TW201216974A - Lactobacillus paracasei and its use - Google Patents

Abstract

A lactic acid bacterium identified as Lactobacillus paracasei, deposited at Bioresource Collection and Research Center of Taiwan with a deposit number BCRC 910482, which is used in the preparation of food or medicine composition to decrease the cholesterol in blood.

Description

201216974 VI. Description of the Invention: [Technical Field] The present invention relates to a lactic acid bacteria (Lactic Acid Bacteria, LAB for short), and more particularly to a bacterium of the genus Lactobacillus which can lower cholesterol in blood. [Previous technique] Cholesterol is an important molecule that constitutes the cell membrane of the human body. φ is used to increase the thermal stability of the cell membrane and maintain its fluid properties. Cholesterol is also a synthetic steroid hormone (Steroid Hormone) and synthetic bile salt (Bile). Salt's raw materials, therefore, cholesterol is closely related to the functioning of human cell physiology, endocrine system, digestive system and so on. There are two sources of cholesterol in the human body: one is taken from the diet and taken into the body. Another source is the self-synthesis of cholesterol in the body. About 70 to 75% of the cholesterol in the body is combined with lipoprotein (Lip0pr〇tein). The form of cholesterol vinegar is present in the blood, and the rest is mainly distributed in the muscles and brain. • Cholesterol is consumed as a raw material for steroid hormones. Another way to remove cholesterol from the body is to synthesize cholesterol into bile salts, secrete bile salts into the intestines to help fat emulsification and promote intestinal absorption of fat, 95%. Bile. The salt is reabsorbed by the intestines and returns to the body to circulate. The remaining 5% of the bile salts are excreted with the food residue. As modern people's diets tend to be more refined, the diet is rich in saturated fatty acids, trans-fatty acids and high-cholesterol foods such as egg yolks, red meat, animal fats, cheese, cakes, chocolate, ice cream, salad dressings and shellfish ( Shrimp, crab, shellfish, cuttlefish, etc., so that the body ingests too much cholesterol, so that too much cholesterol 201216974 alcohol accumulates in the body, the human body can not consume cholesterol, and cause hypercholesterolemia. Therefore, modern people rely on health foods to control the body's cholesterol content. Many scholars have found that in addition to maintaining the normal bacterial phase of the human intestinal tract, lactic acid bacteria prevent extrinsic pathogens and winds (such as Escherichia coli, Scdmonella typhimurium anti-C/ still WZ) sp .) Invasion, prevention or treatment of gastrointestinal diseases, and can activate human immune response, induce the production of cytokines such as IL-12 and TNF-α, or improve hereditary allergy symptoms, alleviate food allergies, lactic acid bacteria also have Reduces the function of cholesterol in the blood. There are two ways in which lactic acid bacteria can lower cholesterol. The first way is to use the enzyme contained in the lactic acid bacteria itself to precipitate bile salts to resist the damage of bile salts to its cell membrane, and the second way is to It is known that the cell membrane of lactic acid bacteria directly adsorbs cholesterol in the intestinal tract. The first approach, which is also the route used by most conventional lactic acid bacteria to lower cholesterol, is based on its own bile salt hydrolase (BSH), which is resistant to the insertion of bile salts into the cell membrane of lactic acid bacteria to maintain the physiology of the lactic acid bacteria. Activity' further reduces cholesterol in the digestive tract. The circulating pathways of cholesterol and bile salts are as follows: Cholesterol in the human body first synthesizes bile acid (Bile Acid, with -COOH), and enters the intestinal tract to become bile salts (Bile Salt, with _COO·), when bile salts and After the amino acid or a small amount of sulfide is combined, a combined bile salt (Conjugated Bile Salt) such as urinary acid (TCA) or Taurodeoxycholic Acid (TDCA) is formed. The combined bile salt has a low pKa value and a high solubility in the intestinal tract, so it is easily reabsorbed by the intestine and returns to the body for circulation. 201216974 - The bile salt hydrolyzing enzyme of the lactic acid bacteria is involved in the circulation of cholesterol and bile salts. The amount of bile salts in the body is reduced, and the body extracts cholesterol in the blood to compensate for the lack of cholesterol to produce bile salts. Bile salt hydrolase can: hydrolyze bound bile salts into the intestine into a non-binding bile salt (Unc〇njugated

