKM3 group (HFD + GKM3).

Figure 12 shows the serum levels of AST and ALT in the control group (ND), high-fat diet group (HFD), and high-fat diet groups of rats administered the lactobacillus GKM3 group (HFD + GKM3).

Figure 13 shows the normal diet control group (ND), high-fat diet group (HFD), and high-fat diet group of rats administered the lactobacillus GKM3 group (HFD + GKM3). The serum triglyceride, cholesterol and High-density lipoprotein cholesterol content.

The fourteenth figure shows the content of ketone bodies in the serum of rats fed the Lactobacillus GKM3 group (HFD + GKM3) in the normal diet control group (ND), high-fat diet group (HFD), and high-fat diet group.

The fifteenth figure shows the serum uric acid content of rats fed the Lactobacillus GKM3 group (HFD + GKM3) in the control group (ND), the high-fat diet group (HFD), and the high-fat diet group.

Figure 16 shows the normal dietary control group (ND), high-fat diet group (HFD), and high-fat diet group of rats administered with Lactobacillus GKM3 group (HFD + GKM3), and their lipid content in dry feces.

Figure 17 shows the airway resistance of rats in the positive control group, the negative control group, the GKM3 low-dose group, and the GKM3 high-dose group under the stimulation of different concentrations of methacholine.

The eighteenth figure shows the blood glucose levels of the mice in the control group and the GKM3 group at the oral glucose tolerance test at 0, 30, 60, and 120 minutes.

The nineteenth figure shows the area under the 120-minute glucose curve of the oral glucose tolerance test in the control and GKM3 mice.

The twentieth graph shows the blood glucose rise values of the control group and the GKM3 group in the oral glucose tolerance test at 0, 30, 60 and 120 minutes.

The twenty-first figure shows the area under the 120-minute glucose rise curve of the oral glucose tolerance test in the control group and GKM3 mice.

"Excluding body fat" as used herein refers to a body that excretes fat from an individual through an excretion system, wherein each body is not restricted to a specific part, such as blood or organs, and the fat is not limited to a specific type , Such as triglycerides or cholesterol.

"Reducing hepatomegaly" as described in this article means that a person's liver weight is reduced. In a preferred embodiment, the weight of the liver is increased due to fat accumulation, which is reduced by the administration of a specific pharmaceutical composition.

As used herein, "anti-inflammatory" refers to alleviating the inflammatory response, specifically to reduce one or more indicators of inflammatory response, such as reducing the amount of cytokines or mediators that cause or enhance the inflammatory response in the serum, reducing the inflammation-related cells' response to hormones Or the secretion of media. In a preferred aspect, it refers to reducing the content of tumor necrosis factor (TNF-α) in the liver.

The term "lower uric acid" as used herein refers to a decrease in uric acid in a body, and specifically refers to a reduction in the amount of uric acid in the blood. In a preferred aspect, it refers to the rise of uric acid in the blood caused by a high-fat diet, which is decreased by the administration of a specific pharmaceutical composition.

As used herein, "allergic improvement" refers to alleviating allergic symptoms, and specifically refers to reducing one or more indicators of an allergic reaction, such as reducing the amount of antibodies in serum, and reducing the secretion of hormones or mediators by allergic reaction-related cells. In a preferred aspect, it refers to reducing airway resistance.

"Lowering blood fat" as used herein refers to reducing the fat in a person's blood. In a preferred aspect, it refers to reducing triglyceride, total cholesterol or high-density lipoprotein cholesterol in the serum.

As used herein, "reducing liver function index" means reducing the levels of aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) in a person's serum. In a better specific form Middle refers to an increase in AST and ALT in the blood caused by a high-fat diet, which is decreased by the administration of a specific pharmaceutical composition.

In the invention, a strain is obtained from the fermentation broth of pickled organic vegetables and fruits, and is named GKM3. GKM3 was sequenced to obtain the full-length gene sequence of GKM3, and it was found that GKM3 contains a plastid. The full-length gene sequence of GKM3 was aligned in the database and identified as Lactobacillus germ. Next, the evolutionary tree of GKM3's pheS and recN sequences was compared with the gene sequences of other strains in the genes or databases of other purchased strains, and the evolutionary relationship between GKM3 and other strains was found from the difference in sequence. The difference is very far, confirming that GKM3 is a novel bacterial lactobacillus species.

After confirming that GKM3 is a novel strain, this strain was deposited with the Institute of Food Industry Development under the name "Lactobacillus plantarum GKM3" on July 14, 2017, and obtained the deposit number BCRC 910787. Confirmation of the survival of the strain was completed on the 26th.

Since GKM3 is a novel strain, the culture conditions of this strain are also studied, and a particularly suitable culture method for GKM3 is provided in the present invention. For selecting the culture medium for culturing the strain, the carbon source and the nitrogen source can be considered. Alternative carbon sources include, for example, glucose, sucrose, lactose, fructose, mannose, sorbitol, glycerol, molasses, or a combination thereof. In a preferred aspect, the carbon source is sucrose. Alternative nitrogen sources include, for example, soy protein, yeast extract, beef extract, casein powder, whey protein powder, fish protein hydrolysate, plant extract protein, or a combination thereof. In a preferred aspect, the nitrogen source is yeast extract. The addition ratio of carbon source or nitrogen source to the total weight of the culture medium will also have an effect on the culture effectiveness, and they are 1 to 10% by weight, respectively. In a preferred aspect, the carbon source or nitrogen source is added in an amount of 3 to 7 wt% relative to the total weight of the medium. In a preferred embodiment, the culture medium comprises sucrose, yeast extract, or a combination thereof, and the addition ratio of the sucrose or the yeast extract is 1 wt% to 10 wt% relative to the total weight of the culture medium.

