JPH0367552A - Stable spore-forming variable cell drug, production thereof and pellet - Google Patents

Abstract

PURPOSE:To obtain the subject drug capable of improving the growth, etc., of domestic animals due to participation in keeping the balance of gut flora and capable of pelletizing by blending a hydrocarbon component derived from grains and tubers with spore-forming viable cells belonging to Bacillus genus. CONSTITUTION:An aqueous dispersion is prepared by using a hydrocarbon component derived from grains (corn powder, rice bran, etc.) and tubers in an amount of 0.01 pts.wt. (preferably 0.1-10 pts.wt.) based on 1 pts.wt. spore- forming cells belonging to Bacillus genus (preferably Bacillus toyoi) containing <=about 10<13>-10<15> viable cell per 1kg cell weight) and subsequently subjected to spray drying, etc., to obtain the objective drug. By mixing the above- mentioned drug into a feed, etc., for animals and pelletizing the resultant mixture by a pelletizer, the subject pellet-shaped feed can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a stable spore-forming live bacteria preparation, a method for producing the same, and a stable spore-forming live bacteria pellet using the preparation.

[Conventional technology]

Live bacteria preparations for feed are feeds in which useful living microorganisms are mixed with feed, and are often used for the prevention and treatment of animal diseases, the promotion of health, etc. Among them, Bifidobacteria, a type of intestinal bacteria.
M) survives in large numbers in the intestines and is known to play a role in preventing intestinal infections, intestinal putrefaction, and diarrhea, so foods and feeds containing live bacteria are commercially available. There is.

Other bacteria belonging to the genus Bacillus are also said to have useful effects, and feeds containing these live bacteria are also known. In other words, food and feed containing such live bacteria are kept in a viable state for humans and animals in order to ensure that the bacteria can survive stably during the manufacturing process, distribution as products, and even before consumption. Various additives are added to live bacterial preparations so that they can enter the body and exert useful effects. Regarding bifidobacteria, asparagine or monosodium glutamate or soluble starch is added to it to withstand high-temperature storage (Special Publication No. 3889/1989), bifidobacterium culture solution, skim milk powder, starch decomposition product, monosodium glutamate, ascorbic acid, etc. Bifidobacterium live bacteria powder with high gastric acid resistance (JP-A-60-188060) obtained by adding acid soda and magnesium salt and freeze-drying, adding raw starch to wet or suspended Bifidobacterium, and freezing. A method for obtaining a powder containing active bifidobacteria that can be stored for a long period of time at room temperature by drying (Japanese Patent Application Laid-Open No. 63-12594), which uses cyclodextrin in which wet or suspended bacterial cells of bifidobacteria are dissolved. A method for producing a bifidobacteria-containing powder that is suspended in a dispersion medium containing bifidobacteria and dried to form a powder, which causes less killing of bacteria during drying, granulation, and storage, and which can be stored for a long time at room temperature (Japanese Patent Application Laid-Open No. 63-251080 No.) etc. are known.

In addition, although it is not used as food or feed, living Fusarium cells are adsorbed onto a zeolite base material and dried naturally, or the same living cells are made mainly of D-sorbitol and a small amount of glutamate is added. There is also known a live bacteria preparation (Japanese Unexamined Patent Application Publication No. 227507/1983) which is prepared by dispersing the bacteria in a dispersion medium and freeze-drying the bacteria to have both the activity and stability of living bacteria.

On the other hand, spore-forming bacteria such as Bacillus form spores and are relatively stable against heat, dryness, and acids, and are useful as live bacteria, so they are used as live bacteria feed (
Japanese Patent Publication No. 53-6211>. Such products include:
Toyoserine (trade name, Bacillus cereu
s var, toyoi, - boat fishing abbreviation Bacill
us toyoi (product name: TOYOSERIN), RAFRIS (
Product name, Bacillus coagulans), Glogen (product name, Bacillus 5ubtil)
is var, natto), paciflor (pac
iflor product name, Bacillus cereus
IP-5832), Bioplus
2B product name, Bacillus licheni
Bacillus formis, Bacillus 5ubtilis
) etc. are known.

