JPH0473984B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a cholesterol-lowering agent and a food or drink containing the same. Today, several drugs, including clofibrate-related preparations, have been proposed as therapeutic and preventive drugs for arteriosclerosis, hyperlipidemia, etc., which are a type of typical adult disease. etc.,
These drugs are not always fully satisfactory, and the desire for more effective drugs is increasing. On the other hand, it is said that 35% of blood cholesterol drugs, which can be a direct cause of these diseases, are absorbed from food, and various nutrients in food have a close relationship with the increase and decrease of blood lipids. So-called dietary therapy, which improves medical conditions by consuming therapeutic foods for a long period of time and changing eating habits, is widely practiced. This can be said to be the most preferable therapy because it can be done at home without worrying about side effects. Polysaccharides obtained by culturing certain microorganisms (e.g., JP-A-57-29292) and dietary fibers obtained from corn fiber (e.g., 1971-36947)
Attempts have been made to add it to foods and drinks as food ingredients. However, it is extremely complicated and difficult to separate and collect the effective parts from the culture medium or raw materials, so it is impossible to provide these to the market at a low price. Therefore, the present invention provides a cholesterol-lowering agent that does not require troublesome steps such as separation, collection, and washing, and can be easily ingested by using a microbial cell culture alone or by adding it to various foods and drinks. The purpose is to provide In other words, the present inventors have discovered that various microorganisms belonging to the genus Streptococcus proliferate well in various edible media containing milk raw materials, carbohydrate raw materials, legume raw materials, or grains as main raw materials, and that the viable microorganisms We discovered that dead bacteria can effectively lower blood cholesterol and triglyceride levels, and that these bacteria originate from so-called intestinal bacteria and are virtually non-toxic when administered orally. This led to the completion of the invention. Hereinafter, the microorganisms, culture medium, manufacturing steps of the cholesterol-lowering agent of the present invention, usage forms, pharmacological effects, and acute toxicity of the agent will be explained in detail. Microorganisms In the present invention, various microorganisms belonging to the genus Streptococcus may be used, and among them, Streptococcus faecium, Streptococcus fuecalis, Streptococcus bovis, Streptococcus evium, and Streptococcus. Suitable examples include Cus duulans, Streptococcus salivarius, Streptococcus miteis, and Streptococcus equinus. Furthermore, specific examples of particularly useful bacterial strains in the present invention are listed below along with their accession numbers.

[Table] Mycological Properties In terms of mycological properties, the microorganisms used in the present invention have the same properties as those shown in the known literature for the same classification of bacteria. That is, the following documents are referred to regarding the mycological properties and culture conditions of the microorganism of the present invention. 1 Bergey's Manual of Determinative
Bacteriology, 8th ed., 490-509 (1974) 2 Int. J. Syst. Bact. 16 114 (1966) 3 Microbiol. Immunol. 25 (3), 257-269 (1981) 4 J. Clin. Pathol. 33 53-57 (1980) 5 J.General Microbiol., 128 713-720
(1982) 6 Applied Microbiol., 23 (6) 1131−1139
(1972) Here, the main mycological properties of the various bacterial strains mentioned above are summarized as follows.