In addition to the components of the culture medium, the culture conditions and the temperature need to be set. The culture temperature can be set from 32 ° C to 42 ° C. In a preferred aspect, the culture temperature is 35 ° C to 40 ° C, and more preferably 37 ° C. The speed can be set from 5 to 50 rpm. In a preferred aspect, the rotation speed is 10 to 35 rpm, and more preferably 20 rpm.

After GKM3 can be successfully mass-produced under specific conditions, its strain characteristics are studied. It is known that for Lactobacillus to function effectively in animals, it needs to be resistant to the gastric acid or choline secreted by the gastrointestinal tract in the animal's digestive system. . Furthermore, after the lactobacillus successfully survives and reaches the desired position in the digestive tract, it needs to have the ability to adsorb gastrointestinal cells before it can compete for adsorption with harmful pathogens in the gastrointestinal tract. If the adsorption capacity for cells is not enough, it will be eliminated by harmful pathogens with strong adsorption capacity, and cannot stay in the gastrointestinal tract to function effectively. Even if no harmful bacteria have been adsorbed on the cells of the gastrointestinal tract to infect the animal body, if the lactobacillus has a high adsorption capacity, subsequent pathogenic bacteria can be eliminated to prevent the gastrointestinal tract from being adsorbed by the harmful pathogenic bacteria and the gastrointestinal tract infection. Based on this, the tolerance of GKM3 to other bacteria in an acid or bile salt environment was compared, and the ability to adsorb animal cells was compared.

In addition to the basic characteristics described above, the present invention also studies the physiological role of GKM3 in animals to develop the medical uses of GKM3. Obesity rats with high-fat diets were measured for their body weight changes, and their organs, adipose tissue, and blood were taken after the rats were sacrificed to study the effects of GKM3 on obesity caused by high-fat diets in animals. Of various physiological aspects, such as: weight, fat, organs, obesity-related indicators in serum, stool, and even the impact on indicators related to inflammation or allergic reactions. The results of these experiments confirm that GKM3 has a relief effect on the multi-faceted pathological response caused by obesity, and can effectively improve allergic symptoms.

Based on the results of the study, it was found that GKM3 is beneficial for improving the health of animals. GKM3 was applied to medical applications and developed into a composition containing GKM3.

In a preferred embodiment, the composition further comprises an additive. The additive may be an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a group thereof Together. The excipient may be selected from sodium citrate, calcium carbonate, calcium phosphate, or a combination thereof. The preservative can extend the shelf life of pharmaceutical compositions, such as benzyl alcohol, parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerin, or a combination thereof. The bulking agent may be selected from lactose, nougat, high molecular weight glycol, or a combination thereof. The absorption enhancer may be selected from the group consisting of dimethyl sulfoxide (DMSO), laurazolone, propylene glycol, glycerol, polyethylene glycol, or a combination thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose / sucralose, neotame, or a combination thereof. In addition to the additives listed above, under the premise of not affecting the medical effect of GKM3, other suitable additives can be selected according to demand.

In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

In a preferred embodiment, the composition is in the form of a powder, a lozenge, a granule, a suppository, a microcapsule, an ampoule / ampule, a liquid spray or a suppository.

The composition of the present invention can be used for animals or humans. Without affecting the effect of GKM3, it can be made into any drug form and administered to the animal or human in an appropriate way according to the drug form.

The following embodiments are described to further explain the advantages of the present invention, but are not intended to limit the scope of rights of the present invention.

Example 1: Collection and isolation of bacteria

Visit farming households in Yilan, Hualien, Taitung and other places to engage in farming, and observe elderly farmers who are more than 75 years old and can still be self-sufficient and flexible in farming. It is inferred that long-term intake of foods containing lactic acid bacteria means long-term exposure to strains with unique probiotic effects, which helps elderly farmers maintain good physiological functions. Therefore, collect samples that may contain lactic acid bacteria in various places, store the samples in sealed sterile bags, and refrigerate them in a mobile refrigerator and return them to the laboratory to isolate and purify the benefits contained in the samples. Raw strain. First, prepare a medium containing different nutrient sources to screen different types of lactic acid strains in the sample. The components and types of the medium include: MRS agar, PCA agar, M17 agar, Rogosa agar, TOS-MUP agar, and MRS agar. 3 % NaCl or MRS agar was added with 0.05% bromocresol green. Next, the collected strains were cultured at 37 ° C. for 48 hours in different types of medium, and then about 5 to 10 single colonies with different appearances of colonies were selected from plates of each medium. Each of these single colonies was cultured in MRS medium at 37 ° C for 16 hours, and then frozen in sterile glycerol and stored at -80 ° C. Subsequent tests for acid resistance, choline resistance and intestinal adsorption of these bacteria were performed. , Or other animal experiments to test physiological activity.