BlleSalt), because the pKa value of the non-binding bile salt is high, the non-binding bile salt is easy to produce a sinking hall in an acidic environment, and the human body is not easy to resorb the precipitated non-cohesive impurities (4) (4), and the non-binding property is difficult. Excreted from the body, so in the bile salt circulation pathway, after the non-binding bile salt is discharged, #cause, the lack of inner salt, the body uses the cholesterol in the blood to make bile salts, thereby reducing the cholesterol content in the blood. . However, it is known that the bile salt hydrolyzing enzyme of lactic acid bacteria lowers cholesterol, and it can remove too much bile salts, causing insufficient bile salts in the body, and the efficiency of the body to absorb fat, fatty acid, etc. is worse. The experience is excreted in response to the gallbladder salt, so that the amount of bile salts in the intestine is insufficient to help the absorption of fat, and then the cholesterol in the blood is extracted to produce bile salts, and the cholesterol content in the blood is greatly reduced, and hypocholesterolemia occurs (Hyp 〇ch〇lesteremic). _ Since lactic acid bacteria containing bile salt hydrolyzing enzymes have the above disadvantages, it is a better choice to use the second-way lactic acid bacteria to lower the cholesterol content in the blood. However, after entering the digestive tract, the lactic acid bacteria have low survival rate and low survival rate, and the lactic acid bacteria have no bile salt hydrolyzing enzyme and cannot resist bile salts in the intestinal tract. After the cell membrane of lactic acid strontium, the double-layer fat-filling of the lactic acid bacteria cell membrane breaks and dies. Therefore, most lactic acid bacteria are not tolerant to bile salts and cannot exist in the digestive tract, so that the lactic acid bacteria cannot be adsorbed. The effect of lowering the cholesterol content in the blood. -5 - 201216974 Therefore, it is necessary to develop a new lactic acid bacteria system having an anti-cell-accommodating ability to enhance the milk _ _ _ _ _ _ _ _ _ _ The main purpose is to provide one kind of bacterium with the ability to reduce the human body. The second purpose of the present invention is to provide a secondary dry lactic acid rod with bile salt tolerance 'to make the vice dry lactic acid: this I month In addition, the objective is to provide a by-product lactic acid stem dry lactic acid dragon with acid tolerance, so that the sub-gamma = the stomach reaches the digestive tract and survives in the digestive tract. In order to achieve the aforementioned object of the invention, the present invention provides a food additive or a pharmaceutical composition for reducing blood cholesterol in the blood. The technical means and the effects that can be achieved by the technical means include: Lactobacillus faecalis (10), which is deposited in the Chinese People's Republic of China Food Industry Development Institute, numbered bcrc 91〇482, the vice The use of the lactic acid bacteria is used to prepare a food composition or a pharmaceutical composition for lowering blood cholesterol. [Embodiment] The above and other objects, features and advantages of the present invention can be made more apparent. 201216974 The preferred embodiment of the invention is accompanied by the accompanying drawings, and the present invention relates to a Lactobacillus bacterium having the ability to adsorb cholesterol in the digestive tract, which is selected from the acid-fermented dairy product to obtain a dry Lactobacillus

'The name given to Lactobacillus paracasei is e/diligence aC_/NPUST-YC-M-〇2, which is deposited in the Hsinchu Food Science and Industrial Development Research Institute of Taiwan, and its registration number is BCRC 910482 (1) Lactobacillus has a bile salt tolerance and acid tolerance 'and can adhere to cells, and has a better ability to lower blood alcohol in the milk, can be placed in the system (10) treatment or prevent blood cholesterol Excessive food composition or pharmaceutical composition. As shown in Fig. 1, the Lactobacillus of the present invention is obtained by the following various methods, including culturing lactic acid bacteria, screening of lactic acid bacteria S2, lactic acid bacteria, S3, and lactic acid bacteria storage S4. The culture S1 is isolated from the lactobacillus contained in a fermented dairy product and cultured. The sample was sampled from a fermented dairy product and placed in a culture medium for enrichment culture. After a suitable incubation time, the culture solution was serially diluted and inoculated into a medium to select a growable strain. The proliferation culture of the present embodiment is to inoculate the acid fermentation milk in a volume ratio of 1:1 (8) to a 0.1% (w/v) protein phage (peptone water) at a temperature of 30 C and a shaking speed of 150 rpm. After culturing for 1 hour, a proliferation culture broth is obtained, and the proliferation culture broth is appropriately diluted with physiological saline, and the diluted proliferation culture broth is inoculated with a MRS solid by smear method.