As mentioned above, the probiotics not only have nutritional effects such as metabolism of nutritional components and fruit synthesis, but also maintain the balance of intestinal flora, which has the effect of preventing disease infection by suppressing the growth of pathogenic bacteria. Its use in compound feeds has become widespread for the purpose of improving the growth of livestock and feed efficiency by maintaining it. Live bacteria agents are effective when living bacteria reach the gastrointestinal tract, so stability in feed,
It must be stable to acids, alkalis, heat, etc.
In particular, spore-forming bacteria belonging to the genus Bacillus meet the purpose and are therefore preferably used as described above.

In addition, pelleted feed has the following advantages: the feed is homogenized and each grain is a complete feed, the utilization rate of nutrients and energy in the feed is improved, palatability is improved, the amount of increased feed is increased, and feed efficiency is improved. It is widely used as feed because it has the following advantages: it has less bulk and is easier to handle, it saves feeding time and labor, and there is less feed being blown away by the wind or wasted by grains. However, since various feed ingredients in the form of skin powder are blended and solidified into a certain shape by applying pressure and steam in a pellet mill, the above-mentioned Regarding the pellet formation of spore-forming bacteria belonging to the genus Bacillus, there is a problem that even heat-stable spore-forming bacteria may be partially destroyed, reducing the survival rate of the microorganisms and therefore losing their efficacy. Ta.

[Problem to be solved by the invention]

In order to produce pelleted feed using a stable spore-forming live bacteria preparation belonging to the genus Bacillus, the present inventors used a conventionally known stabilizer for Bifidobacterium and other live bacteria and a spore-forming live bacteria preparation. Pelletize the combination of
Various attempts were made to obtain stable spore-forming live bacterial preparations by measuring the viable bacteria rate, but none of them yielded satisfactory results because the bacteria were killed by the heat and pressure during the pelletizing process.

However, completely unexpectedly, a spore-forming live bacteria preparation belonging to the genus Bacillus obtained by spray drying, kneading, and ventilation drying using spore-forming live bacteria and carbohydrate components derived from cereals and potatoes has been found to be suitable for use on animals. The present invention was completed based on the discovery that the decrease in the number of bacteria caused by mixing with feed and pelletizing it was significantly improved and stabilized.

That is, the present invention provides a stable spore-forming live bacteria preparation containing a carbohydrate component derived from cereals or potatoes and a spore-forming live bacteria belonging to the genus Bacillus, a method for producing the same, and feed pellets made using the same.

As an example of the spore-forming live bacteria in the present invention, any bacteria belonging to the genus Bacillus that can be used in feed may be used, such as Bacillus toyoi (333 (illus to
yoi (trade name: Toyoserine), Bacillus coagulans (trade name: Lafrys), Bacillus subtilis varietus
Nut (Bacillus 5ubtilis)
var, natto (trade name, Grogen)], Bacillus cereus
IP5832 (trade name Paciflor)), Bacillus lich
eniformis), Bacillus subtilis (Bac
illus 5ubti1is (product name, Biop
lug 2B)). Among these, preferred are bacteria belonging to Bacillus cereus, which belongs to the genus Bacillus, and a particularly preferred example is Bacillus toyoi.

In addition, cereals used as a base for the live bacteria preparation of the present invention.
Examples of carbohydrate components derived from potatoes include components derived from corn, such as corn flour itself;
It may be prepared by crushing the seeds of G. ne (Gramineae) and drying them if necessary, such as commercially available corn flour and corn grits.

Other aS-derived carbohydrates, such as rice-derived components, include defatted rice bran itself, such as rice (Oryza 5ativa Linne).
A mixture of pericarp, seed coat, ectosperm, aleurone layer, etc. obtained during polishing of naked seeds of Gramineae) may be defatted, dried, and crushed if necessary.

Further, examples of starch include corn starch, rice starch, wheat starch, potato starch, sweet potato starch, etc. These are essentially mixtures of amylose and pectin, and therefore, they can be derived from any grain or potato. Starch may also be used.

Examples of flour derived from this earth change include Tri
ticumsativum Lamarck (Gra
It is a product of crushed and, if necessary, dried seeds of Minaae), and examples include wheat, bran, etc.

In addition, carbohydrates derived from soybeans include, for example, Glycin.
e max Merrill (Leguminos
ae) seeds are crushed and dried if necessary, and examples include defatted soybean flour, soybean mill run, and soybean meal.