In the end, it was found that from the samples collected from the mountainous area of Jiaoxi Township, Yilan, the fermentation broth obtained by pickling organically cultivated vegetables and fruits, the isolated strain had excellent acid resistance, bile salt resistance, and intestinal adsorption, and was specifically named GKM3.

Example 2: Gene analysis and identification of strains

First, the GKM3 DNA was collected for sequencing, and the strains were identified by gene comparison. GKM3 was streaked and activated, and a single colony was picked with an inoculation loop and inoculated into the culture medium to enlarge. The fresh bacteria were collected every other day and the gDNA of the strain was extracted by CTAB method. The gDNA quality was confirmed by 0.6% agar colloid electrophoresis. After concentration, the whole-genome analysis of the strain was performed using the third-generation sequencing technology platform PacBio RSII. According to the results of the whole genome analysis, the GKM3 whole genome is 3,076,381 bp in length and contains a 49,415 bp plastid (SEQ ID NO: 1). The whole genome sequence of GKM3 was compared in the whole genome database, and the pheS (SEQ ID NO: 2) and recN (SEQ ID NO: 3) gene sequences of GKM3 were obtained. Next, the pheS gene (SEQ ID NO: 2) sequence of GKM3 was aligned in the NCBI database. The results showed that the pheS gene of GKM3 had the highest genetic similarity with Lactobacillus plantarum, and it was found that the strain type of GKM3 was Lactobacillus germ.

Furthermore, the specific gene fragments of GKM3 and other strains were compared to study the uniqueness of GKM3 in evolution. Three types of Lactobacillus embryos were randomly selected and purchased from the Center for Bioresources Conservation and Research of the Food Industry Development Institute, and their serial numbers were BCRC 12251, BCRC80222, and BCRC80578. After gDNA was extracted by strain activation amplification, the primer pairs shown in Table 1 were used, followed by 35 cycles of reaction at 94 ° C for 3 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 1 minute and 30 seconds. Finally, a polymerase chain reaction was performed under the conditions of reaction at 72 ° C for 5 minutes.

After the reaction, samples were sent for sequencing, and the pheS and recN gene sequences of 3 strains were obtained.

In addition, in addition to the pheS and recN gene sequences of the three strains purchased above, the pheS and recN sequences of the following five strains were downloaded from the NCBI database, which are lactic acid bacterial strains LZ95 (CP012122) and B21 (CP010528) , JDM1 (CP001617), WCFS1 (AL935263), and Lactobacillus paraplantarum L-ZS9 (CP013130).

Aiming at the pheS and recN sequences of the GKM3 of the present invention, 3 types of lactobacillus purchased, and 5 types of strains downloaded from the database, the evolution tree was drawn in Neighbor-Joining mode of Phylogeny's MEGA Bootatrap Test. The alignment results of the pheS sequence are shown in the first figure. The recN sequence is shown in the second figure. According to the results of the first graph and the second graph, Lactobacillus paraplantarum L-ZS9 (CP013130) was used as an out group to compare the similarity of other strains. It was compared with GKM3 and 7 other strains. The distances in the evolutionary tree are farthest apart. In the first figure, GKM3 located at the top is closest to B21 (CP010528) obtained from the database, indicating that the alignment of the two in the pheS gene sequence is the closest in the evolutionary relationship. In the second figure, GKM3 is also located at the top. The strains that are close to it are LZ95 (CP012122) obtained from the database and BCRC 12251 purchased. It is known from the alignment of recN gene sequence that GKM3 is compared with these two strains Closer to other strains. Based on this, a comprehensive comparison of the evolutionary tree obtained from the pheS and recN gene sequences shows that the position of GKM3 in the evolutionary tree can be distinguished from other strains and form a self-aligned line, indicating that GKM3 is different from the existing lactobacillus strains and is a novel lactobacillus Strains.

Example 3: Bacterial Culture

The effect of different culture conditions on the yield of GKM3 was studied. The carbon and nitrogen sources in the fermentation medium for GKM3 were tested using a one-factor test at a time.

Start with the demand for carbon sources and compare the 8 carbon sources: glucose, sucrose, lactose, fructose, mannose, sorbitol, glycerol and molasses under the conditions of GKM3 colony count and IFN-γ for peripheral leukocyte lymphocytes Induced ability to select a carbon source with high yield and physiological activity for GKM3. The MRS medium was added with the above 8 kinds of carbon sources at 3% by weight relative to the total weight of the medium, and the culture temperature was set at about 37 ° C and the rotation speed was about 20 rpm. The best 4 carbon sources and the number of viable colonies formed in the experimental results are shown in Table 2 below. The results showed that the highest number of colonies formed by sucrose culture was 4.2x10 ⁹ cfu / mL, compared with 3.1x10 ⁹ cfu in the control group. There was a significant increase in / mL, indicating that the yield of GKM3 cultured by sucrose was the highest. The number of strains cultured with the above-mentioned four kinds of good-quality carbon sources was adjusted to 1 × 10 ⁹ cfu / mL, and a test of IFN-γ induction efficacy of peripheral leukocyte lymphocytes was performed. The results are shown in Table 2 below. The results showed that GKM3 cultured with sucrose increased the activity of IFN-r by up to 553 pg / mL, compared with 402 pg / mL of the control group, an increase of 32%, indicating that the physiological activity of GKM3 cultured with sucrose was the highest. . Based on the results of tests that simultaneously considered high yield and physiological activity, it was evaluated that sucrose as the carbon source was the most suitable for fermentation culture of GKM3.