Mann Rogosa and Sharp agar plate (MRS for short)

Agar, as shown in Table 1), was cultured at a temperature of 3 〇ΐ under an anaerobic environment and selected for growth. Table 1: MRS solid medium (MRS Agar, 1 liter) Ingredient content (g) Peptone 10 Beef Extract 10 Yeast Extract 5 Glucose (Dextrose) 20 Ammonium Citrate 5 Magnesium Sulfate (MgS04) 0.1 Sulfuric Acid (MnS〇4) 0.05 Dipotassium Phosphate 2 Sodium Acetate 5 Tween 80 1 Calcium Carbonate (CaC03) 5 Agar 15 Table 2: MRS Liquid Medium (MRS Broth '1 liter) Ingredient Content (g) Peptone 10 Beef Extract 10 Yeast Extract 5 Dextrose 20 Ammonium Citrate 5 Magnesium Sulfate (MgS04) 0.1 201216974 Manganese Sulfate (MnS04) 0.05 Dipotassium Phosphate 2 Sodium Acetate 5 Tween 80 1 Carbonic acid (CaC03) 5 This lactic acid bacteria strain S2 is selected from several strains of lactic acid bacteria isolated, and has a cholesterol-lowering ability, and a lactic acid bacteria strain which does not lower cholesterol by bile salt hydrolyzing enzyme (Bsh) is selected. (S2-1) Cholesterol-lowering test The isolated strains of lactic acid bacteria were cultured in a mixture containing 0.2% (sodium thioglycolate, 0.2% bile salt, and 1 〇〇ppm cholesterol microparticles (Cholesterol micelle). MRS liquid medium (MRS Broth, as shown in Table 2), wherein the cholesterol microparticles are prepared according to the method designed by Razin et al. (1980) and Noh et al. (1997): weighing 22 mg of squamous bile ( Phosphatidylcholine) and 10 mg of cholesterol, add 10 ml of sucrose (0.4 M), vortex for 10 minutes after ultrasonic vibration, and let stand for 5 minutes. Repeat twice to obtain 1000 ppm of cholesterol particles. The size of the cholesterol particles is between 0.1 and 1.0. Μιχι. The volume ratio of the strain of lactic acid bacteria to the MRS liquid medium is 1.200 'at a temperature of 37. (: 'After 24 ± 2 hours in an anaerobic environment', centrifugation at 12000 g for 10 minutes to obtain above each experimental group. For the clear liquid, the concentration of cholesterol remaining in the supernatant (the absorbance at a measuring wavelength of 550 nm) was measured by the o-pjithaldialdehyde method, and the MRS liquid medium without the inoculated lactic acid bacteria was used as a control group. Calculated record The ability of acid bacteria to reduce cholesterol 201216974 I, the calculation of public Ρ ΤΓ ΤΓ 乳酸 乳酸 ( ( ( ( ( ( ( ( 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸The method of measuring cholesterol in the test is to take 0.25 mi of the supernatant of each experimental group and add 15% of 95% alcohol to mix evenly, and then add i ml 5〇% (v/v) potassium hydroxide (KOH) to mix at a temperature of 6〇. In a °C environment, react for 1 minute and cool to room/dish. Add 2.5 ml of jjexane and mix for 15 seconds. Add 1 ml of distilled water and mix for 15 seconds. Allow to stand for 1 minute and absorb the upper layer. The organic liquid is poured into a new test tube, and the organic liquid is blown dry with nitrogen at a temperature of 7 〇〇c, and then 2 ml of the phosphoromodialdehyde reagent is added, uniformly mixed, and allowed to stand for 1 minute, and 1 ml of concentrated sulfuric acid is slowly added thereto. Carefully mix and measure the absorbance at 550 nm after standing for 1 minute, which can be used to evaluate the cholesterol concentration of each experimental group. OD^g The higher the absorbance value, the higher the cholesterol concentration of the group. S2 design contains a bile salt and sodium thioglycolate (Sodimnthioglycolat In the environment of e), the ability of the lactic acid bacteria of each experimental group to lower cholesterol was tested. Among them, the lactic acid bacteria of the present invention can reduce the cholesterol of 51.6 ppm in a 24-hour period, and has a good cholesterol-lowering ability. (S2_2) Bile Salt Hydrolase Activity Screening (BSH Assay) Each lactic acid bacteria is also screened for solid medium (BSii)

