JPS6148916B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a method for producing granular microbial cells that are suitable for long-term storage and reach the intestines with less annihilation by gastric juices even after oral ingestion, as well as an apparatus suitable therefor. BACKGROUND ART Conventionally, when preserving many microorganisms such as bacteria and yeast, methods have been used to dry and preserve the microorganisms. For drying, the bacterial cells isolated from the culture solution are left as they are.
Alternatively, starch, lactose, protein, or organic or inorganic salts are added to this, and the moisture content is reduced to 10% or less by low-temperature ventilation drying, vacuum drying, or freeze drying. For example, Bifidobacterium, which is said to be lacking in the adult intestines, can be ingested raw by mixing it with drinks or yogurt, but it is also possible to dry it from a place that is not suitable for storage and mix it with other foods. This is widely practiced, and Japanese Patent Publication No. 58-46293 describes a base tableting formulation containing one or more of starch, starch hydrolyzate, or protein with a moisture content of 4% or less;
A method for producing a tablet confectionery containing Bifidobacterium by mixing freeze-dried Bifidobacterium and compressing the mixture into tablets is disclosed, and Japanese Patent Application Laid-Open No. 14967/1983 discloses a method for producing Bifidobacterium-containing tablets by mixing Bifidobacterium cells and raw starch and freeze-drying the mixture. A method is disclosed for increasing the survival rate of. Dried Bifidobacterium produced by the above method has a considerable shelf life, but since it is basically in powder form and has a large contact area with air, special consideration is required for preservation, and it is difficult to take it into the body. If the bacteria are dispersed uniformly, a considerable number of bacteria will die under the influence of gastric juices, and a small percentage of them will reach the intestines. Furthermore, it is well known that even if lactic acid bacteria, yeast, etc. are molded by granulation, extrusion, etc. and dried by known methods, there are many problems with storage stability. The present inventors have fundamentally solved the drawbacks of the above drying method, and have created dried bacterial cells that are easy to handle, have good storage stability, and can reach the intestines without being killed by gastric juices even after being ingested. As a result of extensive research, we found that instead of simply adhering to the bacterial surface as with conventional drying aids, we suspended the bacterial cells in a protective film-forming solution and reacted with a coagulating salt solution to solidify the bacteria. Focusing on the fact that it is good to form a uniform protective film on the body surface, we first uniformly mix live bacterial cells and a protective film-forming solution, then pour the mixed solution into a coagulating salt solution to coagulate it into a lump. They succeeded in developing a method for separating and drying the coagulated material and coating it with an oil or fat having a melting point above body temperature, as well as an apparatus suitable for this process. As the bacteria used in the present invention, microorganisms for food, drug manufacturing or processing, or general industrial use such as lactic acid bacteria and yeast bacteria can be used, and microorganisms that are ingested orally such as Bifidobacterium are particularly suitable. Examples of bifidobacteria include Bifidbacterium infantis, Bifidbacterium longum, and Bifidbacterium infantis.
Iongum) and Bifidbacterium adlecsentis. The above microorganisms are cultured in a natural or synthetic culture medium containing sugars, salts, and growth factors, and after culture, the cells are usually separated using a centrifuge and concentrated, and the concentrate is diluted with water or physiological saline. Wash and centrifuge again. The above-mentioned washing operation may be repeated 2 to 3 times depending on the degree of concentration of the bacterial cells. The obtained washed bacterial cells can be used as they are, since the culture medium components have been sufficiently removed, resulting in bacterial cells with little off-flavor, or if necessary, they can be further dehydrated before use. The protective film forming solution to be mixed with the bacterial cells contains a substance such as sodium alginate, potassium alginate, pectin, or glucomannan that binds to metal ions such as calcium ions to form a protective film on the surface of the bacterial cells. To this, it is preferable to add substances such as starch, dextrin, sucrose, sodium glutamate, and sodium ascorbate, which have a humidity control function and prevent rapid dehydration during drying and prevent oxidation. Examples of the composition of such a mixture include, for example, 5 to 10 parts of Bifidobacteria cells, 10 to 50 parts of starch, 0.1 to 1.0 parts of sodium alginate, and 0.5 parts of sodium glutamate as solids.