Next, using sucrose as a fixed carbon source, the effects of different nitrogen sources on GKM3 probiotics were tested. The seven nitrogen sources were selected as follows: soybean protein, yeast extract, beef extract, casein powder, whey protein powder, and fish protein hydrolysis. And plant-derived protein. Comparing the observed concentration (OD600) with the count of bacteria after culture, it was found that yeast extract had the best effect on promoting the growth of GKM3. According to this, sucrose and yeast extract were used as the medium component, and the yield was compared with the commercial formula MRS liquid medium. It was found that the newly developed medium formula in the GKM3 fermentation test, the number of viable colony formation reached 4.6 × ^{10 9} colony formation Units / ml (cfu / mL), a 48% increase compared to MRS medium. It is proved that the newly formulated fermentation medium can effectively increase the yield of GKM3 probiotics. GKM3 can be produced in large quantities, increasing the feasibility of commercial application of GKM3.

Example 4: Acid resistance test

Compare the acid resistance of GKM3 to other strains. Four strains of GKM3, BCRC 12251, BCRC 80222, and BCRC 80578 purchased from the Center for Bioresources Preservation and Research of the Food Industry Development Institute were activated. The pH value of the original MRS liquid medium is about pH 6.5. By adding HCl to the MRS liquid medium, the pH of the medium was adjusted to three different pHs: about pH 3.2, pH 2.4 and pH 2.0. The strains were inoculated in the media with different pH values and cultured at 37 ° C for 3 hours, and then the number of colony formation was counted.

The results are shown in the third figure. Under the original pH culture (approximately pH 6.5), the number of bacteria of GKM3 and the other three strains can reach the power of ten. When the pH value was pH 3.2, the bacterial count of all strains decreased slightly, and GKM3 showed no significant difference compared with the other three strains. When the pH value dropped to pH 2.4 and pH 2.0, the bacterial counts of BCRC 12251, BCRC 80222 and BCRC 80578 dropped sharply to about 6th power, which were significantly lower than GKM3 (P <0.05). . According to this, it is learned that the number of viable bacteria of GKM3 is significantly more than that of other strains in an acidic environment, indicating that GKM3 has better acid resistance and better resistance to gastric acid when passing through the stomach.

Example 5: Bile salt resistance test

Compare GKM3's bile salt tolerance to other strains. Four strains of GKM3, BCRC 12251, BCRC 80222, and BCRC 80578 purchased from the Center for Bioresources Preservation and Research of the Food Industry Development Institute were activated. These bacteria were inoculated in MRS liquid medium containing 0.3% bile salt, and after soaking at 37 ° C for half an hour, the number of bacteria was observed and counted.

The results are shown in the fourth figure. In the original MRS liquid culture medium, the unit bacteria number of GKM3 and BCRC 12251, BCRC 80222 and BCRC 80578 are close to 9 x109. In the MRS supplemented with 0.3% bile salt, the bacterial counts of BCRC 12251 and BCRC 80222 were significantly lower than those of GKM3 (P <0.05), while the bacterial count of BCRC 80578 was not statistically significantly different from GKM3. Based on this, it was learned that under the bile salt environment, the number of viable bacteria of GKM3 was significantly more than that of other strains, indicating that GKM3 has better acid resistance and better resistance to bile salts when passing through the digestive tract of the body.

Example 6: Intestinal adsorption test

Compare the intestinal adsorption capacity of GKM3 relative to other strains. Four strains of GKM3, BCRC 12251, BCRC 80222, and BCRC 80578 purchased from the Center for Biological Resources Conservation and Research of the Food Industry Development Institute were activated, and the initial number of bacteria was adjusted to 10 to the power of 7 and added to the cultured HT- 29 cells. The HT-29 cell line is a colon cancer cell cultured in DMEM (Sigma) medium containing 10% fetal bovine serum (HyClone ^™ ), and added with 1 mM sodium pyruvate (sigma), 1% non-essential amino acid (NEAA ) And 1% antibiotics (100 units / mL penicillin and 100 μg / mL streptomycin), and cultured in a 37 ° C, 5% CO2 incubator for about 2 to 3 days. The HT-29 cells to which the strain was added were cultured at 37 ° C for 2 hours, and the non-adsorbed cells were washed with PBS. The HT-29 cells were lysed with 1% Triton X-100 to release the cells adsorbed on the cell surface. And calculate the number of bacteria.

As shown in the fifth figure, the number of bacteria adsorbed on HT-29 cells by BCRC 12251, BCRC 80222, and BCRC 80578 was significantly lower than the number of bacteria adsorbed by GKM3 (P <0.05). Based on this, it can be known that GKM3 is superior to other bacteria in the intestinal adsorption capacity and can effectively stay in the intestine for a long time in order to exert benefits.