These bases can be made into an aqueous suspension by combining, for example, spore-forming live bacteria and one or more bases, and heated in advance to reach a gelatinization temperature of, for example, starchy corn-derived ingredients of 60 to 7.
It is used after being heated to 5° C. or higher, or it is spray-dried without prior heat treatment, or it is kneaded and dried through ventilation. For example, when using a corn-derived component, the corn-derived component is heat-treated at least once in the presence of moisture, kneaded and dried through ventilation, and then powdered using a crushing tool (for example, a speed mill) as appropriate. It may be obtained as a dry powder live bacteria preparation by pulverizing with an oscillator or the like. In addition, when using carbohydrate components derived from grains and potatoes such as rice bran, starch, wheat flour, soybean flour, and potatoes, these may be directly made into an aqueous suspension, or
Alternatively, the heated gelatinized mixture may be mixed with spore-forming live bacteria and spray-dried, or kneaded and dried through ventilation.

Depending on the type of the base used in the present invention, the base used in the present invention may be subjected to treatments such as spray drying or ventilation drying in the form of an aqueous suspension, or treatment such as spray drying or ventilation drying after heating and gelatinization. Although the effect on the stability of the spore-forming live bacteria preparation when pelletized depends on the processing method, it is presumed that the starch component is heated and gelatinized, The effect is also recognized.

The thus obtained dry powder live bacteria preparation is mixed with animal or emergency feed, and the obtained live bacteria mixed feed is pelletized using a conventional pellet molding machine to obtain pelleted feed. As for the feed and additives to be mixed, suitable compounded feed and additives may be used depending on the type of animals and fish such as livestock and poultry to be fed.0 Corn-derived ingredients or defatted rice bran-derived ingredients, starch, and wheat flour used in the present invention The mixing ratio of soybean flour or potato-derived ingredients and spore-forming live bacteria belonging to the genus Bacillus should be at least 0.01 parts by weight of these base ingredients per part of the bacterial weight, and usually 0.01 parts by weight. ~100 parts by weight, preferably 0.1
~IO! It is a quantity part. In addition, approximately 1 per kg of bacterial weight
Contains less than 013 to 1 OIS viable bacteria.

Pelletization of the feed containing live bacteria is carried out by a normal pelletizing method. That is, the above-mentioned dry powder viable bacterial preparation is mixed with appropriate compounded feed and additives, and then pelletized with a die using a pellet wheel (for example, manufactured by Ueda Iron Works, model JlO) while pumping in water vapor. , can be dried to obtain pelleted feed.

It goes without saying that the hardness and size of the pellets vary depending on the type of animal to which they are fed, and can be changed as appropriate. In particular, in Japan, the hardness is soft for chickens, i.e. Monsanto hardness of about 2 to 5 kg, medium hardness for pigs, i.e. Monsanto hardness of about 5 to 7 kg, and hard for rabbits. Monsanto hardness of about 9 to 12 kg is suitable, and pellets may be formed using feeds for these various livestock, poultry and other animals, as well as various fish feeds.

Pelleted feed is produced in this way, and in this pelletizing process, the live bacteria added to the feed are solidified into pellets using steam and high pressure. They are destroyed, denatured by heat, and die to a large extent.

In other words, when spore-forming live bacteria belonging to the genus Bacillus obtained by normal spray drying are mixed with feed to make pellets, the survival rate of viable bacteria varies depending on the hardness of the pellet, but in the case of higher hardness, it is more than 30%. On the other hand, when using a viable bacterial agent mixed with the base of the present invention and spray-dried, a viable bacterial survival rate of 80% or more could be obtained in most cases.

Therefore, assuming a pellet hardness of 8 to 10 kg, approximately 70
% or more, preferably 80% or more of residual bacterial cells is particularly preferably stabilized.