【table】

[Table] Medium Main raw materials for the edible medium that can be used in the production of the cholesterol-lowering agent of the present invention include whole milk, skim milk, skim milk powder, whey, or milk products such as enzyme-treated proteins contained in these. raw materials, honey,
Examples include carbohydrate raw materials such as molasses, soybean raw materials such as soy milk and soy milk whey, and grains such as wheat flour and rice flour. Also, the medium concentration is 0.01 to 5%, more preferably 0.1 to 1.0
It is about %. Cultivation of bacterial cells The conventional method for culturing lactic acid bacteria in fermented milk production may be followed. That is, first, the medium prepared as described above was
Heat sterilize at 110°C for 10-30 minutes. After cooling to approximately 40°C, Streptococcus microorganisms are inoculated at approximately 10 ⁵ cells/ml and cultured aerobically at 37 to 40°C. However, as mentioned above, since various edible media can be used, the culture time may be different from usual. For example, in a medium prepared with skim milk powder and water, the bacterial cell concentration is approximately 10 ⁸ as shown in the experimental example below.
It takes more than 11 hours to reach the bacterial cell concentration per ml, whereas a liquid medium containing trypticase, yeast extract, and tryptose in tap water achieves the same bacterial cell concentration within 6 hours. Pharmacological Effects As shown in the experimental examples below, the cholesterol-lowering agent of the present invention extremely effectively lowers blood cholesterol and triglyceride levels, and its active ingredient, the bacterial cells, is cultured in an edible medium. Therefore, it is not necessary to completely wash and remove inedible components such as heavy metals that are contained in ordinary culture media, and by simply subjecting the culture to freeze-drying, spray-drying, etc., various It becomes possible to freely add it to foods. Therefore, it is extremely easy to treat or prevent diseases such as arteriosclerosis, hyperlipidemia, hyperlipoproteinemia, xanthomatosis, cholelithiasis, hypertension, and diabetes at home in a long-term manner. This makes it possible. The dose of the present agent is usually 10 ⁶ to 10 6 dead bacteria.
10 ¹³ pieces/Kg body weight/day, more preferably 10 ⁸ to 10 ¹¹
body weight/day. Acute toxicity As shown in the experimental examples below, the LD ₅₀ value of the present agent is:
8.9×10 ⁸ to 1.3×10 ¹⁰ for those consisting of viable bacterial cells
cells/mouse (intraperitoneal administration), and in the case of dead bacteria, the number is 6×10 ¹³ cells/mouse (intraperitoneal administration) or more for any type of bacteria. Furthermore, in the case of oral administration, both live and dead bacteria are substantially non-toxic. Destruction treatment of microbial cells In order to make the active ingredients in the microbial cells act more effectively, it is desirable to destroy the microbial cells using an autoclave or ultrasonic waves. Next, part 1
Give an example. Example 1 Each of the above-mentioned microorganisms was inoculated into medium 5 consisting of 5% skim milk powder, and aerobically statically cultured at 37°C for 10 hours to prepare a culture solution with a viable bacterial count of 5 x 10 ⁷ /ml. Autoclave the solution at 115°C for 10 minutes to obtain a suspension of destroyed bacterial cells. Example 2 Culture solution obtained in the same manner as Example 1 was heated to 15KC.
Perform ultrasonic disruption treatment for 60 minutes to obtain a suspension of disrupted bacterial cells. Concentration of Bacterial Cells The solid content of the edible medium according to the present invention is within the range of 0.01 to 5% as described above. When added to food or drink, the flavor of the food or drink itself may be significantly impaired.
Therefore, depending on the type of food or drink to be added, it may be necessary to concentrate the bacterial cells in the culture. Here, the method of concentrating bacterial cells by centrifugation can be considered the most preferable method because the operation is simple and economical. Unbroken cells are
Since it can be obtained as a precipitate in 5 to 10 minutes at 700 to 1000 x g, it can be concentrated to 1/10 to 1/100 using centrifugation conditions within this range. The bacterial culture is dried to a final moisture content of 1~
