GB2138023A - Bacillus licheniformis used in animal feeds - Google Patents

Abstract

The present invention relates to an animal feed additive and feed containing the microorganism Bacillus licheniformis as active ingredient. They have an excellent effect on increase in animal body weight, survival rate of young pig, growth rate of young cattle, egg-laying rate and the like. The present invention further relates to an effective strain of Bacillus licheniformis MN-001, FERM BP-266.

Description

SPECIFICATION Animal feed additive, feed and microorganism employed therefor (Detailed Explanation of the Invention) The present invention relates to a novel animal feed additive and feed. More particularly, the present invention relates to an animal feed additive and feed containing Bacillus licheniformis as available microorganism. And the present invention further relates to a strain of Bacillus licheniformis M N-00 1, FERM BP-266.

Using feeds for animals have been promptly changed in recent years from using mainly selfsupplying feeds to using formula feeds produced in modern factories to result in a very high ratio of the use amount of formula feeds to the total use amount of all feeds nowadays.

For such formula feeds, it is generally employed to use, as supplemental ingredients, various additives, such as amino acids, minerals, vitamins, antibiotics, enzymes, living microorganism preparations etc. with various objects, such as nutrient replenishment, improvements of digestion and absorption, growth promotion, disease prevention etc., besides main ingredients, such as cereal grains, seed cakes, fish meals, oils and fats etc..

However, with respect to the antibiotics, the uses thereof have now a tendency to be subject to restriction and the scope of their use tends to be limited and reduced, because it is apprehended that the remainings and transferrings of antibiotics into the human body may cause allergies or the change of enteric bacterial flora.

As the enzymes, the preparation of lipase, amylase, cellulose, protease etc. and the mixture thereof have been studied, however, it is the present conditions that the definite effect can not be expected because they presents the irregular effects.

And as the living microorganism preparations, those of Bacillus natto, lactic acid bacteria, butyric acid bacteria, Lactobacillus bifidus etc. have been studied, however, although they present a certain effect, it is not yet adequate.

Accordingly, it is the present condition that the perfect animal feed additive is not available.

In consideration of the present condition described above, the present inventors et al have researched in the wide range for seeking spontaneous microoganisms of genus Bacillus useful for creating an animal feed additive, and as a result found unexpectedly that Bacillus licheniformis is not only harmless but also shows various effects on animals. The present invention has completed on basis of this discovery.

The present invention relates to an animal feed additive and feed containing Bacillus licheniformis as useful microorganism.

Bacillus licheniformis used in the present invention belongs to the group of facultative anaerobic bacteria belonging to the genus Bacillus. According to the present invention, not only spontaneous strains but also artificial mutants thereof may be used effectively. Among these strains, a preferable strain is a mutant named Bacillus licheniformis MN-001 which has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, under the deposition number of FERM BP-266.

Bacillus licheniformis strain MN-001 (referred to hereinafter as strain MN-001) shows the following microbial properties: A.Morphological properties: (1) Bacillus being 0.5 to 0.8m wide by 2 to 3m long.

(2) Gram's strain: Positive (3) Formation of elliptical endospores.

(4) Motility: Positive B. Cultural properties: (1) Normal agar: Abundant growth. No production of pigment.

(2) Medium containing molasses and ammonium sulphate as the major components: Abundant growth. No production of pigment (3) Liquid medium containing sodium chloride: 5% salt:Growth 7% salt:Growth 10% salt:Growth C. Physiological properties: (1) Catalase activity: + (2) Growth under anaerobic conditions: + (3) VP(Voges-Proskauer) test: + (4) Growth under high temperature conditions: at 50"C: + at 55"C: - (5) Acid formation from carbohydrates: Glucose: + L-Arabinose: + D-Xylose: + Mannitol: + D-Tagatose: + (6) Argininedihydrolase activity: + (7) Lecithinase activity: + (8) Starch hydrolysis: + (9) Gelatin hydrolysis: + (10) DNA hydrolysis: + (11) Utilization of citrate: + (12) Utilization of propionate: + (13) Nitrate reduction: + (14) Indole formation: + (15) Deamination of phenylalanin: - Bacillus licheniformis useful in the present invention may be cultured in either liquid medium or solid medium by conventional fermentation techniques.

As a medium, various media, such as natural medium, semi-synthetic medium and synthetic medium, can be widely used.

(i) As the carbon source, the following can be utilized: glucose, sucrose, dextrin, glycerol, starch, molasses and the like.

(ii) As the nitrogen source: peptone, meat extract, yeast extract, dry yeast, soybean cake, urea, thiourea, ammonium salts, nitrates and other organic or inorganic nitrogen-containing compounds.

(iii) As the mineral salt: phosphates, nitrates, carbonates, chlorides etc. of magnesium, manganese, potassium, calcium, iron etc.. Besides, amino acids, vitamins, nucleic acids and the compounds related therewith may be added to the medium. According to the conventional fermentation techniques, the microorganism is cultured at a suitable temperature and for an adequate period, perferably, for example, at a temperature of from 20"C to 50"C and for a period of from 1 2 hours to 5 days.

According to the present invention, thus obtained culture liquid may be used with or without isolating cells by repeat washing. Besides, the culture liquid, processed one thereof and isolated cells may be subjected to a frozen drying or spray drying treatment etc. with or without additives to be formulated. And thus obtained formulation may be used as the feed additive or feed of the present invention.

In order to prepare a general animal feed or feed additive according to the present invention, it is enough to formulate the culture liquid directly as such or in diluted form with the following diluent agents: wheat flour, starch, dextrin etc. and raw materials for feed, for example, cereal grains, chaffs and brans such as deoiled rice bran, cakes of seeds rich in oil and fat, and the like.

And if desired, after isolation of cells from the culture liquid by washing, thus isolated cells may be formulated in the same manner as described above.

Thus obtained living microorganism formulation of Bacillus licheniformis is given to animals in the form of animal feed containing it or in the form of feed additive in an adequate amount, permitting the increases in body weight and growth promotion. The typical examples of the animals covered by the feed additive or the feed of the present invention are enumerated as follows: livestocks, such as beef cattle, dairy cattle, young cattle, pigs, young pigs, sheep, goats, horses, rabbits, dogs, cats, etc.; poultries, such as chicken, laying hens, chicken domestic fowls, seed cocks, domestic ducks, geese, turkeys, quails, small birds etc..

In addition, since an amount of administration thereof varies according to the factors, such as the kinds of the intended animals, the ages of the intended animals and the kinds of other feed ingredients, it is difficult to define it without variation. Generally speaking however, the formulation of the present invention is added to the feed with 102 to 105 cells of living Bacillus licheniformis, preferably with 104 to 1012 cells, and more preferably with 105 to 10'0 cells per 1 kg of the feed.

Hereinafter, the present invention will be illustrated in relation to Production examples and Examples.

Production example (Production of living microorganism formulation of Bacillus licheniformis) 2 parts by weight of molasses, 1 part by weight of ammonium sulphate, 0.1 parts by weight of dibasic potassium phosphate and 0.05 parts by weight of yeast extract were added to 100 parts by weight of tap water to prepare nutrient solution. Thus obtained solution was regulated into a pH of 8.0 by the use of 0.1 N hydrochloric acid of 0.1 N sodium hydroxide to prepare liquid medium. One liter of liquid medium was charged in a jar fermenter and sterilized at 121"C for 1 5 minutes. Thus sterilized liquid medium was inoculated with a precultured Bacillus licheniformis strain MN-001, FERM BP-266 followed by the shaking culture for 24 hours.

Then the cells were collected by centrifuging the culture fluid. The collected cells were added with 1 kg of starch and dried to obtain the formulation of Bacillus licheniformis strain MN-001 (referred to hereinafter as MN-001 formulation). This formulation contains 108 cells per 1 kg.

On the other hand, another formulation was also obtained in the same manner as in the operation described above except that starch was replaced with deoiled rice bran.

Example 1 (Breeding of broilers) 120 Broilers (female: 60, male: 60) were divided into three groups respectively consisting of 40 broilers (female: 20, male: 20) and the feeding tests were carried out for 8 weeks by giving such feed prepared by adding MN-001 formulation obtained in Production example (containing 108 cells per 1 kg of starch) to the basic feed of which composition is shown in the Table 1 below. The control group broilers were breeded by giving such feed prepared by adding starch to the basic feed (starch-containing group).

The results are shown in the Table 2.

Table 1 (Composition of basic feed) 0-4 weeks after 5-8 weeks after the breeding the breeding start start Commercially available feed for broilers for the former period 100 0 (Note*) Commercially available feed for broilers for the latter period 0 100 (Note**) (Note) Standard feed for test made by Nippon Haigoshiryo Co., Ltd.

SD broilers for SD broilers for the former the latter period period** Crude protein 2 23 2 19 Crude fat 2 5 2 6 Crude fiber 5 4 c 4 Crude ash ' 6.5 I 6.5 ME(Metabolizable energy) (Kcal/Kg) 23000 23100 Table 2 Body weight Body weight Increase in Feed at start after 8 weeks body weight conversion (g/broiler) (g/broiler) (g/broiler) (Index) (Index) Control (0.1% starch- 40 2280 2240 2.32 containing (100) (100) group) MN-001 formulation-containing 40 2460 2420 2.13 group (0.1%) (108.0) (91,8) Example 2 (Breeding of piglets) 30 weaned piglets of 25-day-old (female: 15, male: 1 5) were divided into three groups respectively consisting of 10 piglets (female: 5, male: 5) and the following feeding tests were carried out for 14 weeks: The first group i.e. control group piglets were breeded by giving such feed prepared by adding deoiled rice bran to the basic feed to which composition is shown in the Table 3 below (deoiled rice bran-containing group). The second group piglets were breeded by giving such feed prepared by adding the Bacillus natto formulation "Glogen 8" (trade name for the product of Eisai Co., Ltd., containing 108 cells/g of starch) to the basic feed. The third group piglets were breeded by giving such feed prepared by adding the MN-001 formulation obtained in Production example (using deoiled rice bran and containing 108 cells/g) to the basic feed.

The results of these breeding tests are shown in the Table 4.

Table 3 (Composition of basic feed) 0-1 2-5 6-14 week weeks weeks Corn 28.1 34.3 65.9 Wheat flour 20 25 Soybean cake 7 8 6.5 Fish meal 7 5 5 Skim milk 27 15 Deoiled rice bran - - 7.3 Wheat bran - - 10 Glucose 10 10 Minerals, Vitamins etc. 0.9 2.7 5.3 (Parts by weight) Table 4 Body weight Body weight Increase in Feed at start after 14 weeks body weight conversion (kg/piglet) (kg/piglet) (kg/piglet) (Index) (Index) Control (0.1% deoiled 7.3 67.9 60.6 2.80 rice bran- (100) (100) containing group) Glogen 8containing 7.3 70.1 62.8 2.69 group (0.1%) (103.6) (96.0) Mn-001 formulation-containing 7.3 73.8 66.5 2.55 groups (0.1%) (109.7) (91.1) From these results, it will be seen that the great increase effects in body weight, feed conversion and digestion ratio can be obtained by adding the living microorganism of Bacillus licheniformis to an animal feed.

Example 3 40 Female-pigs, with body weight of from 1 70 Kg to 1 80 Kg and being with young, were divided into two groups respectively consisting of 20 female-pigs, and the following feeding tests were carried out for 80 days or more (from 60 days before the delivery to 21 days after the delivery): The control group female-pigs were breeded by giving such feed prepared by adding starch to the Highbreed B (trade name for the feed product of Kumiai Haigoshiryo Co., Ltd., and the composition thereof is shown in the Table 1 for reference) (starch-containing group). The test group female-pigs were breeded by giving such feed prepared by adding the MN-001 formulation obtained in Production example (containing starch and 108 living cells/g) to the above available feed.

The results of these feeding tests are shown in the Table 2.

Table 1 Composition of Highbreed B Crude protein 15.0% or more Crude fat 2.0% or more Crude fiber 10.0% or more Crude ash 10.0% or less Calcium 0.6% or more Phosphorous 0.45% or more Digestible crude protein 11-5% or more Total amount of digestible nourishments 72.0% or more Table 2 Control Test group group Feed composition: Formula feeds for breeding female-pig(%) 100 100 Additives (%) Starch MN-001 (0.1) formulation (0.1 Number of tested femalepigs (female-pig) 20 20 Female-pig:: Average body weight (kg/female-pig) 178.0 177.0 Number of delivered femalepigs (female-pig) 20 20 Average number of born piglets (piglets/female-pig) 9.4 9.6 Average body weight of born piglets (kg/piglet) 1.39 1.40 Average number of weaned piglets (piglets/female-pig) 7.7 8.6 Survival (%, average number of weaned piglets/average number of born piglets x 100) 81.9 89.6 Example 4 10 Young seed bulls of Holstein of 1-week-old were divided into two groups respectively consisting of 5 young bulls. On the one hand, the milk substitute for control group was prepared by adding starch to the basic milk substitute of which composition is shown in the Table 1, and on the other hand, that for test group was prepared by adding the MN-001 formulation obtained in Production example (containing starch and 108 living cells/g) to the basic milk substitute.

The feeding tests were carried out as follows: One part by weight of milk substitute for each group described above was respectively dissolved and dispersed in 7 parts by weight of warm water to prepare respectively the feed of milk substitute for each group. Then, each group bulls were breeded by giving respectively the feed of milk substitute above obtained for 4 weeks.

The results of these feeding tests are shown in the Table 2.

By the way, according to the recent breeding method of young cattle in our country, it is breeded by giving the pellet form feed (containing less protein and fat than those in the milk substitute) called Calfstarter in combination with such milk substitute as described above, however, in this Example, in order to examine only the effect of the milk substitute itself, the calfstarter was not used in combination therewith.

Table 1 Division Control Test group group group group Ingredients and Skim milk 70 70 mixing ratio in Whey 10 10 the milk Beef tallow 18 18 substitute Premix of (parts by feed additive weight) (Note 1) 2 2 Starch 0.1 MN-0O1 formulation - 0.1 Lecithin 0.5 0.5 (Note 1) Containing vitamins and minerals etc.

Table 2 Control Test group group Average body weight (Kg/calf): Start of feeding (1-week-old) 45.0 44.8 End of feeding (5-week-old) 72.3 74.1 Average increase in body weight (Kg/calf) 27.3 29.3 Increase index in body weight 100 107.3 Feed intake (Note 2)(kg/calf) 45.7 46.0 Feed efficiency 0.60 0.64 Outbreaking times of diarrhea (times/calf/test period): Muddy + explosive diarrhea 5.0 2.5 Explosive diarrhea 2.5 0.3 (Note 2) Substitute milk (values represented by a conversion into powder form) Example 5 Among 100 White Leghorns with 68 weeks in age, 60 hens that have shown egg-laying ratios during the past 4 weeks were selected, and they were divided into two groups.Then the following breeding tests were carried out for 2 weeks after the preliminary feeding period of 1 week: The control group hens were breeded by giving such feed prepared by adding starch to the "Mylayer 17" (trade name for the feed product for laying hen of Kumiai Haigoshiryo Co., Ltd., and the composition thereof is shown in the Table 1 )(starch-containing group). The test group hens were breeded by giving such feed prepared by adding the MN-001 formulation obtained in Production example (containing starch and 108 living cells/g) to the above available feed.

The results of these breeding tests are shown in the Table 2.

Table 1 Composition of Mylayer 17 Crude protein 17% or more Crude fat 3.5% or more Calcium 2.8% or more Phosphorus 0.55% or more Crude fiber 5.0% or less Crude ash 13% or less Metabolizable energy 2750 Kcal/Kg or more Table 2 Control Test group group Feed composition: Feeds for laying hen (%) 100 100 Additives (%) 0.1 0.1 Number of tested hens 30 30 Average egg-laying ratio (%) 68 75 Average egg weight 59.5 60.0 Average feed conversion 2.78 2.58 (Relative value) (100) (92.8)

Claims (5)

1. An animal feed additive containing a microorganism of Bacillus licheniformis as useful microorganism.

2. An aminal feed additive as claimed in claim t, wherein the microorganism is Bacillus licheniformis strain MN-001 which has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, under the deposition number of FERM BP-266.

3. An animal feed containing a microorganism of Bacillus licheniformis as useful microorganism.

4. Animal feed as claimed in claim 3, wherein the microorganism is Bacillus licheniformis strain MN-001 which has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, under the deposition number of FERM BP-266.

5. Bacillus licheniformis strain MN-001 showing the following microbial properties: A. Morphological properties: (1) Bacillus being 0.5 to 0.8,um wide by 2 to 3ym long.

(2) Gram's strain: Positive (3) Formation of elliptical endospores.

(4) Motility: Positive B. Cultural properties: (1) Normal agar: Abundant growth. No production of pigment.

(2) Medium containing molasses and ammonium sulphate as the major components: Abundant growth. No production of pigment.

(3) Liquid medium containing sodium chloride: 5% salt:Growth 7% salt:Growth 10% salt:Growth C. Physiological properties: (1) Catalase activity: + (2) Growth under anaerobic conditions: + (3) VP (Voges-Proskauer) test: + (4) Growth under high temperature conditions: at 50"C: + at 55"C: - (5) Acid formation from carbohydrates: Glucose: + L-Arabinose: + D-Xylose: + Mannitol: + D-Tagatose: + (6)Argininedihydrolase activity: + (7) Lecithinase activity: - (8) Starch hydrolysis: + (9) Gelatin hydrolysis: + (10) DNA hydrolysis: + (11) Utilization of citrate: + (12) Utilization of propionate: + (13) Nitrate reduction: + (14) Indole formation: + (15) Deamination of phenylalanin: - (16) Special properties: Showing a great producing ability of protease and cellulase out of cells.