JP2006280263A - Bacillus bifidus cell powder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain dry powder comprising Bacillus bifidus cells of good survivability, maintaining probiotic functions that are exerted by the intake of Bacillus bifidus cells including intestine-conditioning activity or intraintestinal harmful bacteria-suppressive activity, immunoregulatory activity, cholesterol level-lowering activity and prophylactic activity. <P>SOLUTION: The Bacillus bifidus cell powder of high survivability is obtained by the following procedure: Ungelatinized starch powder or its suspension and a Bacillus bifidus culture liquid are mixed together so as not to gelatinize into a suspension, which is then spray-dried in fine circular or ellipsoidal droplets through a nozzle or under pressure. When observed with a polarizing microscope, a cross (interference ring) indicating that starch is not gelatinized can be seen. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a bifidobacteria cell powder having good survival characteristics that maintains a probiotic function.

  Bifidobacteria are widely used in the production of foods, and are used when producing dairy products such as fermented milk and lactic acid bacteria beverages. Furthermore, in recent years, probiotic functions, which are bioregulatory functions that are exerted by ingesting live cells such as intestinal harmful bacteria suppression action, immunoregulatory action, cholesterol lowering action, infection defense action, etc. have been revealed one after another. Developments have been made to use bifidobacteria cells themselves or bifidobacteria cultures as materials for health foods and pharmaceuticals. Moreover, these bifidobacteria are used by blending them with livestock and poultry feeds. Until now, there has been a problem that antibiotics have been administered in large quantities for the purpose of health maintenance management of livestock and poultry, and so-called drug-resistant bacteria have occurred, but by administering bifidobacteria with probiotic function There are also reports that the occurrence of drug-resistant bacteria can be suppressed while maintaining the health of livestock and poultry.

  When bifidobacteria cells are administered to humans or animals, they are powdered and used as tablets, capsules, granules, powders, and the like. When bifidobacteria cells are made into tablets, capsules, granules, powders, etc., it is necessary to mix the bifidobacteria cell powder with excipients and other components, but when trying to mix the powders uniformly If the particle diameter of the powder to be mixed is not uniform or the particle shape is different, a so-called classification phenomenon occurs and the powder cannot be uniformly dispersed. In addition, when bifidobacteria cell powder is to be encapsulated in a capsule, seamless capsule, or soft capsule with a small particle shape, the bifidobacteria cell powder must be encapsulated in a small space. When it is round or oval, the bifidobacteria cell powder is uniformly and efficiently dispersed in the capsule. However, in the case of a powder prepared by freeze-drying Bifidobacteria cells and mechanically pulverizing them, the average particle size is irregular even though the particle size is uniform to some extent. There is a drawback that it is difficult to uniformly and efficiently enclose an irregularly shaped powder in the space. The same applies to tableting with a tableting machine. Furthermore, since a block-like solid is formed, there is a disadvantage that a powder with a uniform particle size cannot be obtained unless further steps such as mechanical pulverization are performed, and even if pulverization is performed, the particle shape becomes linear. Does not become round or oval.

On the other hand, the bifidobacterial cell powder obtained by spray drying was incorporated into an excipient such as starch and the particle shape became circular or oval, and was prepared by drying and mechanically grinding by a method such as freeze drying. There is an advantage that the bifidobacteria cells are uniformly and efficiently dispersed in the capsule from the powder having a linear particle shape. In addition, spray drying is much cheaper in energy and equipment than freeze drying in which a sample is frozen and dried in a reduced pressure state.
Therefore, in order to prepare bifidobacteria cell powder, spray drying is overwhelmingly more advantageous than freeze-drying in terms of ease of use and preparation cost, and preparation of bifidobacteria cell powder by spray drying is A lot has been done so far.

Bifidobacteria cells are widely used in foods, health foods, pharmaceuticals, and feeds. However, as one of the possibilities for expanding the use of Bifidobacteria cells, Attempts have been made to increase the persistence. In general, the probiotic function refers to a useful bioregulatory function that is exerted on an ingested human or animal by supplying live bacteria to the human or animal. Therefore, in fermented milk, fermented milk lactic acid bacteria beverages, and lactic acid bacteria beverages as fermented foods, the number of viable bacteria is defined as a product standard, and the maintenance of the number of lactic acid bacteria including bifidobacteria is required as a condition for product establishment. Therefore, maintaining the viable count in these products is an important issue.
It is also important to keep the probiotic function from being inactivated by maintaining the viable cell count and at the same time ingesting the above-mentioned bifidobacteria cells.