Example 7: Animal Experiment I

The test animals were ordered by BioLASCO Taiwan Co., Ltd., Taiwan from 18 male Wistar rats that were 6 weeks old and weighed between 201-225 g. The animals were kept in stainless steel squirrel cages, and the temperature of the animal room was controlled at 22 ± 2 ° C, humidity was controlled at 60-80%, and light and darkness were twelve hours each (07: 00-19: 00 for the light period; 19: 00- 07:00 is the dark period). This experimental procedure was approved by Chung Shan Medical University Animal Care Committee (IACUC AHPP-Groval NO: 1199). During the experiment, rats were given free access to feed and distilled water.

After the test animals were fed with normal adult rat feed and drinking distilled water to adapt to the environment for one week, they were randomly divided into three groups of 6 each, as follows:

1. Normal diet (ND)

2.High-fat diet (HFD)

The feed formula is based on AIN93G, and the fat ratio is adjusted. The feed contained 68% solids, 7% soybean oil and 25% lard.

3. GKM3 (HFD + GKM3) was given in the high-fat diet group

GKM3 was orally administered at a dose of 102.8 mg / kg rat / day for 6 weeks, except for the same feed formula as the high-fat diet group.

Prior to the start of the experiment, the initial body weight of the rats was recorded, and then the weight was finely scaled every two days. Rat feces were collected on the third day before the sacrifice and fasted 12 hours before the end of the test. After using carbon dioxide (CO2) to sacrifice, record its final body weight and calculate the weight change. Blood was collected from rat veins. The blood was collected in a serum separation tube (BD Vacutainer, Plymouth, UK), centrifuged at 4000 rpm for 10 minutes to remove the serum, aliquoted into a microcentrifuge tube and stored in a -80 ° C refrigerator for analysis. use. Remove organ tissues (heart, lungs, liver, spleen, and kidneys) and adipose tissues (perirenal fat, parasacral fat, mesenteric fat, groin fat, and extraperitoneal fat). Wash and wipe with physiological saline, and record each organ. And the weight of adipose tissue. After weighing, it is covered with aluminum foil paper, frozen with liquid nitrogen, and stored in -80 ° C refrigerator for experimental analysis.

The obtained experimental data were analyzed using SPSS computer statistical software. The analysis of variance was analyzed by PROC ANOVA and Duncan ’s multiple range test. P <0.05 was considered significant difference.

Weight change

From the starting weight and the final weight, the amount of weight change was calculated by the following formula 1.

Formula 1: Amount of weight change (g) = [final weight (g)-initial weight (g)]

The initial weight, final weight, and weight changes of GKM3 (HFD + GKM3) given to the normal diet group (ND), high-fat diet group (HFD), and high-fat diet group are summarized in Table 3:

As shown in Table 3 according to Table 3, there was no significant difference in starting weight between the groups (p> 0.05). In the high-fat diet group, the final weight was significantly higher than in the normal diet control group (p <0.05). Compared with the high-fat diet group, the obese rats induced by the high-fat diet and given GKM3 had their final weight. Significant reduction (p <0.05). The same trend can also be seen from the change in body weight. The group fed GKM3 has a smaller change in body weight than the group without feeding, showing that GKM3 can reduce the weight gain caused by a high-fat diet.

2. Organ weight

The weights of organs in each group: heart, liver, spleen, lung and kidney are shown in Table 4 below.

According to Table 4, rat organ weights did not change significantly in heart, spleen, lung and kidney. For the liver, the weight of the high-fat diet group was higher than that of the normal diet group, and the group fed GKM3 significantly decreased compared with the high-fat diet group (p> 0.05). The results are plotted as the seventh figure. According to this, taking GKM3 can effectively reduce the weight of the liver and slow down the hepatomegaly caused by a high-fat diet.

3. Fat weight

Divide the weight (mg) of each fat by the final body weight (g / rat), calculate the fat mass of each part, aggregate it into Table 5 below, and draw it as shown in the eighth figure.

From this result, it can be seen that compared with the high-fat diet group, each fat weight of the group fed with GKM3 decreased significantly. This result indicates that GKM3 can still effectively reduce the accumulation of various body fats in a high-fat diet.

If the removed adipose tissue is divided into two types: visceral fat and subcutaneous fat, perirenal fat, para-fat fat, and mesenteric fat are visceral fat, and groin fat and extraperitoneal fat are classified as subcutaneous fat. General as the total fat mass. The amount of visceral fat and subcutaneous fat is calculated by the following formulas 2 and 3, the total fat amount is calculated by the following formula 4, and the calculation results are aggregated as shown in Table 6 below, and plotted as the ninth figure.

Formula 2: visceral fat mass (mg / g rat) = [peri-renal fat mass (mg) + para-fat fat mass (mg) + mesenteric fat mass (mg)] ÷ final body weight (g)

Formula 3: Subcutaneous fat mass (mg / g rat) = [inguinal fat (mg) + extraperitoneal fat mass (mg)] ÷ final weight (g)

Formula 4: Total fat mass (mg / g rat) = [peri-renal fat mass (mg) + parasacral fat mass (mg) + mesenteric fat mass (mg) + inguinal fat (mg) + extraperitoneal fat mass (mg) ] ÷ Final weight (g)

According to the ninth figure, the weight of visceral adipose tissue in obese rats induced by a high-fat diet was significantly higher than that in the normal diet group (p <0.05). The weight of visceral fat and subcutaneous adipose tissue in the high-fat diet combined with GKM3 group was significantly lower than that in the high-fat diet group (p <0.05). This result indicates that GKM3 effectively reduces the accumulation of various fats caused by a high-fat diet regardless of visceral fat or subcutaneous fat.