〔Effect of the invention〕

As described above, in the present invention, spore-forming bacteria, preferably spore-forming bacteria belonging to the genus Bacillus, are combined with carbohydrate components derived from corn flour, defatted rice bran or starch, wheat flour, soybean flour, and cereals and potatoes such as potatoes. The spore-forming live bacterial preparation is powdered using a spore-forming microorganism preparation that can be used as animal, emergency feed, or animal feed! ! Even if it is added together with drugs and pelletized, it is possible to maintain the same number of viable bacteria as before pelletization, making it easy to use in feed and having a high economic effect. It is added to fish feed at 0.2 to 1X10' pieces/kg feed weight, and as an animal drug at 5X10' to 2X10'' pieces/kg feed weight.

〔Example〕

The present invention will be specifically described below with reference to Examples, but the present invention is not limited thereto.

Back box 1 Bacillus toyoi (toyoserine) spore viable bacterial cell suspension (
Bacterial count 2. Suspend corn flour (manufactured by Sunny Maize ■) in the composition shown in Table 1 in 1 x 10"/m1 (10% bacterial cell mass) and dry by spray drying (KC-50 model, manufactured by Okawara Processing Machine ■). Then, powdered live bacteria preparations were obtained.

In addition, spray drying is performed at a ventilation temperature of 150°C and an exhaust air temperature of 90°C.
The rotation speed of the atomizer was 10.00 rpm.

In addition, as a control product-1, calcium carbonate powder (Colocalso WB, manufactured by Shiraishi Calcium Co., Ltd.) was used as a base and spray-drying was performed to obtain a powdered viable bacterial preparation.

Fruit 14 Lu Approximately 1.1 x 10 Lu for the test product obtained in Example 1 in various feed
Mix uniformly (matshu) so that the number of bacteria is 6, and pound pellets using a die with a diameter of 3.5 mm while sending steam with a pellet l' Qle (specially manufactured by Ueda Iron Works, model JIO). The product was obtained by drying.

The number of bacteria in the pellet was measured and compared with the number of bacteria in Matushu to examine stability. In addition, since the hardness of pellets varies depending on the feed, the feed was selected to have a graded hardness. Hardness increases (A); rabbit feed (NR
T-I S: Nisseiken (?ll), medium hardness (B); pig feed (artificial milk late feed 2 Japan compounded feed ■), hardness relatively small (C); chicken feed (Feed for young chicks 2 Japan Compounded Feed In) was used.

The hardness of the pellets was measured using a Monsanto hardness meter. The results are shown in Table 2, compared to the decrease in the number of bacteria after making pellets for the control product, the decrease in the number of bacteria was slightly improved for test product 1-d, and for test products 1-a, 1-b, and l. -c was clearly improved. This was particularly noticeable for pellets with high hardness and relatively hardness.

From the above, powder preparations obtained by spraying 0.01 parts or more, preferably 0.1 parts or more of cornflour per 1 part of the bacterial weight can be used with viable bacteria in the pelleting process with feed. It was confirmed that the stability was improved.

Suspend 2 g and 20 g of jitsu corn flour in 200 mJ of water and heat (85°C) to gelatinize the liquid in advance, cool it, add 20 g of Bacillus toyoi cells (21
11 pieces/g), knead well, spread on a flat plate, and dry with ventilation at room temperature or 80°C.

This dried material was crushed with a speed cle and passed through a sieving machine (32 mesh oscillator) to obtain a viable bacterial preparation.

Also, 20g of corn flour and 209mj of water! For the suspension, 20 g of bacterial cells were added, and the mixture was mixed.
A formulation was prepared in the same manner as above. The results are shown in Table 3.

Blood loss side [-( Regarding the test product obtained in Example 3, pellets were produced in the same manner as in Example 2, the number of bacteria was measured, and the number of bacteria was compared with that of mafush to examine the stability of viable bacteria. Ta.

The results are shown in Table 4 for test products 3-a, 3-b, and 3- using a liquid made by heating corn flour to form a paste.
In case of c, the stability of the bacteria during the pelleting process was clearly improved compared to the results of the control product, but for the preparation prepared by simply suspending cornflour in water and drying, as in test product 3-d, the bacteria were significantly improved. No improvement in stability was observed. From the above, it has been found that in the case of corn flour, heating the solution to form a paste-like aqueous solution and using it for drying with bacterial cells has the effect of stabilizing viable bacteria during pelletization.