  However, many Bifidobacteria are vulnerable to various environmental stresses such as acid, temperature, pH, salt concentration, water activity, bile acid, etc., and their use is often limited when actually used in the above products. . As mentioned above, spray drying is more advantageous than freeze drying in terms of particle shape and cost of bifidobacteria cell powder, but bifidobacteria are weaker to heat, and when spray drying, liquid is made into fine particles and turned into hot air. As a result, the survival rate decreased, and the probiotic function of bifidobacteria was often impaired.

  As a general method for obtaining bacterial powder such as lactic acid bacteria, there can be mentioned a starch gelatinized bacterial preparation method (for example, see Non-Patent Document 1). In this method, gelatinized starch is added to the cell suspension as an excipient, and a protective agent such as L-cysteine, L-lysine or sodium glutamate is added and dried under reduced pressure. According to this method, the viable cell count was 7.2 log / g by the vacuum drying method (as a control experiment, 9.6 log / g by freeze-drying and 8.4 log / g by spray-drying). Although this invention shows the use of starch as an excipient, all "gelatinized starch" is used.

  Also, by improving the spray nozzle of the spray dryer, the lactic acid bacteria and bifidobacteria suspension were submicronized so that the particle diameter of the suspension of lactic acid bacteria and bifidobacteria was 10 μm or less, and the survival rate was improved by spray drying. There is a report that it has become possible to prepare fungus powder (see, for example, Patent Document 1). The gist of the present invention is to make the particle diameter of the suspension of lactic acid bacteria and bifidobacteria submicron by devising a spray nozzle. In the specification of the present invention, pregelatinized starch is shown as one of the binders that can be incorporated into the cell fluid, but this is “gelatinized starch”.

  Furthermore, regarding the spray drying conditions for Bifidobacteria cells, gelatinized starch and Bifidobacteria cells were mixed and spray-dried, and this was generated when Bifidobacteria cells were incorporated into the starch network. There is a report that the persistence was good (for example, see Non-Patent Document 2). According to this report, the survival rate of bifidobacteria was about 10% when gelatinized starch was not used, but about 30% when gelatinized starch was added and spray-dried. It is stated that it has risen.

Although various methods for preventing the decrease in the survival rate of bifidobacteria have been disclosed in this way, none of the methods show a result that the survival rate has been significantly improved. It is complicated because it involves changes in itself and processes.
Japanese Patent No. 3363438 Mitsuoka Chitose, Bifidobacteria research, published by Nihon Bifidobacteria Center, (1994) K. O'Riordan, D.C. Andrews, P.A. Conway, J.A. Appl. Microbiol. 91: 1059-66 (2001)

The present invention maintains a probiotic function that is exhibited by ingesting bifidobacterial cells such as intestinal regulation, intestinal harmful bacteria suppression, immunoregulatory, cholesterol lowering, and infection protection. It is an object of the present invention to obtain a dry powder containing bifidobacteria having good persistence.
In addition, if the particle size of the Bifidobacteria cell powder is non-uniform or the particle shape is different, classification may occur when mixing with other components, and uniform dispersion will not be possible, and attempts to encapsulate in the capsule It is an object of the present invention to solve the disadvantage that it is difficult to uniformly and efficiently encapsulate an irregularly shaped powder in a minute space when molding and tableting.