Assay Plate) was cultured for 12 〇 ± 2 hours at 37 ° C in an anaerobic environment, and opaque precipitates were observed around the colonies during the culture period. The 'BSH Assay Plate' was formulated with MRs solid medium. The ingredient 'addition of 0.5% taurodeoxycholic acid and 〇.〇37% calcium sulphate is prepared. When the lactic acid bacteria have a sturdy | raw, the colonies around | appear impervious to 201216974. The lactic acid bacteria are cultured in the BSH Assay plate by the smear method, and the opaque precipitate is not produced around the colony of the lactic acid bacteria of the present invention or at the bottom of the medium, and thus the lactic acid bacteria are preliminarily determined that the lactic acid bacteria do not have BSH activity, so the milk (4) There is no money related to the activity of the coffee-to-coffee. Afterwards, the continuation of the fine activity of the lactic acid S of the present invention will be confirmed. The identification of the lactic acid bacteria S3 is based on the appearance of the lactic acid bacteria, physiological characteristics, or sub-characteristics, and the like. In this embodiment, the lactic acid bacteria are subjected to Gram staining (Gram, Catalase Activity, and molecular identification of 16S rDNA), and the staining diagram of the lactic acid bacteria is judged as a leather-positive bacteria and a catalase reaction test. For the negative and 16S rDNA partial sequence and other identification results, it is determined that the lactic acid (4) of the present invention is - Lactobacillus parasiticus (Lact〇baci'nusparacase〇, the accession number is BCRC 9i〇482, the sub-dry lactic acid rod 8 series-short rod g , New Power (she magnetic y), the detailed identification results are compared with the strains of the nCBI database, such as Annex 1. The preservation of the lactic acid bacteria S4' system makes the sub-dry (four) acid system stable in low temperature environment Long-term storage 'for subsequent use. The Lactobacillus paracasei of the present invention was inoculated to the MRS solid medium, and a single colony was selected and transferred to a liquid culture medium (as shown in Table 2) after incubation for 24 hours at a temperature. Temperature 3rt, anaerobic culture for 18 ± 2 hours, cold; east preservation; after the end of the culture 'the MRS liquid medium is better to add anti-; east agent (such as glycerol) 'where the volume ratio is 3 :], temporary storage Yu·Talking The temperature preservation chamber is to be used later. In order to confirm that the secondary lactic acid rod 8 of the present invention has the ability to lower cholesterol by adsorption and its ability to withstand bile salts and acid, the following 201216974 T ° type of bacillus The ability to reduce cholesterol in different growth stages, (7) the bile salt of the lactobacillus: the acid tolerance of the acid bacillus, the secondary touch bis (5) the bile salt hydrolyzing enzyme unit of the secondary lactic acid rod S Activity and (6) lactic acid rod _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Vari, AN0VA) and Duncan's multivariate field test (Duncan, s singular Range Test) for multiple comparisons of differences in experimental data. (1) The ability of the secondary secretory Lactobacillus to grow in different stages and to lower cholesterol - please refer to Table 3 and Figure 2, which is the case of the LH·6 group of sterol in the different culture period of the present invention. Wherein, each of the tissue culture medium is a MRS liquid medium containing 〇.3% biliary-alcohol cholesterol, and after culturing for 24 hours at a temperature of 3 rc, the biliary concentration of the MRS liquid medium in each group is measured by the dish benzene two-way method. , wherein, the sterol concentration check line is shown in the third _, according to the 胆固醇 axis of the cholesterol concentration and the Υ axis of a regression equation γ = 〇〇〇 296 X + 0.0002 (R2 = 0.9945) 'to the regression The equation calculates the actual value of cholesterol corresponding to 〇〇55.1. The different cultures in this example are purely referred to as activation of the Lactobacillus paracasei, starting from the start time of the culture, respectively at the second hour, the 12th hour, the 24th hour, the 36th hour, the 48th hour, and the The cholesterol concentration test was carried out for 72 hours, respectively, in the group of 3_丨~3_6, and the cholesterol-lowering test of the above conditions was carried out.

