The present invention relates to a food composition and a pharmaceutical composition, particularly to a hepatoprotection food composition and pharmaceutical composition with strains of lactic acid bacteria.
The liver is the largest organ in the human body, performing very important functions, including 1. Blood circulation: circulating blood from the hepatic artery to the whole body, participating the immunological mechanism through the reticuloendothelial system thereof, storing blood and regulating blood volume; 2. Excretion: generating bile to the intestines, wherein the bilirubin conjugates, cholesterol and cholic acid, which are excreted by the liver, together with the bile to form bile salts, and removing the materials that the liver cells separate from the blood, such as heavy metals, bromsulphalein, and alkaline phosphatase etc.; 3. Metabilism: participating the metabolism of carbohydrates, proteins, lipids, minerals and vitamins, and generating heat; 4. Protection and detoxification: removing the foreign bodies in the blood through endocytosis of Kupffer cells, lowering the toxicity of poisonous materials through bonding, methylation, oxidation and reduction, driving out the poisonous materials through the excretion system, and removing ammonia from the blood, especially the ammonia from the portal vein; 5. Hematopoiesis and blood coagulation: forming blood in the embryonic stage, also forming blood in the adulthood in some special conditions, generating coagulants, such as fibrinogen, prothrombin and heparin, and destroying aged red blood cells. Therefore, it is very important for a person to maintain the normal functions of the liver.
Ethanol (alcohol) is an important hepatitis-inducing factor. Long-term alcoholism may lead to alcoholic hepatitis. The symptoms of alcoholic hepatitis include discomfort, hepatomegaly, ascites, and higher values of liver function indexes. Slighter alcoholic hepatitis only leads to higher values of liver function indexes. Severer alcoholic hepatitis shows symptoms of severe liver inflammation, jaundice, prolonged prothrombin time, and liver function failure. The most severe cases can be unconscious, accompanied by an increase in bilirubin values and prolonged prothrombin time. Severe alcoholic hepatitis has a mortality rate as high as 50% within 30 days from the appearance of these symptoms.
The anatomy of the non-alcoholic fatty liver disease (NAFLD) is very similar to that of alcoholic hepatitis (lipid droplets and inflammatory cells). However, the patients of NAFLD is free of alcoholism. A similar but moderate disease is steatosis hepatis, also called the fatty liver. 80% of the patients suffering obesity also suffer steatosis hepatis. Lipid droplets appear all over in the anatomy of the liver of the patient suffering steatosis hepatis. However, no inflammation appears in the anatomy of the liver of the patient suffering steatosis hepatis. Steatosis hepatis may further evolve into hepatitis, hepatic cirrhosis, or even hepatoma.
So far, only few strains of lactic acid bacteria have been experimentally confirmed to have the effect of protecting livers. The functionality of lactic acid bacteria to health is not based on the species of bacteria but dependent on the specificities of strains. The strains favorable to health are called the probiotics (Guidelines for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002:1-7).
Accordingly, it is an urgency to develop a nutrient that is safe and can be used for a long time to protect livers. In general, lactic acid bacteria are safe for human beings. Therefore, finding out lactic acid bacteria strains and its inactive strains able to protect livers becomes a target the manufacturers are eager to achieve.
The present invention provides a food composition and pharmaceutical composition with strains of active or inactive lactic acid bacteria, which can protect livers and relieve liver inflammation and fatty livers.
In one embodiment, the present invention provides a food composition with strains of lactic acid bacteria, which comprises lactic acid bacteria strains able to protect livers, and a physiologically-acceptable excipient, diluent or carrier. The lactic acid bacteria strain is at least one isolated lactic acid bacteria strain selected from a group including a TSP05 strain of Lactobacillus plantarum (CGMCC No. 16710), a TSF331 strain of Lactobacillus fermentum (CGMCC No. 15527), and a TSR332 strain of Lactobacillus reuteri (CGMCC No. 15528). The abovementioned lactic acid bacteria strains are deposited in China General Microbiological Culture Collection Center (CGMCC).
In another embodiment, the present invention provides a pharmaceutical composition with strains of lactic acid bacteria, which comprises lactic acid bacteria strains able to protect livers, and a pharmaceutically-acceptable excipient, diluent or carrier. The lactic acid bacteria strain is an at least one isolated lactic acid bacteria strain selected from a group including a TSP05 strain of Lactobacillus plantarum (CGMCC No. 16710), a TSF331 strain of Lactobacillus fermentum (CGMCC No. 15527), and a TSR332 strain of Lactobacillus reuteri (CGMCC No. 15528). The abovementioned lactic acid bacteria strains are deposited in China General Microbiological Culture Collection Center (CGMCC).
Below, embodiments are described in detail in cooperation with the attached drawings to make easily understood the objectives, technical contents, characteristics and accomplishments of the present invention.