As a result of intensive studies, the inventors of the present invention have found that by mixing a bifidobacteria culture solution and an ungelatinized starch powder or an aqueous suspension of starch in a suspended state so as not to gelatinize, an irregular starch The present invention was completed by successfully obtaining a bifidobacteria cell powder having a uniform particle size, a uniform particle shape, and a good survival rate that does not contain crushed materials.
Further, in the present invention, when preparing a dry powder containing bifidobacteria cells having a probiotic function, a culture solution of bifidobacteria and a non-gelatinized starch powder or suspension are used. This is a method for producing a bifidobacteria cell powder having good survival characteristics, characterized in that the suspension is mixed so as not to gelatinize, and the suspension is spray-dried.
As a prior art, it is disclosed that powdered gelatin powder or suspension and lactic acid bacteria culture liquid are mixed and spray-dried. However, starch powder or suspension without gelatinization and Bifidobacterium culture liquid are disclosed. The method of the present invention, in which the powder is suspended and spray-dried so as not to gelatinize, reduces the survivability while maintaining the merit of spray-drying, in which a powder having a uniform particle size and a constant particle shape is obtained. This produces a special effect of preventing the disadvantages of spray drying.

  When starch is suspended in water and heated, the starch granules absorb water and gradually expand, and when heating is continued, the starch granules eventually disintegrate and dissolve. This phenomenon is called gelatinization. Starch is composed of amylopectin, which is sparingly soluble in water, which forms the skeleton, and water-soluble amylose, which is contained in the starch. Gelatinization of starch is caused by water molecules entering the gaps between the amylopectin in the crystalline structure. It is thought that the structure is loosened and each branch spreads into the water. The gelatinization temperature varies depending on the starch raw material, but is usually 60 to 80 ° C. In the present invention, the starch and water are mixed at room temperature (about 25 ° C.) below the gelatinization temperature to obtain a suspension in which starch is not gelatinized, and this is mixed with a cell culture solution of Bifidobacterium. And spray drying. Non-gelatinized starch powder and Bifidobacterium cell culture solution may be mixed and spray-dried. When the bifidobacteria cell powder of the present invention obtained as described above was observed with an optical microscope, no bifidobacteria cell was observed, and two powders of starch powder and bifidobacteria cell powder surrounded by starch were mixed. It is in a state. When water was added in this state, bifidobacteria were observed. Therefore, it is considered that the surface of the bifidobacteria is covered with starch.

  The present invention also provides a non-gelatinized starch powder or suspension and a bifidobacteria culture solution mixed so as not to be gelatinized, and this suspension is finely rounded with a nozzle or pressure. Or, in order to spray-dry into elliptical droplets, the particle size of the bifidobacteria cell powder is a normal distribution, and when observed with an optical microscope, the starch powder and the bifidobacteria cell powder covered with starch are They do not contain irregular shaped starch fragments, and all consist of almost spherical particles. The shape of the bifidobacteria cell powder of the present invention is almost the same as that of the conventional method in which it is mixed with gelatinized starch and spray-dried. When the bifidobacteria cell powder of the present invention is observed with a polarizing microscope, the bifidobacteria cell powder is Among the fungus body powders, the starch powder can have a characteristic cross (interference ring) indicating that the starch is not gelatinized.

  The bifidobacteria powder obtained by the conventional method obtained by mixing the bifidobacteria suspension with gelatinized starch and spray-drying was observed with an optical microscope. Similarly, the bifidobacteria were not observed, and the cells were observed when water was added in this state. The shape is almost the same as that of the bifidobacteria cell powder of the present invention, but when observed with a polarizing microscope, the cross-linked (interference ring) cannot be seen when the mixture is spray-dried by mixing with gelatinized starch.

The bifidobacteria powder produced according to the present invention is powdered by freeze-drying, mixed with starch that has not been gelatinized through a mechanical grinding process, or powdered by freeze-drying and then pasteed. The difference between the powder mixed with non-converted starch and mechanically pulverized can be confirmed by observation with an optical microscope as described above, whether it is linear powder, spherical or elliptical.
Moreover, it differs from what was obtained by the conventional method which mixed the Bifidobacterium suspension with gelatinized starch and was spray-dried by observing the presence or absence of a cross (interference ring) with a polarizing microscope as described above. Can be confirmed.