2.0 parts, and 0.5 to 1.0 parts of sodium ascorbate. In addition, as the coagulating salt solution, a substance that has the function of binding with the main component of the protective film forming solution to form a film on the surface of the bacterial cells is used, and in practical terms, it does not kill the bacterial cells and does not harm the human body. Calcium lactate, harmless
A neutral solution of about 1% at 5 to 25°C, such as calcium chloride, is used. An example in which Bifidobacterium Iongum is separated in the manner described above to produce Bifidobacterium granules will be described below with reference to the accompanying drawings. In the figure, 1 indicates a raw material mixing tank, which is equipped with a stirrer 2 to uniformly mix bifidus cells, sodium alginate, starch, sodium ascorbate, and sodium glutamate in the above ratio. At this time, the viscosity of mixture A is usually about 200 cp. The resulting mixture A is then transferred via pump 3 to mixture storage tank 4 . The storage tank 4 is a closed type, and the upper part has a pipe 6 communicating with an air compressor 5 as a pressure regulating device.
and a pressure regulating valve 6' are provided to keep the appropriate pressure at all times, e.g.
Extrusion pressure from the storage tank 4 is applied at a pressure of 3 kg/cm ² . Also, a stirrer 7 is installed at the bottom of the storage tank 4.
This prevents bacterial cells, starch, etc. from settling.
Further, a pipe 8 is connected to a desired position in the storage layer 4, and the other end of the pipe 8 is opened to a large number of distribution pipes 9.
A large number of nozzles 10 are attached to this distribution pipe 9 facing downward, and the inner diameter of the nozzles 10 is 1 mm or less.
In particular, the range of 0.2 to 0.5 mm is good. Furthermore, each nozzle 10 is preferably provided with a valve 11 so that the flow rate can be adjusted or the injection can be stopped. With the above configuration, the inside of the reservoir 4 is pressurized by the air compressor 5, so the mixture A is injected from the tip of the nozzle 10, but the injection amount depends on the pressure of the air compressor 5 and the adjustment of the valve 11. Therefore, it is possible to adjust the amount of ejection, but instead of the air compressor 5, a pump or other device that can adjust the amount of ejection may be provided in the middle of the pipe 8. A coagulation liquid tank 12 is provided below the nozzle 10, and a solution of calcium lactate or calcium chloride is put therein to serve as a coagulation aid B. Also, an overflow gutter 13 is provided on one side, and a coagulation liquid storage tank 14 is provided on the other side.
The coagulating liquid B can be supplied through the metering pump 15 and the liquid level is approximately 3.
Position it ~10cm below. Therefore, the mixture A from the nozzle 10 is always injected under constant conditions, solidifies in a short period of time in the coagulation liquid tank 12, becomes granular, and sinks. At this time, since the bacterial cells in mixture A are uniformly suspended in the sodium alginate solution, the surfaces of the particles first come into contact with calcium ions and instantly form a scarlet film, and the calcium ions gradually penetrate inside the bacterial cells. Also forms a film, so the whole solidifies. The extrusion pressure from the nozzle 10 is preferably adjusted by the diameter of the nozzle 10, the composition of the mixture A, and the distance between the nozzle 10 and the liquid surface. Granules of desired size up to large granules can be produced.
If the extrusion pressure is too strong or the distance between the nozzle and the liquid surface is insufficient, the mixture A cannot be made into granules but becomes continuous rod-shaped, so care must be taken. In this way, the mixture A becomes coagulated particles C that do not stick to each other and settles downward. In order to take out the coagulated particles C, a screen 16 of 24 to 32 meshes is run in a direction perpendicular to the flow of the coagulated liquid B, and pulleys 17, 17...
A gutter 18 is provided on the discharge side of the screen 16, and water is sprayed from a nozzle 19 provided above for cleaning. Therefore, the coagulated particles C are washed, and when the screen 16 further rotates, the direction changes downward and falls into the container 20. However, the coagulated particles C that still adhere to the air blowing nozzle 21 provided on the back side of the return side of the screen 16 It is removed by a further air flow and collected in the container 20 in its entirety. The recovered coagulated particles C contain a large amount of water and have poor preservability if left as is, so they are then dried. For drying, a vacuum drying method, a freeze drying method, or a ventilation drying method using an inert gas or the like can be employed, and the moisture content after drying is preferably about 1 to 5% in the form of granules. The granules obtained by the above method have the structure shown in FIG. 3 with the oil and fat layer D removed, and the surfaces of the bacterial cells A and starch B are coated with a calcium alginate film C, and the inner diameter of the nozzle 10 is 2 to 3 mm thick. Although it has a structure that has expanded to twice the diameter, as can be seen from the figure, the coating is complete and uniform, and the coating layer is extremely thin compared to the conventional mechanical mixture of bacterial cells and drying aid. Not only does it have significantly improved preservative power, but it is also convenient to handle because it is in granular form. Next, Table 1 shows the number of viable bacteria when the bacterial granules obtained in Example 1 were wrapped in aluminum foil and stored at room temperature and 37°C.