Actual side 1j- Bacillus toyoi bacterial suspension (number of bacteria 2.lXl0"7m
l: bacterial cell mass 10%) and defatted rice bran (manufactured by Nippon Seishin Oil ■)
were suspended and kneaded with the composition shown in Table 5, and spray-dried according to Example 1, and ventilation-dried according to Example 3 to obtain powdered live bacterial preparations.

Blood Loss Agent q For the test product obtained in Example 5, pellets were produced in the same manner as in Example 2, and the number of bacteria was measured and compared with the number of bacteria in Matushu to examine the stability of viable bacteria.

As the results are shown in Table 6, for defatted rice bran,
In both the ventilation drying and spray drying methods, the stability of the bacteria in the pelletizing process of the powdered viable bacterial preparations obtained was good without being impaired. From the above, it was found that defatted rice bran also improves the stability of live bacteria in the pelletizing process, similar to corn flour.

Fruit 14m Bacillus toyoi (toyocerin) spore viable bacterial cell suspension (
Bacterial count 2.7 x 1 OIO/m1: bacterial cell amount 10%) and starch (
Corn starch (made by Wako Pure Chemical Industries, Ltd.) was suspended in the composition shown in Table 7 below and dried by spray drying (KC-50 model, manufactured by Okawara Processing Machine) to obtain each powdered viable bacterial agent. I got it.

In addition, spray drying is performed at a ventilation temperature of 150°C and an exhaust air temperature of 90°C.
The rotation speed of the atomizer was 10.00 rpm.

In addition, as a control product-2, calcium carbonate powder (Colocalso WB, manufactured by Shiraishi Calcium Co., Ltd.) was used as a base and spray-drying was performed to obtain a powdered viable bacterial preparation.

Skilled criminal 11jL Approximately 1 liter of each type of feed was added to the test product obtained in Example 7.

Mix uniformly (mash) so that the number of bacteria is 1X10', and use a pellet mill (manufactured by Ueda Iron Works, model Jlo) to pound pellets using a die with a diameter of 3.5 mm while supplying steam, and dry. and got the product. The number of bacteria in the pellets was measured and compared with the number of bacteria in the matshu to examine stability. In addition, since the hardness of pellets varies depending on the feed, the feed was selected to have a graded hardness. Hardness increases (A); rabbit feed (NRT-I S
: Nisseiken H)), medium hardness (B); pig feed (artificial milk late stage feed 2 Japan compounded feed ■), hardness relatively small (C): chicken feed (feed for young chicks) ; Japan Compounded Feed ■> was used.

The hardness of the pellets was measured using a Monsando hardness meter.

The results are shown in Table 8 below, as compared to the decrease in the number of bacteria after pelletizing the control product, the decrease in the number of bacteria was improved in all cases, and especially for those with high hardness, test products 7-a,
7-bs? -c was clearly improved. From the above, powder preparations obtained by spraying using 0.01 part or more, preferably 0.1 part or more of starch per 1 part of bacterial weight can be used to reduce the amount of viable bacteria in the pelleting process with feed. It was confirmed that the stability was improved.

Wheat seeds were crushed into a suspension of Bacillus toyoi (toyoserine) spores in the same manner as in Example 7, and dried wheat flour, protein content, 9.3-9.5% thin blade powder (Diamond) was added. : Nippon Seifun Special) was suspended in the composition shown in Table 9 below and dried by spray drying to obtain each powdered viable bacterial agent.

The test product obtained in Example 9 was pelleted in the same manner as in Example 8, the number of bacteria on the pellet was measured, and the results were compared with the number of bacteria on Matsushi to examine stability. , similar results were obtained with the starch of Example 8.

As shown in Table 10 below, good results were obtained.

Soybean seeds were defatted to a suspension of Bacillus toyoi (toyoserine) spores in the same manner as in Example 7, dried, and ground (8
Soybean flour (Sunrichf 1o)
ur Showa (trade name) manufactured by Showa Sangyo ■ was suspended in &ltc shown in Table 11 below and dried by spray drying to obtain each powdered probiotic agent. Breasts I again! 11-
e (Pharma t.

se: Import and sales agency; Iwaki ■, De Melkind
ustrie Veghel bv) was suspended in water and spray-dried in the same manner as above.

Fruit 1 □□□ILL The test product obtained in Example 11 was pelleted in the same manner as in Example 8, the number of bacteria on the pellet was measured, and the stability was examined by comparing it with the number of bacteria on Matsushi.