The bifidobacteria cell powder obtained by the present invention has the following effects.
(1) The bifidobacteria cell powder spray-dried using the non-gelatinized starch is excellent in survivability compared with the bifidobacteria cell powder spray-dried using the gelatinized starch.
(2) Compared to freeze-drying, the particle size of the bifidobacteria cell powder is uniform and the particle shape is the same, so classification that occurs when mixed with other components does not occur and uniform dispersion can be achieved. Moreover, when it is going to enclose bifidobacteria cell powder in a capsule and a tablet, the cell of a bifidobacteria can be uniformly and efficiently disperse | distributed to the micro space in a capsule and a tablet.
(3) Bifidobacteria cell powder can be obtained by spray drying, which requires much less energy and facilities than lyophilization.
(4) When it is prepared by freeze-drying, a block-like solid is formed, so that powder with a uniform particle size cannot be obtained unless mechanical grinding and other steps are further performed. It is possible to obtain a powder having a uniform particle size which does not contain irregular shaped starch fragments.
(5) A bifidobacteria cell powder having a uniform particle size and the same particle shape can be obtained using a normal spray drying apparatus, regardless of a method such as improvement of a spray nozzle or drying under reduced pressure.

  Bifidobacteria used in the present invention include Bifidobacterium longum, B. breve, and B. animalis used in fermented milk. ), B. pseudolongum, and the like. However, the bifidobacteria to be used is not limited to these species.

  The bifidobacteria used in the present invention can be cultured using various media such as a milk medium, a medium containing milk components, and a semi-synthetic medium. Examples of such a medium include reduced skim milk medium, BL medium, MRS medium and the like obtained by reducing skim milk powder and heat sterilization. In addition, these bifidos can be used in plant materials such as milk and soybeans, and in liquid media prepared by adding appropriate nutrient sources such as yeast extract, peptone, vitamins, minerals, etc., or by causing proteolytic enzymes to act on them. Incubate the fungus.

  Culture methods include normal liquid media, those in which the upper layer of the liquid media is replaced with inert gas or carbon dioxide, neutralization culture with a constant pH, batch culture, continuous culture, etc., and an appropriate fraction size. Can be mentioned by removing the lactic acid and acetic acid which are the culture products of bifidobacteria using the separation membrane, but the culture method is particularly limited to these methods as long as the cells grow well. It is not limited.

  After completion of the culture, the cell culture solution is sufficiently mixed with starch powder or suspension not gelatinized so that the starch does not gelatinize, and is subjected to spray drying before the starch precipitates. Non-gelatinized starch suspension is a mixture of starch powder in water or warm water below the gelatinization temperature. In addition, a bacterial cell culture solution concentrated to an appropriate concentration by centrifugation or membrane separation, or a bacterial cell that has been washed an appropriate number of times, preferably 1 to 3 times, with an appropriate washing solution such as physiological saline or sterilized water. The body culture solution can also be spray-dried before the starch precipitates by mixing well with non-gelatinized starch powder or suspension so that the starch does not gelatinize. After washing the cells and mixing with a suitable spray-drying protective agent, mix well with non-gelatinized starch powder or suspension to prevent starch from gelatinizing and spray-dry before starch precipitates You can also.

  The starch used in the present invention is not particularly limited, and any starch may be used as long as it is widely used, such as potato, sweet potato, onion, wheat, rice, tapioca. it can. These starches can be used alone or in combination.

  The spray drying conditions are preferably a spray pressure of 50 to 200 kPa, a hot air temperature of 60 to 150 ° C, and an exhaust air temperature of 30 to 120 ° C. Examples of the nozzle for spraying include a centrifugal nozzle and a two-fluid nozzle, but are not particularly limited as long as they are used for normal spray drying.

The viable count of bifidobacteria contained in the powder obtained according to the present invention is usually 1 × 10 ⁸ cells / g to 5 × 10 ¹³ cells / g, preferably 1 × 10 ¹⁰ cells / g to 5 × 10 ¹² cells. / g.

  The bifidobacteria cell powder spray-dried using the non-gelatinized starch of the present invention is superior in viability to the bifidobacteria cell powder spray-dried using the gelatinized starch. In the present invention, the culture solution of bifidobacteria and the non-gelatinized starch powder or suspension are sufficiently mixed so that the starch does not gelatinize. To cover the surface of bifidobacteria. When spray-drying with non-gelatinized starch, it is better for survival. When spray-drying with non-gelatinized starch, some starch granules are gelatinized to surround the cells. This is probably because the heat applied to the cells is used for gelatinization of the starch, and the cells are less damaged by the heat. On the other hand, when spray-dried using gelatinized starch, which is a conventional method, starch has already been gelatinized, so heat from spray-drying is directly applied to the cells, and the cells are damaged by heat and survive. Is considered to be low.