Even when reducing the amount to about 3%, it may be desirable to perform centrifugation as a pretreatment. Drying of Culture The bacterial cell culture obtained by the method described above is dried by an appropriate means. The drying method is selected depending on the usage type, such as freeze drying, spray drying, foam-mat drying, centrifugal thin film drying, or foam drying. An example is shown below. Drying Example 1 Cultivate microorganisms for 8 hours according to the method of Production Example 10,
Bacterial cells are collected from the culture solution by centrifugation, suspended in 10% skim milk, and then dispensed into ampoules (bacteria number concentration: 10 ⁹ /ml). The ampoule is cooled to -30°C, frozen, dried under vacuum, and sealed. Live bacteria can be stored for a long time at about 5°C. Moisture content is 2%. Drying Example 2 A culture solution in which microorganisms were cultured for 10 hours according to the method of Production Example 2 was neutralized with sodium bicarbonate and dried at 110℃.
Sterilize by heating for 10 minutes, then concentrate for a short time at 85°C in a thin-film falling type vacuum concentrator to reach a solid content of 50%. This is emulsified at 57℃ using a 100Kg/cm ² single-stage homogenizer, uniformly blown with N ₂ gas using a microporous glass blower, and then cooled to 13℃ and dried to form a porous foam. Obtain a dry concentrate. When pulverized, it has excellent condensation properties, and therefore becomes a beverage additive that can be added to tea, coffee, etc. and easily dissolved. Usage form The cholesterol-lowering agent of the present invention is composed of bacterial cells obtained from an edible medium, and as mentioned above, various processing steps can be performed relatively easily, so it can be used in various forms when used. can be taken. In other words, the bacterial cell culture as it is,
Alternatively, it can be consumed as food or drink simply by adding additives such as seasonings, or it can be consumed as is or after the above-mentioned concentration, drying, etc. It can also be added to foods. For example, when obtaining lactic acid drinks, fermented milk, soft drinks and other foods characterized by sourness, the culture can be added as is, but the flavor of the tea, seasoning, or other product itself must not be impaired. For foods, we remove as much of the culture medium as possible and concentrate the bacterial cells to make it possible to ingest the prescribed dose.
In addition, the form of use of the agent of the present invention will depend on the nature or characteristics of each food to which it is added and the dose of the agent to be ingested, such as tea, etc., which requires drying for convenience of preservation. It is chosen. Next, examples of production, pharmacological effects, acute toxicity, and food composition of the cholesterol-lowering agent according to the present invention will be shown. Example 1 (Production Example) A production example of the cholesterol-lowering agent according to the present invention is shown below. Production example 1 Skim milk powder prepared to have a weight concentration of 10%
Heat sterilize at 110°C for 10 minutes to obtain Streptococcus fuecalis ADV9001, Streptococcus faecium ADV1009, Streptococcus duulans ADV3001, and Streptococcus evium AD2003 individually to a viable bacterial count concentration of approximately 10 ⁵ cells. /ml and statically cultured at 37°C. Figures 1 and 2 show changes in viable bacteria concentration and pH changes.The maximum viable bacteria concentration for all four strains was approximately 10 ⁸ cells/ml, and in skim milk powder,
It was shown that it could grow well. Also, Streptococcus fuecalis
In those inoculated with ADV9001, the PH decreased to PH4.2, and in those inoculated with Streptococcus faecium ADV1009, the pH decreased to PH4.4 and the milk curdled. In those inoculated with AD2003, the pH did not decrease much and the milk did not curd even after one week of culture. Next, use the bacterial cell culture solution cultured for 10 hours using the above method.
The bacterial cells were concentrated to 1/10 by centrifugation at 3000 rpm for 15 minutes, and when this was spray dried, a granular white powder was obtained. Production Example 2 Commercially available milk was heat sterilized at 110°C for 10 minutes, and the above four strains were inoculated and cultured in the same manner as in Production Example 1. The viable cell count concentration and pH after 24 hours are shown in Table 3. These four strains proliferated well in commercially available milk. Regarding the curdled milk, the results were similar to those in Production Example 1. Next, the culture solution of the bacterial cells cultured in the above method for 10 hours was treated in the same manner as in Production Example 1 to obtain a white powder. Production Example 3 Commercially available soybeans were soaked in water overnight, then ground, water was added in an amount 8 times the weight of the soybeans before soaking, heated at 80°C for 10 minutes, and filtered through a cloth to obtain soymilk. This at 120℃