As a result, similar results were obtained for soybean flour as for the starch of Example 8. Also, milk 1! No effect was observed for (11-e).

The results are shown in Table 12 below, and good results were obtained.

11. Add 2 g each of starch (see Example 7), wheat flour (see Example 9), soybean flour (see Example 11), and hydroxypropyl methyl cellulose (TC-5: manufactured by Shin-Etsu Chemical Co., Ltd.) to 200 g of water.
ml suspension and heating (85℃) to gelatinize the solution in advance, and add 20g of Bacillus toyoi cells (25xlO
10 pieces/g), knead well, spread this on a flat plate, and dry with ventilation at room temperature. This dried material was crushed in a speed mill and passed through a sieving machine (32 mesh oscillator) to obtain a viable bacterial preparation.

The results were as shown in Table 13 below. Note that carboxymethylethylcellulose was also not stabilized like hydroxypropylmethylcellulose.

Regarding the test sample obtained in Example 13, a pellet was produced in the same manner as in Example 8, the number of bacteria was measured, and compared with the number of bacteria in the matshu, the stability of viable bacteria was determined. I looked into gender.

The results are shown in Table 14 below, and the test products 13-a, 13-b, and 13-c, which used a liquid made by heating each base to form a paste, had clear results when compared with the results of the control product. The stability of bacteria in the pellet process was improved. No significant improvement in stability was observed for 13-d.

Claims (19)

[Claims] (1) A stable spore-forming live bacteria preparation containing at least a carbohydrate component derived from cereals or potatoes and a spore-forming live bacteria belonging to the genus Bacillus. (2) The preparation according to claim 1, wherein the grain is corn. (3) The preparation according to claim 1, wherein the grain is rice, wheat, or soybean. (4) The preparation according to claim 1, wherein the carbohydrate component is a starchy or other polysaccharide-containing component. (5) The preparation according to claim 1, wherein the carbohydrate component derived from grains or potatoes is obtained by heat-treating the carbohydrate component derived from grains or potatoes at least once in the presence of moisture. (6) The carbohydrate component derived from cereals and potatoes is at least 0.0% per part by weight of spore-forming live bacteria belonging to the genus Bacillus.
2. The formulation according to claim 1, wherein the amount is 0.01 parts by weight or more. (7) A method for producing a stable spore-forming bacteria preparation, which comprises drying in the presence of at least a carbohydrate component derived from cereals or potatoes, a live spore-forming bacteria belonging to the genus Bacillus, and a non-toxic aqueous medium. (8) The production method according to claim 7, wherein the grain is corn. (9) The method according to claim 7, wherein the grain is rice, wheat, or soybean. (10) The method according to claim 7, wherein the carbohydrate component is a starch or other polysaccharide-containing component. (11) The production method according to claim 7, wherein the carbohydrate component derived from grains or potatoes is obtained by heat-treating the carbohydrate component derived from grains or potatoes at least once in the presence of moisture. (12) The production method according to claim 11, wherein the carbohydrate component derived from cereals or potatoes has been previously heat-treated. (13) The manufacturing method according to claim 11, wherein the heating is dry heat. (14) Carbohydrate components derived from cereals and potatoes are at least 0% per part by weight of spore-forming live bacteria belonging to the genus Bacillus.
．． 8. The method according to claim 7, wherein the amount is 0.01 parts by weight or more. (15) A pellet containing as an active ingredient a spore-forming live bacteria preparation containing at least a carbohydrate component derived from cereals or potatoes and a spore-forming live bacteria belonging to the genus Bacillus. (16) The pellet according to claim 15, wherein the carbohydrate component of the grain/potato is obtained by heat-treating the carbohydrate component derived from the grain at least once in the presence of moisture. (17) The pellet according to claim 16, wherein the carbohydrate component derived from cereals or potatoes has been previously heat-treated. (18) The pellet according to claim 16, wherein the heating is dry heat. (19) Carbohydrate components derived from cereals and potatoes are at least 0% per part by weight of spore-forming live bacteria belonging to the genus Bacillus.
．． 16. The pellet according to claim 15, wherein the pellet contains 0.01 parts by weight or more.