The bifidobacteria cell powder obtained by the present invention can be used as it is as a health food having a probiotic function, and tablets such as sugar-coated tablets and tablets, powders, granules, capsules, etc. It is also possible to process and use it.
In addition, the bifidobacteria cell powder of the present invention is fermented milk prepared by adding lactic acid bacteria to a milk as a starter alone or mixed with lactic acid bacteria, and fermented milk prepared by adding these powders to a fermentation mix. Can be used for Furthermore, it can be used for various dairy products, meat products, marine products, desserts, dressings, health foods, noodles, beverages and other various foods and drinks.
Furthermore, the present invention can be used by being added to livestock feed such as cattle, pigs and chickens and pet food such as dogs and cats. It can also be used as a starter for promoting silage fermentation.

  Hereinafter, the bifidobacteria cell powder of the present invention will be described in detail with reference to Examples and Test Examples, but these are merely illustrative, and the present invention is not limited thereto.

Bifidobacterium longum FERM P-10657 strain was cultivated at 37 ° C for 16 hours in BL medium (Mitsuoka Chichiho, edited by Bifidobacterium, published by Nihon Bifidobacteria Center, 1994). Concentrated twice. This culture concentrate and an equal amount of non-gelatinized 10% tapioca starch suspension are mixed so as not to be gelatinized at 25 ° C., and spray-dried at an inlet temperature of 100 ° C. and an outlet temperature of 50 ° C. in a spray dryer, A Bifidobacterium cell powder of the present invention was obtained. The number of viable bacteria in the powder was 1.4 × 10 ¹¹ cells / g.

[Test Example 1]
The bifidobacteria cell powder obtained in Example 1 was suspended in isopropanol, and the particle size distribution of the powder was measured with a laser particle size distribution analyzer (Shimadzu SALD-2000J). As a control, it was mixed with the same amount of 10% tapioca starch suspension mixed at 70 ° C. and gelatinized with the culture concentrate, and spray-dried under the same conditions as in Example 1 in a spray dryer. The particle size distribution of the bifidobacteria cell powder was measured.
The results are shown in FIG. The bifidobacteria cell powder of the present invention shows a normal distribution. The particle size distribution of the bifidobacteria cell powder using the gelatinized starch suspension as a control also showed a normal distribution.
When observed with an optical microscope, the bifidobacteria cell powder of the present invention consists of almost spherical particles, and is observed when the dried powder is mechanically micronized by a method that does not prepare droplets such as freeze-drying. It did not contain any linear, irregular crushed material. Furthermore, when the bacterial cell powder was observed with a polarizing microscope, a characteristic cross (interference ring) indicating that starch was not gelatinized could be seen. The bifidobacterial cell powder using the gelatinized starch suspension as a control has almost the same shape as that of the present invention, but a cross (interference ring) could not be seen.

Bifidobacterium longum FERM P-10657 strain is inoculated into BL medium (Mitsuoka Chichiho, Bifidobacterium research, published by Japan Bifidobacteria Center, 1994) supplemented with lactose to a final concentration of 1%, While maintaining the pH at 6.0 with NaOH water, the cells were cultured at 37 ° C. for 16 hours. The culture was concentrated 20 times in a centrifuge. Suspend and mix the same amount of non-gelatinized 10% potato starch suspension at 25 ° C in this culture concentrate so that it will not gelatinize at 25 ° C, and spray it with a spray dryer at an inlet temperature of 125 ° C and an outlet temperature of 60 ° C. It dried and the bifidobacteria cell powder of this invention was obtained. As a control, it is mixed with the same amount of 10% potato starch suspension mixed and gelatinized at 70 ° C in the culture concentrate, and spray-dried under the same conditions in a spray dryer to obtain a Bifidobacterium cell powder. It was. The number of viable cells in the Bifidobacterium cell powder of the present invention is 8.5 × 10 ¹¹ cells / g, the control is 1.6 × 10 ¹¹ cells / g, and the cell of the present invention mixed with an ungelatinized starch suspension The survival rate was more than 5 times higher than the bifidobacteria powder using the gelatinized starch suspension.
When the bifidobacteria cell powder of the present invention was observed with an optical microscope, it consisted only of substantially spherical particles and did not contain linear, irregularly crushed material. Furthermore, when the cell powder was observed with a polarizing microscope, a characteristic cross (interference ring) indicating that starch was not gelatinized could be seen.
As a control, the bifidobacteria cell powder using the gelatinized starch suspension had almost the same shape as that of the present invention, but a cross (interference ring) could not be seen.