—12 — 201216974 Test time (Hr) 0 12 24 36 48 72 Viable count (Log CFU/ml) 8.10 8.74 8.75 8.01 6.27 5.05 Cholesterol concentration (ppm) 184.91 138.04 91.94 85.79 87.33 ^. 93.86 Please refer to Figure 2 'The X axis is the group of different culture periods, the Y1 axis is the logarithm of the viable counts of each group (Log CFU/ml), the line graph in the field should be 'the other Y2 axis is the cholesterol concentration of each group (ppm) , corresponds to the long bar graph in the figure. As shown in the line graph and the bar graph, the 3-2 group and the 3-3 group having the highest training period of the Log Phase and the Stationary Phase have the highest ability to lower cholesterol, and the 3_4 is the highest. There was no significant difference in the cholesterol-lowering ability of the 3-6 group, which represents that the ability of the Lactobacillus paracasei to lower cholesterol in the present invention is better in the growth phase and the stationary phase. From this, it can be confirmed that the Lactobacillus paracasei of the present invention has a better ability to adsorb cholesterol during the growth period and the stationary period of the growth period. (2) The characteristics of the bile salt and cholesterol lowering of the Lactobacillus brevisii, please refer to Table 4 and Figure 4, which are the catechins of the present invention. In the case of cholesterol lowering in groups 4-6, the medium of each group was MRS liquid medium containing 10 〇ppm of cholesterol, and after 24 hours of culture, the concentration of the biliary domain of the MRS liquid in each group was measured by the linacaldehyde method. The actual supplement does not (5) the concentration of bile salt is different from the MRS liquid sorrow medium containing f 0%, 0.1%, 〇, 2%, 0.3%, 〇.4% and 5%.5% bile salt The group contains the bribes, and the concentration of cholesterol in the MRS liquid medium containing the present invention is included in the present invention. The concentration of cholesterol in the MRS liquid medium containing the present invention is as follows, and the cholesterol is contained in the CRS liquid medium of the present invention. The number of viable cells in the medium (VC-100 ppm) and the cholesterol-free medium (VC-0 ppm). The first table: different bile salt concentration groups 4-1 4-2 4-3 4-4 〇 0.1 0.2 0.3

Choi-Blank Cholesterol concentration (ppm) 7.11 9.01 8.20 8.07 7.93 7.72 88.66 92.79 61.99 46.75 51.83 48.98 95.48 94.69 100.4 85.48 86.91 82.94 : The number of viable bacteria in each group of the different biliary 4-group 'Y1 axis system Long bar graph, another Y2 axis system corresponds to the line graph in the corresponding figure (4), VC-100Ppm, empty... μ 1 bar graph oblique line group is the solid line of the invention 崎崎ΓΓ崎线线 represents the old-〇&gt ;, and the virtual fungus Chol-Blank in the (tetra) sterol environment. The ability of the bile salt of the present invention to test the number of viable bacteria and the increase of cholesterol lowering _ bile salt by the ability of not containing the number of viable bacteria in the environment of the cholesterol, = 201216974 even in the environment containing cholesteric alcohol, even _ up to ° = xlG or more live s number compared to other conventional lactic acid bile salt tolerance; in addition, 'the invention = ^ ^ 5% of the environment, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ppm). From this, it can be confirmed that the lactobacillus of the present invention has the ability to record heterosis and to absorb lysine in an environment containing cholesterol. (3) Acid tolerance of the Lactobacillus paracasei

Refer to Table 5 for the survival of the MRS liquid medium of the present invention, which is cultured in the environmental pH of pH 3.0 and 7. The acidity test values were 5-1 group pH 3.0 and 5-2 group pH 7.0, and cultured for 3 hours at a temperature of 3rc and a shaking speed of 80 rpm, and then cultured in an appropriate sequence to MRS solid medium to obtain each. The viable count of the group and the acid tolerance of the Lactobacillus paracasei of the present invention was calculated. X

Table 5: pH values of different environments Acid tolerance (%)

7-35+0.14 8.65±0.02 85.00+1.66 It is confirmed by the test of (3) acid tolerance that the crepe lactic acid of the present invention has the ability to resist the pH value of the stomach, thereby improving the enterococci of the present invention into the intestine Survival rate in the road 'further to achieve cholesterol reduction