It was heat sterilized for 10 minutes, and the four strains from the previous example were inoculated and cultured in the same manner as in the previous example. The viable cell count concentration and pH after 24 hours are shown in Table 3. All four strains grew extremely well, with 24
Within hours the soy milk curdled. Production Example 4 Mix 1 volume of commercially available molasses (manufactured by Dainippon Sugar Co., Ltd.) and 9 volumes of water, adjust the pH to 7 with 1N NaOH,
It was heat sterilized at 110°C for 10 minutes. The previous four strains were inoculated and cultured in the same manner as in the previous example. Viable cell count concentration after 24 hours and
The pH is shown in Table 3. All four strains proliferated, but especially
Streptococcus fuecalis ADV9001,
Streptococcus faecium ADV1009 grew well. Production Example 5 When the four strains from the previous example were inoculated into a liquid medium prepared as below in the same manner as in the previous example and cultured statically at 37°C under aerobic conditions, all the strains grew well and about 6 hours after inoculation. A viable cell count concentration of 5×10 ⁷ cells/ml or more was obtained. Next, the culture solution obtained by culturing for 8 hours according to the above method was centrifuged (4000 rpm, 10 minutes) to concentrate the bacterial cells to 1/100, and this was freeze-dried to obtain a solid material. Composition of liquid medium Distilled water 1 part Trypticase 25g Yeast extract 15g Tryptose 10g Production example 6 Skim milk powder adjusted to a weight concentration of 0.8% was heat sterilized at 110℃ for 10 minutes, and the four strains from the previous example were transformed into live bacteria. Inoculate so that the number concentration is approximately 10 ⁵ cells / ml,
It was statically cultured at 37°C. Table 3 shows the viable cell count concentration and pH after 24 hours. Production Example 7 Commercially available milk was diluted 10 times, heat sterilized at 110°C for 10 minutes, and the previous 4 strains were inoculated and cultured in the same manner as above. The viable cell count concentration and pH after 24 hours are shown in Table 3. Next, the culture solution obtained by culturing for 10 hours according to the above method was centrifuged (4000 rpm, 10 minutes) to concentrate the bacterial cells to 1/100, and then a freeze-dried product was obtained. Production Example 8 Soybean milk with a solid content of 0.6% was heat sterilized at 120°C for 10 minutes, and the four strains from the previous example were inoculated and cultured in the same manner as in the previous example. The viable cell count concentration and pH after 24 hours are shown in Table 3. The culture solution obtained after 10 hours by the above method was centrifuged in the same manner as in the previous example, and this was spray dried to obtain a granular powder. Production example 9 1 volume of commercially available molasses (manufactured by Dainippon Sugar Co., Ltd.) and water
After mixing 11 volumes and adjusting the pH to 7 with 1N NaOH,
It was heat sterilized at 110°C for 10 minutes. The four strains in the previous example were inoculated and cultured in the same manner as in the previous example. The viable cell count concentration and pH after 24 hours are shown in Table 3. Next, the culture solution obtained by culturing for 10 hours by the above method was subjected to ultrasonic disruption treatment (15KC, 60 minutes) to obtain a disrupted bacterial cell suspension. Production Example 10 When the four strains from the previous example were inoculated into a liquid medium prepared as below in the same manner as in the previous example and cultured statically in an aerobic manner at 37°C, all strains proliferated well and about 6 hours after inoculation. A viable cell count concentration of 2×10 ⁷ cells/ml or more was obtained. The culture solution obtained by culturing for 8 hours using the above method was heated at 115°C for 10 minutes in an autoclave.
Next, this disrupted bacterial cell suspension was centrifuged (4000 rpm, 15 minutes) to obtain a lyophilized fraction containing concentrated disrupted bacterial cells. Composition of liquid medium Distilled water 1 part Trypticase 6g Yeast extract 3g Tryptose 2g