4. Liver lipid content

Lipid analysis was performed on rat liver. The cut liver tissue was weighed, and after adding an extraction solvent (chloroform: methanol ratio of 2: 1 (v / v)), it was ground with a homogenizer. The ground liver tissue was filtered into a covered test tube, and the extract solution was quantified to 10 mL. Then, 2 mL of 0.05% calcium chloride (w / v) was added, and the mixture was centrifuged at 3500 × g and 4 ° C. for 3 minutes to remove the supernatant. Next, the ratio of chloroform: methanol: water (3:48:47 (v / v / v)) was quantified to 12 mL, and the mixture was centrifuged at 3500 × g and 4 ° C. for 3 minutes to remove the supernatant. Then, first add methanol to quantify to 10mL, and then use extraction solvent (chloroform: methanol 2: 1 (v / v) ratio) to quantify to 25mL, then collect the sample In the bottle, store at -20 ° C for analysis experiment samples. The total lipids, triglycerides, and cholesterol contained in this liver analysis sample were analyzed using a commercially available reagent group.

Experimental results of rat liver total lipid, triglyceride and cholesterol contents The experimental results are shown in Table 7 below, which is plotted on the tenth figure.

According to the results of this experiment, it was found that in the content of total lipids and triglycerides, the group given the high-fat diet alone was significantly higher than the normal diet control group (p <0.05). In terms of total lipids, triglycerides, and cholesterol, the high-fat diet combined with GKM3 was significantly lower than the high-fat diet alone (p <0.05). This result indicates that GKM3 effectively reduces lipid accumulation in the liver caused by a high-fat diet.

5. Liver inflammation substance content

Enzyme-Linked ImmunoSorbent Assay was used to determine the TNF- α concentration in the liver. Ice-cold saline was used to wash away excess blood from the liver tissue. The liver tissue was placed on a funnel and the saline solution was dripped and weighed. Next, an appropriate amount of a buffer solution (0.32M sucrose, 1 mM EDTA, 10 mM Tris-HCl) with a pH value of 7.4 was added, and then the liver tissue was homogenized with a homogenate (homogeneate) of 10%. The homogenate was further centrifuged at 13,600 × g for 30 minutes at a high speed, 50 μL of the supernatant was taken, detected by a TNF- α ELISA kit, and the absorbance (A) was measured at 450 nm with a spectrophotometer.

The experimental results of TNF- α concentration in rat liver are shown in Figure 11. As shown in the results, the concentration of TNF- α in the liver of rats in the high-fat diet group was significantly higher than that in the normal diet group. Compared with rats in the high-fat diet group, the concentrations of TNF- α in the liver of the obese rats induced by the high-fat diet combined with GKM3 significantly decreased. The results show that taking GKM3 reduces the concentration of TNF- α in the liver, and GKM3 has the effect of reducing the inflammatory response in the body.

6. Liver index

Serum obtained from rat venous blood was separated by centrifugation, and Aspartate Aminotransferase (AST) and Alanine Aminotransferase (Alanine Aminotransferase) were measured using a commercially available analysis kit (Diasys Co Ltd., Holzheim, Germany). , ALT) concentration, the experimental results are shown in Table 8 below, which is plotted as the twelfth figure.

The results showed that the serum levels of ALT and AST in the high-fat diet group were significantly higher than those in the normal diet control group (p <0.05), indicating that the degree of liver damage was higher. In contrast, the group of GKM3 combined with high-fat diet-induced obese rats significantly reduced serum ALT and AST concentrations compared to the high-fat diet group (p <0.05). This result shows that taking GKM3 can reduce the concentrations of ALT and AST in serum, and GKM3 effectively improves liver function.

7. Serum lipid content

Serum obtained from rat venous blood by centrifugation. Triglyderide, total cholesterol, and high-density lipoprotein cholesterol in serum were measured using a commercially available analysis kit (Diasys Co Ltd., Holzheim, Germany). (HDL-cholesterol) concentration, the experimental results are shown in Table 9 below, and are plotted in order as the thirteenth figure.

The results showed that the triglyceride in the serum of the high-fat diet group was significantly higher than that of the normal diet group (p <0.05). Compared with the high-fat diet group, the triglyceride content of the obese rats induced by the high-fat diet combined with GKM3 was significantly reduced.

The total cholesterol content of obese rats on a high-fat diet is about 148.35 (mg / dL). After taking GKM3, the total cholesterol drops to about 90.55 (mg / dL), showing that taking GKM3 can also reduce the total cholesterol in the serum of a high-fat diet. concentration.

High-density lipoprotein cholesterol in the serum of rats in the high-fat diet group was significantly lower than that in the normal diet group (p <0.05). Compared with the high-fat diet group, the high-fat diet-induced obese rats combined with the GKM3 administration group had a slightly higher HDL cholesterol content. This result indicates that taking GKM3 can increase the concentration of high density lipoprotein cholesterol in serum.