S —15 a 201216974 effect. From this, it can be confirmed that the Lactobacillus paracasei of the present invention has acid tolerance and can reach the intestinal tract through the stomach to adsorb cholesterol in the intestinal tract. (4) Cell adsorption capacity of the Lactobacillus paracasei As shown in Annex I, the adsorption of the Lactobacillus of the present invention to cells was carried out in May. In this example, the cell adsorption was carried out by Caco-2 cells. The Caco-2 cell line was purchased from the Bioresource Conservation and Research Center of the Hsinchu Food Industry Research Institute under the number BCRC 60182. The Caco 2 cells were cultured for 15 days to differentiate into colonic cell polarization, brush borders, and hydrolyzing enzymes. Therefore, Caco-2 was selected for (4) cell adsorption capacity test. The Caco-2 cell line is in DMEM medium [Dulbecco, s Modified Eagle Medium, whose composition contains 15% fetal bovine serum (Fetai Bovine Serum ' FBS), 0.1% non-essential amino acid (Nonessential)

Amino Acid, NEAA), 100 U/ml penicillin G (Penicillin G) and 100 pg/ml streptomycin] were cultured and subcultured in a 9 cm culture dish at 37 ° C. The ventilation capacity of 5% carbon dioxide was set, and the culture solution was changed after about 36 to 48 hours, and the cell adsorption capacity test was carried out after the cells were cultured for 15 days.

The Lactobacillus paracasei of the present invention was cultured for 18 hours, and after removing the MRS liquid medium, the number of bacteria of the lactic acid bacteria liquid was adjusted to ixi 〇 8 CFU/ml by PBS buffer solution (Phosphate Buffer Saline), and in addition, Caco- was removed. After 2 cells of DMEM medium, inject 10 ml of lactic acid bacteria solution into a Petri dish carrying Caco-2 cells, incubate at a temperature of 37 ° C, set aeration with 5% carbon dioxide for 1 hour, and then PBS 201216974 The slow larvae of the CaeG_2 _ three times the silk is not adsorbed to the fine lactic acid bacteria on the cells, added 5 ml of methanol and placed at a temperature of 4 for 2 minutes to fix the cells and lactic acid bacteria in the culture dish, then carry out the leather Lan's staining, observed under the 4 〇〇χ optical brewing mirror

The case of Caco-2 cells. As shown in Annex 2, after the incubation of the 400x optical microscope, the lactic acid (four) of the present invention has a cell count of 156±14 cells, which can be proved. The sub-dry lactic acid bacterium of the present invention has the ability to adsorb cells. (5) Unit activity of the bile salt hydrolyzing enzyme of the Lactobacillus paracasei, as shown in Tables 6 and 5, is the bile salt hydrolyzing enzyme unit activity of the lactic acid bacteria of the side of the present invention. In this example, the bile salt hydrolase activity per gram of bacteria (1〇9 CFU/ml) was calculated by BSH Ninhydrin Assay. The Lactobacillus paracasei of the present invention was subjected to anaerobic culture in a MRS liquid medium containing 0.3% (w/v) of bile salts, and after 18 hours, the culture liquid of i blood was taken for the BSH Ninghai quasi-test. In this example, 1 ml of the culture broth was removed by centrifugation (6 〇〇〇 Xg, 5 minutes) to remove the MRS liquid medium, and then ppb buffer solution (Potassium Phosphate Buffer, 0.287 g Κ2ΗΡ04 and 0.456 g ΚΗ2Ρ04 was used to quantify distilled water. Wash to 3 ml to 500 ml, adjust pH to 6.5), add 500 μΐ Reaction Mixture to temperature 37. (: Anaerobic incubation for 30 minutes, then add 15% (w/v) Trichloroacetic acid 500 μΐ to stop the reaction 'by centrifugation (12000xg '1〇 minutes), then take 5〇〇μ1 supernatant Add 500 μΐNinghai quasi-reagent to the glass test tube at a temperature of i00°c water 201216974 ==min, and after natural cooling for 3 minutes, measure the sputum D57〇' with Taurine as the base. j μιη〇1β Taurine represents a bile brine, and the enzyme unit activity 'calculates the green sputum d57. The production of the dragon sir is known to the sequel to the sputum as shown in the figure γ = 0.0177 X+ 0.0223 (R2 -0.9996), wherein 'the axis of tau is the amount of taurine produced, and the axis of γ is 0%'. The unit activity of the corresponding bile salt hydrolyzing enzyme is calculated by the enthalpy of the cow.