[Table] Example 2 (Pharmacological effect) Hereinafter, an experimental example regarding the pharmacological effect and acute toxicity of the cholesterol-lowering agent according to the present invention will be shown. Experimental Example 1 The culture of Production Example 10 above was freeze-dried without cell destruction treatment, and this was used in normal and germ-free mice (male).
16 weeks old, average weight 19 g; 10 rats in each group) and normal rats (16 weeks old, male, average weight 232 g; 10 rats in each group) were added to the diet equivalent to ¹⁰ viable bacteria and allowed to ingest ad libitum. The animals were kept on diet alone for 4 weeks. Next, arterial blood was collected from the inferior aorta of these mice and rats and centrifuged to obtain a serum sample, which was then treated with Cholescuit (trade name; manufactured by Kanto Kagaku Co., Ltd., Zurkowski method) and triglyceride TG Wako (trade name; manufactured by Wako Pure Chemical Industries, Ltd.). Cholesterol and triglyceride levels in serum samples were measured using the acetylacetone extraction method. The composition (weight %) of the diet is as follows. Composition of the diet Casein 20 Soybean oil 10 Wheat starch 61 Minerals 4 Vitamin mixture 2 Filter paper powder 3 The results obtained are shown in Table 4. In the table, "cholesterol load" or "fructose load" refers to the addition of 1% cholesterol to the feed. This shows the case of using feed supplemented with wheat starch or wheat starch completely replaced with fructose, and the numerical values are the reduction rates compared to the non-administered group. Experimental Example 2 The freeze-dried product obtained in Production Example 10 was used in regular rats (16-week-old male, average weight 235 g; 10 rats in each group).
Normal and germ-free mice (male 16 weeks old, average weight 18 g;
(10 animals in each group) were orally ingested an amount equivalent to ^10.10 dead bacteria every day for 4 weeks. Arterial blood was then collected from the inferior aorta of these rats, and cholesterol and triglyceride levels were measured in the same manner as in Experimental Example 1. Diet was given in the same manner as in Experimental Example 1. The results are shown in Table 5. Experimental Example 3 The culture of Production Example 10 was subjected to continuous centrifugation at 12,000 rpm, the bacterial cells were collected, washed with physiological saline, and suspended in physiological saline to obtain 50 ml of bacterial solution (10 ¹¹ /ml).
obtained from normal rats (male 18 weeks old, average weight
238g; 15 mice in each group), normal and germ-free mice (18 weeks old male, average weight 31g; 10 mice in each group) for 12 weeks, 10 ¹¹
The drug was orally administered daily, and the reduction rates of serum cholesterol and triglyceride were measured in the same manner as above. The results are shown in Table 6. In addition, in the table, "cholesterol load" or "fructose load" indicates the case where 1% cholesterol was further added to the above feed or the feed where wheat starch was completely replaced with fructose.
The numerical value is the reduction rate compared to the non-administration group. Experimental Example 4 The viable bacterial cell saline suspension of Experimental Example 3 was further washed twice with physiological saline, and then washed with physiological saline (0.85%
50ml of bacterial suspension obtained by suspending in NaCl aqueous solution)
(10 ¹¹ /ml) at 115°C for 10 minutes to obtain a bacterial cell suspension. This was carried out using normal rats (male 18 weeks old, average weight).
246g; 15 mice per group) and germ-free mice (male, 18 weeks old,
After orally administering 10 to ¹¹ mice (average body weight: 30 g; 10 animals in each group), they were kept for 12 and 8 weeks, and the rate of reduction in blood cholesterol and triglyceride was measured using the same method as described above. The results are shown in Table 7.