8. Serum ketone bodies

Serum obtained from rat venous blood by centrifugation, and ketone bodies were measured using a commercially available analysis kit (Denka Seiken Co Ltd., Taipei City, Taiwan). The experimental results are shown in Table 10 below and plotted as the fourteenth figure.

The results showed that the serum ketone body content of rats in the high-fat diet group was significantly higher than that in the normal diet group (p <0.05). The group of obese rats induced by high-fat diet combined with GKM3 significantly reduced the increase of serum ketone body concentration induced by high-fat diet (p <0.05) and decreased The ketone body content of obese rats induced by high-fat diet was about 27.8%. This result shows that taking GKM3 reduces the ketone body concentration in the serum of a high-fat diet.

9. Serum uric acid

Serum obtained from rat venous blood by centrifugation, and uric acid was measured using a commercially available analysis kit (Diasys Co Ltd., Holzheim, Germany). The experimental results are shown in Table 11 below and plotted as the fifteenth figure.

The experimental results showed that the serum of rats in the high-fat diet group had significantly higher uric acid content than the normal diet group. Compared with the high-fat diet group, the group with high-fat diet combined with GKM3 significantly reduced the serum uric acid concentration (p <0.05) to the level of the normal diet group. This result indicates that administration of GKM3 is effective for reducing uric acid in serum.

10. Fecal fat excretion

Rat feces collected on the third day before the sacrifice were used to determine the total lipid content. Dry feces were collected by weighing and ground, and an extraction solvent (chloroform: methanol ratio of 2: 1 (v / v)) was added, followed by shaking and filtering with filter paper. Next, add 0.05% calcium chloride to the filtrate, mix and centrifuge at 3500 xg, 4 ° C for 5 minutes, remove the supernatant, quantify to 25mL with the extraction solvent, and store at -20 ° C for subsequent analysis experiments sample. Take 10 μL of this fecal lipid extract as a sample, dry it with a reduced pressure concentrator, and then add a reagent from a commercially available reagent group. After 1000 μL of mixing, water bath at 37 ° C for 5 minutes. Next, the absorbance at a wavelength of 500 nm is measured with a spectrophotometer, and the standard solution absorbance is converted to obtain the fecal lipid concentration. The experimental results are shown in Table 12 below and plotted as the sixteenth figure.

The results showed that the total fecal lipids in the high-fat diet group were significantly higher than those in the normal diet group (p <0.05). However, in the high-fat diet combined with GKM3, the excretion of lipids in feces was significantly increased compared with the high-fat diet group (p <0.05). This result indicates that the administration of GKM3 to obese rats caused by a high-fat diet has the effect of increasing lipid excretion by feces.

Example 8: Animal Experiment II

A total of 36 BALB / c male mice were raised from 4 to 5 weeks. After breeding for a week, they began to experiment. First randomly divided into the following three groups, 12 in each group.

1. Positive control group: no sensitization, feeding with 0.9% physiological salt water, and no feeding with GKM3.

2. Negative control group: sensitized, fed with 0.9% physiological salt water, and without GKM3.

3. GKM3 group: sensitized, fed 0.9% physiological salt water and GKM3.

According to the above groups, physiological saline and 0.2ml of GKM3 were continuously fed for four weeks, and allergy was induced by subcutaneous injection of OVA antigen in the third and fourth weeks, and sensitization with OVA antigen in the nasal cavity and trachea in the fifth week, two days later Perform airway resistance tests in a non-invasive manner.

Respiratory resistance test: Whole-body plethysmogragh (WBP) produced by Buxco was used to measure the non-invasive resistance change in the respiratory tract of mice. The resistance change was based on the computer measured value during the breathing process of the mouse to obtain a lung. Function indicator Penh (enhanced pause) value, the higher the value, the greater the airway resistance. The mice were first placed in an animal cabin and the basal Penh value of the mice was measured for three minutes. The mice were then exposed to aerosolized 0.9% NaCl for three minutes, and the Penh value was measured for three minutes. The mice were then exposed to a nebulized non-specific tracheal contraction stimulant, methacholine (3.125 mg / ml) for three minutes, and the Penh value was measured for three minutes to gradually increase the concentration of acetamidine Base (methacholine), the concentrations are 6.25mg / ml, 12.5mg / ml, 25mg / ml, 50mg / ml, repeat the above steps, and use Penh to represent all the obtained values and average them. The experimental results are shown in Figure 17.

Judging from the results, the positive control group had the lowest airway resistance under the stimulation of methacholine because of no sensitization, while the airway resistance of the other two groups increased significantly, indicating that the sensitization was indeed successful. Compared to mice that were not fed with GKM3 after sensitization (negative control group), the group fed with GKM3 after sensitization was stimulated by a progressive concentration of the respiratory stimulant methacholine at various concentrations of B Methacholine has a significantly lower stimulation response (p <0.05). This result indicates that GKM3 has the effect of improving respiratory pressure caused by allergens.