Live bacteria count bovine acid production unit cell weight per unit weight (Log CFU/ml) Oimole) BSH active BSH activity '----------(U/109 CFU) (U/mg) ~_9A4±0A2 6.6112.57 Q.16±Q.〇6 0.08±0.Q3 < Generally lactic acid bacteria with bile salt hydrolase activity, the bile salt hydrolase activity per unit number of bacteria is at least 0.3 u/1〇9 CFU or more, In the sixth table, it can be seen that the bile salt hydrolyzing enzyme activity diagram of the Lactobacillus paracasei of the present invention is lower than Gl U/1G9 CFU), and the (1) different growth period and its ability to lower cholesterol and (2) the bile salt resistance and The test of cholesterol-lowering characteristics and the like confirmed that the Lactobacillus paracasei of the present invention differs from the conventional lactic acid bacteria by the BSH cholesterol-lowering method contained therein, which lowers cholesterol by adsorbing cholesterol to the cell membrane, and thus the Lactobacillus paracasei of the present invention does not There is a problem that hypocholesterolemia occurs when the concentration of cholesterol in the blood is too low due to excessive precipitation of bile salts. (6) For the animal test for the cholesterol-lowering of the Lactobacillus paracasei, refer to Table 7 and Figures 7 and 8, and after inducing the obesity of the experimental animal with high-calorie feed, the lactic acid of the present invention is administered. Bacillus, —18 — 201216974 Evaluate the efficacy of this Lactobacillus paracasei in cholesterol reduction in animal experiments. The experimental animal of the present example was selected for the animal test at a four-week-old Spmgeu_Dawley (SD) money of the Specific Path〇gen (SpF) grade, and the SD rat was purchased from Lesco Biotech Co., Ltd. The animal test system of this example was designed into four experimental groups, the 7_丨 group was the control group, the 7-2 group was the high-calorie control group, the 7_3 group was the high-calorie experimental group _ medium dose and the 7-4 group was the calorie test. Group - high dose. The 7-1 group is a diet for feeding, which contains 23.4% protein, 72.1〇/〇 carbohydrate and 4.5% fat, and contains 3.3 kcal of calories per gram of feed; 7-2~7-4 group is high. The calorie feed contains 21% protein, 48% carbohydrate, 30.8% fat and 0.2% cholesterol, and contains 4.47 kcal of heat per gram of feed, wherein the 7-3 group and the 7-4 group are respectively fed. The Lactobacillus paracasei (with a feeding volume of 0.5 ml) was administered at 2xl08 CFU/ml and lxl〇9 CFU/ml. Table 7: Animal test group group 7-1 7-2 7-3 7-4 (Control) (HEC-C) (HEC-M) (HEC-H) Feed ingredients 100% Basic feed 44% condensed milk, 4% corn oil, 4% lard, 0.2% cholesterol and 48% basal feed induction period 69.7a 70.0 a 76.7 a 72.5 a total cholesterol (mg/dl) 68.1 b 58.3bc 49.0c 47.0c 1悤 cholesterol (mg/dl) Cholesterol reduction 1.6 11.7 27.7 25.7 (mg/dl) 1-19 — 201216974 Weight (g) 486.3 476.1 d 440.2de 433.6de Total cholesterol in the induction period, total cholesterol in the test period and body weight experiment The same letter (a~e) indicated by the data represents no significant difference. The feeding temperature of the SD rats of this example was 22 ± 2 〇 c, and the relative humidity was 50 to 60 /. 'The light period is 12 hours for four consecutive groups of animals in the first two weeks of white basal feed foraging (stable period)' the third week from the 7_2 group, the 7_3 group and the 7-4 group began to feed high-calorie feed (induction Period), and from the twelfth week, the 7_3 group and the 7-4 group daily tube, high dose of lactic acid bacteria 5 m to end the 21st week to end the animal test (test period). / Qing refer to Tables 7 and 8, there is no significant difference in the total cholesterol levels during the induction period of each group, and the total cholesterol and body weight measured after the test again, although the 7-1 group and the 7-2 group There was no significant difference, but there was a tendency for the total alcohol in the 7_3 group (10) 7_4 group with medium and high doses of lactate to decrease. According to the study by Miao (1997), the metabolic coefficient of the rat relative to the human body is 6.25 ′. Therefore, the ingestion dose per kilogram of body weight of the rat obtained in the animal test = the human body's body weight (5). In the present embodiment, the dose of the dose test group is about 2xl 〇 8cFu / Kg, and an adult weighing 60 kg is calculated according to the above formula, and the adult takes about 7.5 χ 1 〇 1 〇 CFU (ie 1.25 x 10 CFU/Kg). According to the national standard CNS3〇58 “fermented milk”, the fermented milk is prepared to be more than 7 cFU/ml per liter of live bacteria, and the mother is drinking the fermented milk (l 〇xl〇7 CFU). /ml or more) about 7500 ml, and, in general, commercially available fermented milk, the number of viable bacteria per ml is about ~8~9 CFU/ml or more', and the commercially available fermented milk per person per day (1〇χ1〇8) The effect of the present invention can be achieved by using about 75 to 750 ml of ~9 CFU/ml or more. 201216974 The dose of the experimental dose group of the present invention is about 1 〇xi〇9 fFU/Kg'. According to the above formula, an adult weighing 6 〇 ^ ^, the adult takes about 3.75X1011 CFU (ie 6.25) Χΐ〇9 CFU/Kg), about 375~3750 ml of fermented milk containing live g Ox Euro 9 CFU/ml or more per person per day, so that it can be proved that the Lactobacillus Rats do have the ability to reduce cholesterol in the blood of rats. - As described above, the Lactobacillus paracasei of the present invention can be grown in the environment containing cholesta, and the effect of the lactic acid bacillus having the effect of adsorbing cholesterol can further reduce the absorption of cholesterol in the human digestive tract. The sub-dry Lactobacillus can pass the acid test with low acidity (ρΗ 3 stomach, and it has the effect of adsorbing cholesterol in the digestive tract after reaching the intestine, so it can be widely used as a food composition or drug composition for adsorbing cholesterol in the digestive tract. Things such as health food, medical preparations or processed foods; the deputy dry = cups can be used as a separate sister and other ingredients (such as fine, nutritious ingredients, made into various product forms, such as (four), capsules , beverage, powder or other contact system processed into a dairy product 'can be administered in various ways to effectively adsorb the alcohol in the host digestive tract; _ dry lactic acid = can be attached to the cell' to stay in the host's intestinal environment to increase its The effect of inhaling S-sterol reduces the amount of cholesterol absorbed by the host digestive tract and further reduces the amount of cholesterol in the blood. Thereby, the side-by-side Lactobacillus strain of the present invention The ability to adsorb cholesterol in the digestive tract, and reduce the content of the blood cap of the human body. The field of the invention, the J. bacillus, the cell membrane system has the bile salt, so that the sub-dry _ acid lake can survive the correction. It has the effect of enhancing the adsorption of cholesterol in the digestive tract by the dried Lactobacillus strain. The Lactobacillus paracasei of the present invention has acid tolerance, and the cup of milk of the pair of cheese reaches the digestion through the moon. In the middle, the effect of promoting the adsorption of cholesterol in the digestive tract by the Lactobacillus paracasei is achieved. The use of the Lactobacillus paracasei of the present invention is to apply the sub-dry lactic acid (IV) as described above to the preparation of the cholesterol-lowering alcohol product to control the human body. Absorbing too much cholesterol 'reducing the amount of cholesterol in the blood is an effect of the present invention. Although the present invention has been disclosed by the above-described preferred embodiments, it is not intended to limit the present invention, and those skilled in the art can devote themselves to the spirit of the present invention. And within the IlL circumference, various modifications and modifications to the above embodiments are still the present invention, and the scope of protection of the present invention is The definition of the scope of patents shall prevail. [Simplified description of the drawings] Figure 1 · Flow chart of the treatment of Lactobacillus in the trunk of the present invention. Alcohol Γ 偷 偷 偷 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸 乳酸Figure 3: Cholesterol sulphate method for the calibration of the sulphur benzene method. Figure 4 · The hair _ secret lactic acid (four) is not _ sterol fold line and silk map. % now secretly lowering the gallbladder line diagram Figure 5: Benja's deputy secretive lactic acid turned bile salt trans-enzyme activity fold Figure 6: Dirty Ninghai quasi-display coffee inspection line diagram 201216974 Figure 7: animal test weight line diagram. Figure 8: total cholesterol test in animal test Bar graph. Annex I: Identification results of Lactobacillus paracasei according to the present invention. Annex II: Cell adsorption micrograph of Lactobacillus paracasei of the present invention. [Key element symbol description] [None]

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Claims (1)

2〇1216974 VII, _ 谤 Patent Fan®: Although! K. bacillus, is a deposit, the Chinese people's food industry development research, the registration number is BCRC 9J0482. 2 HSS:== -twenty four -