【table】

【table】

【table】

[Table] Experimental Example 5 S. faecium was produced by the method of Production Example 10 above.
ADV1009 was cultured, the culture was freeze-dried using the same method, and this was applied to hyperlipidemic rats (male 18
Amount equivalent to 10 to 11 dead bacteria was added to the diet shown in Table 2 for 2 weeks and orally ingested to mice (10 ^{to 10} in each group, 1 week old, average weight 233 g, reared on a cholesterol-loaded diet, every day) for 2 weeks. Next, the cholesterol levels in the arterial blood of these rats were measured in the same manner as in Experimental Examples 1 to 4. The results are shown in Figure 3. Changes in the reduction rate of blood cholesterol and triglyceride in rats were observed depending on the culture time. Experimental example 6 ICR mouse (male 6 weeks old, average weight 30.0±0.5
g) and the physiological saline used in Experimental Example 3 above.
0.5ml suspension per mouse 9×10 ⁹ , 9×10 ⁸ , 9
An equivalent amount was administered intraperitoneally to three stages of bacterial counts (10 mice in each group) of ×10 ⁷ bacteria, and the mice were observed for 14 days to see if they were alive or dead. Table 8 shows the LD ₅₀ values (number of bacterial cells/mouse) calculated according to the Behrens-Karber method. In addition, in the case of dead bacteria, no matter which type of bacteria it is,
LD ₅₀ value is 6×10 ¹³ cells/mouse or more (intraperitoneal administration)
Naturally, it was completely non-toxic when administered orally. Table 8 S. faecium ADV1009 6.3×10 ⁹ S. fuecalis ADV9001 3.8×10 ⁹ S. evium AD2003 4.2×10 ⁹ S. dudulans ADV3001 8.9×10 ⁹ Example 3 (Food formulation example) The cholesterol-lowering agent according to the present invention is It can be taken alone or as is by adding additives such as seasonings and fragrances, but it is also possible to add it to various foods and use it as a preventive medical food. Extremely spacious. Examples of food formulations using this agent are shown below. Food composition example 1 (Powdered soup) Ingredients Mixture (g) Cooked bean powder 3600 Wheat flour 135 Dried yeast powder 90 Dried onion 90 Salt 11 White koshiyo 91oz MSG 12 Powdered bay leaf 3 Powder of production example 2 300 Food composition example 2 (Ochazuke) Paste) Raw materials Mixture (g) Grain 2.5 Paste 0.75 Seasoning granules 3.3 Freeze-dried product of production example 10 0.3 Food composition example 3 (Curry roux) Raw materials Mixture (g) Beef tallow 40 Sugar ester 0.5 Flour (weak) 31.7 Salt 10 Sugar 2 MSG 1 Skim milk powder 1.5 Curry powder 6.2 Onion powder 1.6 Caramel 0.5 Adipol beef (powder) 5 Dried foam from Production Example 6 0.8 Food composition example 4 (Hamburger steak) Raw materials Mixture (g) Minced meat 20 Plant protein 10 Onion 40 Eggs 10 Bread 10 Baked flour 3 Salt 1 Koshiyo 0.4 Margarine 5 MSG 0.8 Dry foam from Production Example 9 0.5 Food composition example 5 (Fruit milk drink) Ingredients Mixture (g) Skimmed milk 70 Fruit juice 5 Citric acid 0.3 Sugar 10 Pigment 0.1 Flavoring 0.2 Stabilizer 0.4 Culture of Production Example 1 14 As an example of the cholesterol-lowering agent of the present invention, which is the easiest way to use it at home and can be one of the forms that best meets the purpose of the present invention, there is Additives may be mentioned. This manufacturing method involves concentrating the culture and drying it as a foam, as shown in the example of the drying process for the bacterial culture.
Alternatively, it is characterized by drying using a form-mat drying method. The powder obtained by this method is used in an amount equivalent to 5 x ¹⁰⁷ to 5 x ¹⁰⁹ microbial cells per person (50 to 50 for the additive processed in Drying Example 2).
When added to tea, coffee, yogurt, juice, etc., it quickly mixes or dissolves.
Does not impair the flavor or appearance of beverages. Therefore, the use of the drug in this form makes it possible to easily carry out long-term treatment and prevention of arteriosclerosis, etc., without significantly changing daily eating habits. Furthermore, as shown in an example below, by adding an appropriate amount of the drug to therapeutic food each time, dietary therapy can be more effectively carried out. An example of a food for the treatment of hyperlipoproteinemia Breakfast: Coffee or tea (added the equivalent of 3 x 10 ₉ bacteria) Bread (rye and wheat) 30g Margarine 10g Cottage cheese 60g Snacks Skimmed milk yogurt ( Add 80mg of the spray-dried product from Production Example 6) 150g Kunetske 8g Margarine 5g Lunch Tonkatsu Pork 100g Oil 5g Carrot dish Carrot 150g Margarine (Add 24g of the spray-dried product from Production Example 8)
(mg added) 5g Potatoes 60g Pears 100g Dinner Black tea (added the equivalent of 2 x 10 ₉ bacteria) Whole grain bread 50g Margarine 10g Ham 40g Tomato salad Tomato 100g Onion 10g Oil 3g

[Brief explanation of drawings]

Figure 1 shows the number of viable bacteria in the medium, and Figure 2 shows the number of viable bacteria in the culture medium.
FIG. 3 is a diagram showing changes in PH over time. In the figure, A is S. duurans ADV3001, B is S. evium AD2003,
C represents S. faecium ADV1009, and D represents S. faecalis ADV9001. FIG. 3 is a diagram showing changes in blood cholesterol and triglyceride reduction rates in hyperlipidemic rats depending on culture time of bacterial cells. The solid line A represents the cholesterol reduction rate, and the dashed line B represents the cholesterol reduction rate.
indicates triglyceride reduction rate.

Claims (1)

[Claims] 1 selected from the group consisting of Streptococcus faecium, Streptococcus fuecalis, Streptococcus evium, Streptococcus salivarius, Streptococcus duulans, Streptococcus miteis, and Streptococcus equinus. Obtained by culturing one or more types of microorganisms in an edible medium whose main raw material is one or more types selected from the group consisting of milk raw materials, carbohydrate raw materials, legume raw materials, and cereals. A food or drink characterized by containing a culture as a cholesterol-lowering active ingredient.