Example 8: Animal Experiment III

Twenty male C57BL / 6JNarl mice were purchased from the Experimental Animal Center of the National Experimental Research Institute. Mice were randomly divided into groups for one week observation period, and 5 mice were raised in each box. The litter is made of wood shavings (Aspen) for experimental animals from NEPCO (Warrensburg, NY, USA). and Shredded Aspen Shavings), use after sterilization, and change twice a week. The feeding method is arbitrary food and drink. The breeding method was carried out according to the conventional experimental animal management method. The environment was set to a temperature of 23 ± 2 ° C, a relative humidity of 50 ± 10%, and 12 hours of light / dark alternately. This experimental design was approved by the Animal Experiment Management Group of the Food Industry Development Institute (Dongguanzi No. 106-10).

The twenty mice purchased were divided into two groups, the control group and the GKM3 experimental group, with 10 mice in each group. The GKM3 experimental group was fed with a continuous dose of 500 mg / kg mouse dose and a volume of 0.1 mL / 10 g body weight / day for fourteen days, while the control group was fed with water. Body weight, feed intake and water intake were measured once a week during the feeding period. Fourteen days later, an oral glucose tolerance test was performed. After fasting the mice for 16 hours, blood samples were collected from the tails, and fasting (0 minutes) blood glucose values were measured using a Glucosure II blood glucose meter (ApexBio Inc., Taiwan) with a blood glucose test strip (glucose oxidase method). Next, each mouse was fed with a glucose solution of 2 g / kg, and blood glucose concentrations were measured at 30, 60, and 120 minutes, respectively. After the oral glucose tolerance test was completed, the mice were sacrificed and their liver, kidney, and spleen were weighed.

The experimental results are expressed by Mean ± SEM, and the experimental results of the GKM3 experimental group and the control group are compared by Student's t-test. * And ** indicate p <0.05 and p <0.01 with the water control group, respectively. The results of changes in mouse weight and overall weight gain are shown in Table 13 below; average daily food intake and water consumption are shown in Table 14 below; mouse organ weights are shown in Table 15 below; mice were tested for oral glucose tolerance The blood glucose values are shown in the following table 16 and plotted as the eighteenth and nineteenth charts; the blood glucose rise values of the oral glucose tolerance test are shown in the following table 17 and plotted as the twentieth and twenty-first charts ;.

Table 13: Changes in weight and weight gain

From the above Tables 13-15, it is known that the body weight and weight gain of the GKM3 group ((grams and percentages), water intake and feed intake, organ weight and relative weight ratio after 14 days of tube feeding, compared with the control group There was no statistically significant difference ((p> 0.05).

From Tables 16 and 17 above, in the oral glucose tolerance test, the 30-minute blood glucose value of the GKM3 experimental group was significantly lower than that of the control group, and the 120-minute area under curve (AUC120min) of the GKM3 group was significantly lower. It was also slightly lower than the water control group (p <0.05). Regarding blood glucose increments, the 30-minute blood glucose increase (i30min) and the area under the 120-minute blood glucose increase curve (iAUC 120min) in the experimental group were significantly lower than those in the water control group. Taken together, it is shown that GKM3 has the potential to improve glucose sensitivity at a dose of 500 mg / kg.

[Biological Material Storage]

Food Industry Development Institute; July 14, 2017; Deposit Number BCRC 910787

<110> Grape King Biotech Co., Ltd.

<120> A lactobacillus germ, composition, culture method and blood lipid lowering, liver function index lowering, uric acid lowering and / or Inflammatory uses

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 49415

<212> DNA

<213> Lactobacillus plantarum strain GKM3

<400> 1

<210> 2

<211> 1047

<212> DNA

<213> Lactobacillus plantarum strain GKM3

<400> 2

<210> 3

<211> 1695

<212> DNA

<213> Lactobacillus plantarum strain GKM3

<400> 3

<210> 4

<211> 25

<212> DNA

<213> Artificail primer

<400> 4

<210> 5

<211> 27

<212> DNA

<213> Artificail primer

<400> 5

<210> 6

<211> 29

<212> DNA

<213> Artificail primer

<400> 6

<210> 7

<211> 23

<212> DNA

<213> Artificail primer

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Claims (8)

A Lactobacillus germium strain is used for preparing a pharmaceutical composition for improving blood lipid and / or reducing liver function index. The registration number of the Lactobacillus germium is BCRC 910787. The use according to claim 1, wherein the aforementioned Lactobacillus embryonicus has acid resistance, alkali resistance and / or cell adsorption capacity. The use according to claim 1, wherein the aforementioned pharmaceutical composition comprises an additive selected from the group consisting of an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a combination thereof. The use according to claim 1, wherein the aforementioned pharmaceutical composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food. The use according to claim 1, wherein the pharmaceutical composition is in the form of a powder, a tablet, a granule, a suppository, a microcapsule, an ampoule, a liquid spray or a suppository. The use according to claim 1, wherein the aforementioned lactobacillus germ is cultured in a medium; wherein the medium contains sucrose, yeast extract or a combination thereof, and the addition ratio of the sucrose or the yeast extract relative to the total weight of the medium is 1% to 10% by weight. The use according to claim 1, wherein the improvement of blood lipids is to reduce triglycerides, reduce total cholesterol, increase high-density lipoprotein cholesterol, or reduce ketone bodies. The use according to claim 1, wherein reducing the liver function index is reducing the content of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in the serum.