The present invention will be described in detail with embodiments and attached drawings below. However, these embodiments are only to exemplify the present invention but not to limit the scope of the present invention. In addition to the embodiments described in the specification, the present invention also applies to other embodiments. Further, any modification, variation, or substitution, which can be easily made by the persons skilled in the art according to the embodiment of the present invention, is to be also included within the scope of the present invention, which is based on the claims stated below. Although many special details are provided herein to make the readers more fully understand the present invention, the present invention can still be practiced under a condition that these special details are partially or completely omitted. Besides, the elements or steps, which are well known by the persons skilled in the art, are not described herein lest the present invention be limited unnecessarily. Similar or identical elements are denoted with similar or identical symbols in the drawings. It should be noted: the drawings are only to depict the present invention schematically but not to show the real dimensions or quantities of the present invention. Besides, matterless details are not necessarily depicted in the drawings to achieve conciseness of the drawings.
The freeze-dried cultures of the strains of lactic acid bacteria mentioned in the specification are deposited in China General Microbiological Culture Collection Center (CGMCC) of Chinese Academy of Sciences (NO. 1West Beichen Road, Chaoyang District, Beijing 100101, China). The details thereof are listed in Table.1.
Lactobacillus plantarum
Lactobacillus fermentum
Lactobacillus reuteri
The three lactic acid bacteria strains listed in Table.1, which are respectively the TSP05 strain of Lactobacillus plantarum, the TSF331 strain of Lactobacillus fermentum and the TSR332 strain of Lactobacillus reuteri, were found to be able to protect livers and prevent from liver inflammation.
The present invention provides a food composition, which comprises lactic acid bacteria strains able to protect livers, and a physiologically-acceptable excipient, diluent or carrier. The present invention also provides a pharmaceutical composition, which comprises lactic acid bacteria strains able to protect livers, and a pharmaceutically-acceptable excipient, diluent or carrier. The lactic acid bacteria strain is at least one isolated lactic acid bacteria strain selected from a group including a TSP05 strain of Lactobacillus plantarum (CGMCC No. 16710), a TSF331 strain of Lactobacillus fermentum (CGMCC No. 15527), and a TSR332 strain of Lactobacillus reuteri (CGMCC No. 15528). The abovementioned lactic acid bacteria strains are deposited in China General Microbiological Culture Collection Center (CGMCC). In one embodiment, the lactic acid bacteria strains are active or inactive strains.
In the embodiment of the food composition, the physiologically-acceptable excipient, diluent or carrier may be a food. For example, the food may be but is not limited to be dairy food, tea, coffee, chewing gum, a tooth-cleaning candy (such as an oral strip, a chewable tablet, or, jelly sweets) or a combination thereof. The dairy food may be fermented milk, yoghurt, cheese, or powdered milk etc. The pharmaceutical composition may be in form of an oral dosage, such as a tablet, a capsule, a solution, or a powder.
In the embodiments of the food composition and the pharmaceutical composition, the number of the lactic acid bacteria strains may be over 106 CFU (Colony-Forming Unit), preferably 1010 CFU.
It should be explained: documents point out that the lactic acid bacteria strains belonging to an identical specie are not all favorable for hepatoprotection. For example, Xingrong Zhao, et al. used high fat diet to generate obese mice and used the obese mice to investigate the effect of lactic acid bacteria strains on obesity and fatty livers induced by high fat diet (PLoS ONE 7(2): e30696. doi:10.1371/journal.pone.0030696). The experimental result shows that only the LP28 strain of Pediococcus pentosaceus can inhibit rising of body weight and reduce triacylglycerol and cholesterol in livers and that the SN13T strain of Lactobacillus plantarum and the heat killed LP28 strain do not have the same effect. Yi Qiao et al. used high fat diet to generate obese mice and used the obese mice to investigate the effect of the L3 strain and L10 strain of Lactobacillus reuteri on inflammation and fat accumulation. The experimental result shows that only the L3 strain can improve liver hypertrophy and hepatic steatosis and that the L10 strain does not have the same effect. Therefore, the present invention does not extensively claim all lactic acid bacteria strains but only claims the three strains deposited in China General Microbiological Culture Collection Center (CGMCC), which are respectively the TSP05 strain of Lactobacillus plantarum (CGMCC No. 16710), the TSF331 strain of Lactobacillus fermentum (CGMCC No. 15527), and the TSR332 strain of Lactobacillus reuteri (CGMCC No. 15528).
The taxonomic characteristics of the strains are identified with the 16S rDNA sequencing analysis and the API bacterial identification system. The morphologies and general properties of the strains are listed in Table.2.
Lactobacillus
plantarum
Lactobacillus
fermentum
Lactobacillus
reuteri
The lactic acid bacteria strains collected by the Applicant are preserved in a solution containing 20% glycerol at a temperature of −80° C. Before usage, the strains are activated two times at a temperature of 37° C. for 24 hours with an MRS broth (DIFCO) containing 0.05% cysteine. The lactic acid bacteria strains used in experiments include a TSP05 strain of Lactobacillus plantarum, a TSF331 strain of Lactobacillus fermentum, and a TSR332 strain of Lactobacillus reuteri. The TSP05 strain of Lactobacillus plantarum is sourced from pickled vegetables; the TSF331 strain of Lactobacillus fermentum and the TSR332 strain of Lactobacillus reuteri are sourced from intestines of healthy chickens.