Bifidobacterium longum FERM P-8743 strain is inoculated into BL medium (Mitsuoka Chitoshi, Bifidobacterium research, published by Japan Bifidobacteria Center, 1994) supplemented with lactose to a final concentration of 1%, While maintaining the pH at 6.0 with NaOH water, the cells were cultured at 37 ° C. for 16 hours. The culture was concentrated 20 times in a centrifuge. Suspend and mix the same amount of non-gelatinized 10% potato starch suspension at 25 ° C in this culture concentrate so that it will not gelatinize at 25 ° C, and spray it with a spray dryer at an inlet temperature of 125 ° C and an outlet temperature of 60 ° C. It dried and the bifidobacteria cell powder of this invention was obtained. As a control, the same amount of 10% potato starch suspension mixed and gelatinized with the culture concentrate at 70 ° C was mixed and spray-dried under the same conditions in a spray dryer to obtain a Bifidobacterium cell powder. It was. The viable cell count in the Bifidobacterium cell powder of the present invention is 8.7 × 10 ¹¹ cells / g, the control is 5.9 × 10 ¹¹ cells / g, and the cell powder of the present invention mixed with non-gelatinized starch is viable. The residual rate was 1.4 times higher than the bifidobacteria cell powder using gelatinized starch.
When the bifidobacteria cell powder of the present invention was observed with an optical microscope, it consisted only of substantially spherical particles and did not contain linear, irregularly crushed material. Furthermore, when the Bifidobacterium cell powder of the present invention was observed with a polarizing microscope, a characteristic cross (interference ring) indicating that starch was not gelatinized could be seen. The bifidobacteria cell powder using gelatinized starch as a control has almost the same shape as that of the present invention, but a cross (interference ring) could not be seen.

  50 g of bifidobacteria powder obtained in Example 1, 140 g of lactose, 8 g of sugar ester, and 2 g of carboxymethylcellulose were mixed and compressed by a compressed tablet manufacturing machine (y-5010-Q, manufactured by Fuji Yakuhin Kikai Co., Ltd.) ˜3 tons) to produce tablets.

  40 g of bifidobacteria cell powder obtained in Example 2 was added to and mixed with 40 g of an equal mixture of vitamin C and citric acid, 45 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose. The mixture was packed in a stick-shaped bag to produce 1.5 g of nutritional health food per bag.

  Inoculate yogurt mix (2% skim milk powder added to raw milk, heated at 100 ° C for 10 minutes) with 0.1% bifidobacteria powder obtained in Example 1 and 1% Lactobacillus acidophilus skim milk culture Then, after filling into a paper cup, it was cultured at 37 ° C. for 6 hours to prepare fermented milk.

The harvested alfalfa material grass is lightly dried, cut into 15 to 25 mm with a mount cutter, and 1 part of the bifidobacteria cell powder obtained in Example 2 and 5 parts of the Lactobacillus acidophilus culture powder are mixed, This bacterial cell mixture was inoculated at 10 ⁵ cfu per 1 g of material grass. Polysilos were stored at 30 ° C. for 10 days to prepare silage.

The particle size distribution of the bifidobacteria cell powder (invention product) obtained in Example 1 and the bifidobacteria cell powder (control product) using gelatinized starch is shown.

Claims (3)

  Bifidobacteria powder having good survival characteristics, characterized by containing non-gelatinized starch and not containing irregularly shaped starch crushed material.   The food-drinks or feed containing the bifidobacteria cell powder of Claim 1.   A bifidobacteria characterized by mixing a culture solution of bifidobacteria and a non-gelatinized starch powder or suspension so that the starch is not gelatinized, and then spray-drying the suspension. Manufacturing method of fungus body powder.

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