[Table] The granules obtained by the method of the present invention can be ingested as is, mixed with other foods, or used for processing other foods or for industrial purposes. It is possible. After oral ingestion, the bacterial cells are coated with calcium alginate, so they are unlikely to be killed by gastric juice, but to further reduce the chance of death, the bacterial granules can be coated with oil or fat. The oils and fats used cannot be the low melting point oils conventionally used for coating microorganisms, but rather high melting point oils that do not melt at human body temperature, for example 37℃~
It is a hydrogenated oil with a melting point of 43℃. Coating with fats and oils can be done by conventional methods, but it is convenient to use a fluidized bed granulation coating device. This device sends gas from below to make the granules flow while spraying and coating them with oil and fat.The coating significantly improves the resistance to gastric juices, and most of the bacteria reach the intestines. An example of this will now be shown with an experimental example. The experiment was carried out using a fluidized bed granulation coating device Flow Coater FLO-5 model (trade name) for bifidobacteria granules produced by the method of Example 1, and 800g of hydrogenated oil with a melting point of 40℃ was added to 2Kg of bifidobacteria granules at 45℃. This was done by melting and spraying into an air stream at 50°C. As shown in Figure 3, the resulting coating has a layer of oil and fat on the surface of the granules of bacterial cells A and starch B, and this coating is coated with artificially prepared gastric juice (salt 0.2%, (containing 0.32% pepsin and adjusted to pH 1.5 with hydrochloric acid) and reacted with stirring in a constant temperature bath at 37° C. for 1 to 5 hours. After reaction
The number of viable Bifidus bacteria was determined using a standard method at pH 7.0. The results are shown in Table 2.

[Table] Values.
The number of viable bacteria obtained as shown in the above table fluctuates within experimental error, and it can be said that there is virtually no dead or sterilized number. Furthermore, when it reaches the small intestine, it is digested into a fat film by digestive juices, and the granules also disintegrate, so some of the bacteria may die due to the influence of the digestive juices. In tests conducted by the present inventors using artificial intestinal fluid, the mortality rate was about 20%, so most of the bifidus can reach the large intestine alive. The above experiment is about Bifidobacterium,
The method of the present invention can be applied to the drying of many microorganisms including lactic acid bacteria and yeast, and since the obtained bacterial cells are thinly and uniformly coated with a protective film such as calcium alginate, they can be stored as is. Even when added to neutral or acidic high-moisture foods, it does not lose its shape, is less affected by the moisture in the food, and maintains its activity for a longer period of time than conventional dried bacterial cells. It is possible. This will be explained below using examples. Example 1 Bifidbacterium Iongum was cultured anaerobically at 37°C for 18 hours in a skim milk medium digested with thanasinase (trade name), and after culture, the bacterial cells were concentrated using a centrifuge. Add physiological saline to the concentrated solution to almost the original volume, perform centrifugation again, and wash. Repeat the washing twice to obtain about 700 g of viable bacterial cells (about 140 g as solid matter) per 10 medium. Obtained. To 700 g of the above-mentioned viable cells were added 200 g of potato starch, 20 g of monosodium glutamate, and 10 g of sodium L-ascorbate, and then 2.00 ml of a 2.5% sodium alginate solution dissolved in advance was added and the whole was mixed uniformly. The above-mentioned liquid mixture was heated using the device shown in Figure 1 with an inner diameter of 0.3 mm.
The mixture was continuously extruded at a pressure of 1.3 kg/cm ² at a distance of about 5 cm from a 1.0 mm nozzle toward the surface of a coagulation liquid tank containing 1% calcium lactate, and coagulated into granules. Scoop this up with a 32-mesh wire mesh screen, spray it with water, thoroughly dehydrate it, and place it on a metal tray for 5 minutes.
It was spread to a thickness of ~10 mm and freeze-dried. The average particle size of the obtained granules was approximately 0.9 mm, and the specific volume was 2.75 ml/
The yield was 340 g, white granules with a moisture content of 2.0%. The above Bifidobacterium granules contain 5.8 Bifidobacterium
It contained 10 ⁸ pieces/g, maintained its activity for a long time when stored in a sealed container, and was suitable as an additive to foods such as yogurt. Example 2 PH containing 10% milk whey protease decomposition product
Using the medium of 6.2, Streptococcus thermophilus was cultured at 40°C for 4 hours, and centrifuged and washed in the same manner as in Example 1 to yield approximately 700 g of bacterial cells (solid matter) per 10 medium. About 150g) was obtained. The above bacterial cells were mixed with a protective film forming solution in the same manner as in Example 1, granulated and freeze-dried, with an average particle size of approximately
1.0mm white granules with a specific volume of 2.75ml/g and a moisture content of 2.0%
Obtained 350g. The above granules contained 1.2×10 ¹⁰ cells/g, and after being packaged in aluminum foil with no air permeability or moisture permeability, a storage test was performed, and the results shown in Table 3 were obtained.

[Table] Example 3 Cells of Bifidbacterium Iongum obtained by the method of Example 1
For 700dl, 400g potato starch, 20g monosodium glutamate, sodium L-ascorbate
10 g of 5% rhotomexyl pectin (100 ml) was added and mixed uniformly. The above mixed solution was prepared in the same manner as in Example 1, and the inner diameter was 0.4 mm.
A 1% calcium lactate solution is injected from a height of 5 cm from a nozzle, granulated, scooped out with a 32-mesh screw, washed with running water, thoroughly dehydrated, and freeze-dried. Average particle size: 1 mm, specific volume: 3.0 ml/ g, moisture 2.0%
530 g of white granules were obtained. The number of Bifid bacteria in these granules was 2.0×10 ⁸ cells/g, making them suitable for long-term storage. Example 4 Saccharomyces cerevisiae (Sac.
Cellevicae) was cultured in cane molasses medium at 30℃ with aeration, centrifuged and washed to produce approximately 700 dl per 1 kg of molasses.
Live bacterial cells were obtained. The above bacterial cells were mixed with 200 g of cornstarch and 1000 ml of 5% rhomethoxyl pectin, extruded from a height of 7 cm from a nozzle with an inner diameter of 0.5 mm in the same manner as in Example 1, granulated, and scooped out with a 32-mesh screen. Wash thoroughly with water and dry in vacuum to determine the average particle size.
Approximately 450 g of granules of 1.2 mm and 5.0% moisture were obtained. These granules did not lose their fermentation power even after long-term storage.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the apparatus, FIG. 2 is a cross-sectional view of the coagulation liquid tank of FIG. 1, and FIG. 3 is an enlarged cross-sectional view of coated granules. 1... Mixing tank, 4... Storage tank, 6, 8... Piping,
10...Nozzle, 12...Coagulation liquid tank, 14...Coagulation liquid storage tank, 16...Screen, 20...Container,
A...Mixture, B...Coagulation liquid, C...Coagulation particles.

Claims (1)

[Claims] 1. Mix viable bacterial cells and a protective film-forming solution, inject the mixed solution into a coagulating salt solution to coagulate it, take out the resulting coagulated product, dry it, and bring it to a melting point above body temperature. A method for producing bacterial cell granules, characterized by coating them with oil and fat. 2. The method for producing bacterial cell granules according to claim 1, wherein the viable bacterial cells are Bifidobacterium. 3. Claim 1, characterized in that the mixture of viable bacterial cells and a protective film forming solution contains 5 to 10 parts of bacterial cells, 10 to 50 parts of starch, and 0.1 to 1.0 parts of sodium alginate in terms of dry weight ratio. A method for producing bacterial cell granules. 4. A storage tank for storing viable bacterial cells and a protective film forming solution;
a nozzle communicating with the storage tank and hanging downward;
It consists of a coagulation tank provided so that the liquid level is located below the nozzle, and a device for adjusting the ejection amount of the nozzle, and the nozzle has an inner diameter of 1 mm or less, preferably 0.2 to 1 mm.
0.5 mm, and a distance between the tip of the nozzle and the liquid level of the coagulation tank is about 3 to 10 cm.