CB7B/6N mice are fed with alcohol-containing diet and used in experiments for evaluating the effects of the lactic acid bacteria strains on the alcohol-induced liver tissue damages. The liver function indexes, such as alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum, are measured. Triacylglycerol in livers is also measured to learn the relation of triacylglycerol and fatty livers. Aspartate transaminase (AST) exists in livers, cardiac muscles, muscles, and erythrocytes. Alanine aminotransferase (ALT) mainly exists in hepatocytes. While these cells are necrotized by various factors, transaminases in cells are released to blood. Therefore, while a person suffers hepatitis, myocardial infarction, muscle inflammation or hemolysis, the value of AST may rise. The extent of cellular damage may be evaluated via inspecting the extents of transaminase rising. Similarly, ALT rising is exactly induced by liver inflammation. While livers are damaged by drugs, alcohol, viruses or hypoxia, the extent of hepatocyte damage may be learned via examining the extent of AST or ALT rising in blood tests.
Firstly, 44 C57BL/6N mice are divided in 6 groups, include a control group, an Alcohol group, an “Alcohol+TSP05” group, an “Alcohol+TSF331+TSR332” group, an “Alcohol+TSP05+TSF331+TSR332 ((Alcohol+Mix-LAB)” group, and a “TSP05+TSF331+TSR332 (Mix-LAB)” group. The control group and the Mix-LAB group are fed with LIEBER-DECARLI REGULAR LIQUID DIET CONTROL. The alcohol group and the “alcohol+lactic acid bacteria” groups are fed with LIEBER-DECARLI REGULAR LIQUID DIET ETHANOL. The control group and the Alcohol group are tube-fed with water through gastric tubes two times every day. The “Alcohol+TSP05” group, “Alcohol+TSF331+TSR332” group, “Alcohol+TSP05+TSF331+TSR332 ((Alcohol+Mix-LAB)” group, and “TSP05+TSF331+TSR332 (Mix-LAB)” group are tube-fed with the tested lactic acid bacteria strains. The experiments are undertaken over a span of 4 weeks. Blood are collected every two weeks during the experiments to analyze alanine aminotransferase (ALT), aspartate transaminase (AST), triacylglycerol and total cholesterol. The mice are sacrificed in the fourth week. The liver tissues thereof are taken out for analysis of triacylglycerol, glutathione, glutathione peroxidase, etc.
Glutathione is a very important antioxidant in human bodies, usually called “the mother of all antioxidants”. Owing to age, irregular living habit, and poor dietary habit, glutathione may decrease gradually. The concentration of glutathione in the liver indicates the antioxidant ability of a person. Glutathione peroxidase is the generic term of an enzyme group having the activity of peroxidase, functioning to detoxify in organisms and protecting organisms against oxidation. Glutathione peroxidase reduces the peroxides of lipids into corresponding alcohols, reduces free hydrogen peroxide into water, and catalyzes glutathione into the oxidized form thereof.
According to the operational manual of the QuantiChrom™ Glutathione Assay Kit, add liver tissue to phosphoric acid buffer solution by a ratio of 1:4, and mix them to form a homogeneous liquid. Take 200 μL of the homogeneous liquid, and use a refrigerated centrifuge to process the homogeneous liquid at a speed of 14000 rpm and a temperature of 4° C. for 10 minutes, and then take out the supernatant liquid ready for test. Next, prepare Blank and Calibrator: add 100 μL ddH2O and 100 μL Calibrator to a 96-well plate, and add 200 μL ddH2O to the Blank and Calibrator wells and mix them uniformly. Take 120 μL Sample and 120 μL Reagent A into a 1.5 mL centrifuge tube, and undertake oscillation mixing. If turbidity appears, centrifugalize the mixture liquid of Sample/Reagent A at a speed of 14000 rpm for 5 minutes. Next, add 200 μL mixture liquid of Sample/Reagent A into the 96-well plate, and add 100 μL Reagent B to the 96-well plate, and then tap the plate to make them mix uniformly. Next, place the plate statically at an ambient temperature for 25 minutes. Next, measure the absorption of OD412 nm by the liquid to work out the concentration of glutathione according to the following formula:
GSH (μM)[(ODsample−ODBlank)(ODCalibrator−ODBlank)]×100×n
wherein ODsample is the light absorption of Sample, ODBlank is the light absorption of Blank, and ODCalibrator is the light absorption of Calibrator, and n is the fold of dilution.
Refer to
It is concluded from the abovementioned experiments: the lactic acid bacteria strains-containing food composition and pharmaceutical composition of the present invention can protect livers and prevent from liver inflammation and fatty livers. Further, the present invention can enhance antioxidant ability of livers and protect livers against alcohol-induced oxidation. The hepatoprotection lactic acid bacteria strains of the present invention is free of side effects and thus a new choice for liver protection.
The embodiments have been described above to demonstrate the technical thoughts and characteristics of the present invention to enable the persons skilled in the art to understand, make, and use the present invention. However, these embodiments are only to exemplify the present invention but not to limit the scope of the present invention. Any equivalent modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention.