DE1492907C - Process for the production of complementary feed - Google Patents

Description

The invention relates to a process for the production of supplementary feed by cultivating Bacillus subtilis or Bacillus natto in an aqueous, carbon, nitrogen and inorganic nutrient source containing nutrient medium with aeration and agitation at an initial pH of 6.0 to 8.0 and a temperature of 30 to 40 ° C.

It has been customary for some time to feed nutrients, such as vitamins, amino acids, antibiotics, to feed. Adding enzymes or preventive agents against diseases. The addition is usually made in Form of so-called supplementary feed, which has gained great importance.

From U.S. Patents 2,906,626, 2,738,274 and 2,132,621, and from the British patent 939 017 is e.g. B. the production of complementary feed is known, with one belonging to the strain Bacillus subtilis Microorganism in carbohydrates, nitrogen compounds, nutrient salts and the like Culture media at pH values of around 5.5 to 7.5 and temperatures of around 25 to 40 ° C as a submerged culture is grown with aeration and stirring, whereupon the obtained culture is concentrated or dehydrated and mixed with animal feed. However, as is generally the case with the manufacture of substances usual by microorganisms, a complete growth of the microorganism is sought. It has evidently always been regarded as essential that the accumulation of enzymes or antibiotic substances reaches the maximum after the microorganism has fully grown.

In contrast, the invention consists in that the cultivation between the beginning and middle of the logarithmic Growth phase is terminated, the entire culture immediately afterwards at a pH value from 4.0 to 8.0 subjected to a heat treatment for 1 to 3 hours at a temperature of 50 to 80 ° C and then dried to a powder or added directly to a feed.

This results in a highly effective, stable feed that can be used as an additive to feed for poultry and other animals.

This result is all the more surprising since it was found that the effect of the culture product a microorganism belonging to the strain Bacillus subtilis or Bacillus natto not as additional feed is constant and changes depending on the respective production method of the same and in some cases no effect or even a delay in the growth of the animals was observed can be. So it is essential that breeding is stopped at a certain stage and that obtained culture product or the culture broth under the specified specific conditions of a Is subjected to heat treatment.

Preferably the cultivation is carried out at a time between 2 and 4 hours after inoculation of the culture medium aborted.

The reason for this phenomenon is likely to lie in the fact that - as stated - in the overall bräuchlichen organisms of Bacillus subtilis (as described, for example, as N strain of Bacillus subtilis are known.) Used for the production of amylase and protease a substance is formed in the culture solution and the cells which, if tracked to animals, promotes or stimulates their growth. The formation of this substance now takes place at a much earlier stage than when the accumulation of enzymes, such as amylase or protease, or an antibiotic can be observed in large quantities. It has also been found that the formation of the enzyme continues even if the cultivation is continued until the growth of the microorganism enters the last part of the logarithmic growth phase, but that on the other hand a factor is also formed in the culture solution which has an inhibiting influence exerts on the growth of animals. This means that in the time from the beginning to the middle of the logarithmic growth phase of the bacteria, a factor or substance forms and accumulates inside and outside the cells, i.e. in the filtrate of the culture, which promotes the growth of the animals, while in During this period, the formation of the factor that inhibits growth is negligible.

It was also found that the promoting effect of the product on the growth of the animals is still increases if the culture broth containing the cells obtained in this specific period of time falls below is heated under certain conditions of pH, time and temperature.

The microorganisms used in practicing the invention belong to Bacillus subtilis and Bacillus natto. It is known that this also includes the strains that amylase and protease in produce large quantities and are used industrially, such as. B. an N strain of Bacillus subtilis (Hagihara, 1958), an N-strain (Boyer et al., 1960), an R-strain (Hagihara, 1958), a K-strain (Oishi et al., 1963), a Η strain (Nishimura et al., 1959) and the Sawamura strain and the SN strain of Bacillus natto, as well as those strains which produce the above enzymes to a very small extent and often for genetic studies are used, such as B. the Marburg strains No. SB-15 (nests and others, 1961), no.160 (Saito et al., 1961), no.168 (Burkholder et al., 1947), No. 30 (Ephrati-Elizur et al., 1961) and No. W-23 (Thome, 1962). These tribes are that Well known to those skilled in the art and readily available from various breed collections.

According to the invention, such a microorganism is grown. It can either be a festival culture process or a submerged culture method with agitation and aeration can be used. However that is Method of submerged culture more suitable because the different ones in the practice of the invention the conditions to be observed can be checked easily and safely;

Any nutrient medium can be used, such as those used for the cultivation of microorganisms of the Bacillus subtilis and Bacillus natto are known. As a carbon source, starch, corn flour, dextrin, Serve glucose and sucrose. Not only inorganic nitrogen compounds, such as ammonium chloride, ammonium sulfate, diammonium phosphate and ammonium nitrate are used but beans such as soybeans, corn steep liquor, skimmed milk powder, casein are also suitable and amino acids. Inorganic salts are also required.

Industrially preferred is a nutrient medium composed of a suitable combination of 3 to 20% alkali extract of defatted soybeans, 0.5 to 10% corn steep liquor, 1 to 10% starch, 1 to 8% Corn flour, 1 to 5% corn bran and 1 to 5% bran with the addition of a small amount of inorganic Salts.

At the start of cultivation, the pH of the nutrient medium is 6.0 to 8.0 or preferably 6.5 to 7.2. The temperature can be 30 to 40 ⁰ C or preferably 35 to 38 ° C.

The culture time is of great importance for the invention, as has already been explained above. in the in general, the transition from one growth phase to the next is noticeably influenced by the individual strain, the conditions of the preculture, the size of the inoculation material, the aeration speed, the type of stirring, the nutrient medium, the pH value and the temperature. In the case of the strains used according to the invention, the transition of the growth phases but essentially the same tendency if the other conditions are the same. Therefore, a more detailed explanation should be based on the strains of Bacillus subtilis which are used for the industrial production of amylase and protease. Under the characteristic, reproduced in Example 1 cultivation conditions begins within 1.5 The logarithmic growth phase begins up to 2 hours after the vaccination, after about the 6th hour the last part of the logarithmic growth phase, followed by the stationary phase.

Usually such extracellular enzymes and antibiotics as amylase and protease are formed even in the stationary phase. However, in order to obtain a cultivated product according to the invention, that can be used as additional feed, must, as already described above, the breeding between the beginning and the middle of the logarithmic growth phase of the bacteria are ended, that is, in this example about between the 2nd and 4th hour after inoculation.

It has been found that no substantial benefit is achieved when used as supplementary feed when the cultivation product or the culture broth as such or after drum drying or spray drying is used, but that the effect can be increased to a considerable extent if only heating under specially selected conditions and then drying takes place. The ^, indicates that the cultivation is terminated at the stage described above and then the pH of the medium, as necessary, adjusted to 4.0 to 8.0. The cultivated product is kept at 50 to 80 ° C for 1 to 3 hours warmed up. Preferably, this heat treatment is carried out within 1 to 2 hours at one temperature carried out from 55 to 75 ° C.

If this heat-treated product or the culture broth is in liquid by means of the centrifuge and insoluble solid is separated, the active ingredient is in both portions. It is therefore not necessary in practice to separate them from one another. The heat-treated product is therefore absorbed as such or after concentration on the animal feed. At will the heat-treated growth product can also be dried using a drum dryer or spray dryer without it being absorbed on a solid feed or the like. When drying will the temperature of the product kept at 50 to 80 ° C if possible.

The additional feed obtained according to the invention can be used as an addition to the usual feed for hens, pigs, . Rats and cows as well as other poultry and other animals can be used.

The amount in which the additive of the invention is added to the animal feed depends on the form of use (Liquid, concentrate or dry powder) as well as the purpose; in general but are about 0.02 to 1 percent by weight (as solid), based on the weight of the basic forage, used.

For example, about 0.02 to 0.05% will increase the percent weight gain and feed conversion of poultry added, 0.02 to 1% to increase the percentage of egg production, 0.02 to 0.1% to increase the percentage weight gain of cows and pigs, 0.05 up to 0.2% to increase the percentage of milk yield and 0.1 to 0.5% to improve the taste.

B e i s ρ i e 1 1

A culture medium (10001) with a pH of 7.0, which contained 5% alkali extract from defatted soybeans, 4% starch, 2% lactose, 1% corn steep liquor and 1% (NHJ ₂ HPO ₄ , was used in

. a culture tank (main tank) with a capacity from 2000 1 filled.

An N strain of Bacillus subtilis was grown for 5 hours at 37 ° C. in 50 1 of the nutrient medium described above cultivated with stirring and aeration in an incubator with a capacity of 1001 and used as an inoculation material.

The cultivation in the main tank was carried out with stirring with the aid of a paddle stirrer at 190 rev / Min. And by introducing 1000 liters of sterile air per minute at 37 ° C. The dimensions of the stirrer was the following:

= 0.7

Length of the wings
■ Diameter of the tank

Width of the wings
= 0.08 ■ diameter of the tank

The cultivation was carried out with stirring and aeration, as described above, within 4 hours, then 150 liters of the obtained broth were taken for each sample.

The samples (used in study groups 1 to 13 in Table 1 below), which were produced by heating the culture broth to 55 to 100 ^° C. for a period of 1 to 3 hours and then drying in a drum dryer, and the sample (used in of a study group 14), which had been obtained by heating the same culture broth to 45 ° C. for 2 hours after the end of the cultivation and by drying it in a drum dryer, were added to the basic feed and for feeding experiments on 100 Rockhorn F ₁ chickens (1 day old ) used per group for 5 weeks. The results are shown in Table 1 / In each case, the amount of dry product added was 0.03 percent by weight, based on the basic feed. A common forage was used, which consisted mainly of corn, defatted soybean meal and fish meal, had an energy content of 2.1 cal / g and 22% crude protein (CP), 8% fish meal, 3% alfalfa meal, 20 g / t of an anti

biotics and 0.01% furazolidone. This mixture makes an ideal feed door poultry and contains all the necessary growth factors.

Table 1

Relationships between heat treatment conditions and weight gain percentage the chick

groups
number temperature
( ⁰ C) warmth
treatment
time Weight
increase
in % 1 55 1 103.5 2 55 2 104.2 3 55 . 3 105.1 4th 60 1 106.1. 5 - . 60 2 107.0 6th 60 3 , 106.8 7th 70 1 105.8 8th 70 2 105.6 9 70 3 105.3 10 80 1 105.0 11th 80 2 104.8 12th 80 3 104.3 13th 100 1 104.4 14th 45 2 101.2 control - - 100

B e i s ρ i e 1 2

A K strain of Bacillus subtilis was found in a

Aeration of 250 l / min, cultured with stirring at 37 ° C, and 250 l of the in Example 1 used nutrient medium in a culture tank with a capacity of 5001.

It was cultivated for 3 hours and the temperature

'Temperature increased to 60 ° C at the end.

The culture broth was kept at this temperature for 2 hours, then 250 kg of bran were added Adsorption added and the mixture dried to a powder at 45 ° C in the air. A. Experimental feed was prepared by adding 1% of this powder to the base forage described below would. 10 albino rats (3 weeks old) were used for the experiments. The results are in Table 2 reproduced.

Composition of the basic forage:

Defatted soybean meal 20.0%

Cod liver oil 1.0%

Bran 20.0%

Corn 55.0%

CaHPO ₄ 1.0%

Vitamin B substances, 0.1%

Minerals 0.1%

NaCl '0.5%

McCollam salt mixture 2.0%

Methionine 0.2%

Vitamin B ₁₂ 0.1%

table

Influence on weight gain in rats

Group number Lining C.
Weight gain
(G) ΐ
Weight gain Weight gain
(G) r-
Weight gain 1 (control) ...
2 Forage
Forage 160.7 100 85.0 100 + 1% addition 180.0 112 129.3 152.1

B e i s ρ i e 1 3

An N 'strain of Bacillus subtilis was grown in the manner described in Example 1. 501 the culture broth were in each case after the 2nd hour (start of the logarithmic growth phase), the 4th hour (middle of the logarithmic growth phase), the 6th hour (last part of the logarithmic Growth phase), the 10th hour (entry of the stationary phase) and the 16th hour removed and Heated to 60 ° C for 2 hours. The heat-treated broth was absorbed in 50 kg of bran, at 45 ° C air dried and ground. One day old Rockhorii Fj ((J) chicks became experimental Raised for 8 weeks with the basic forage used in Example 1, and it was found that a remarkable Influence on the percentage weight gain was achieved when one in the middle or supplementary feed taken from the culture broth before the middle of the logarithmic growth phase was used but that an inhibiting influence on the growth of the animals was recorded, if fractions later removed from the culture broth were used. In this case it had after of the 5th week, ground forage used an energy content of 2.6 cal / g and contained 20% crude protein. The addition of supplementary feed was 0.5 percent by weight, based on the basic feed. The results are listed in Table 3. .

7 8

Table 3
Influence on growth when supplementary feed obtained in different culture stages is added

Group additive 1 week Weight gain (%) 6 weeks 8 weeks 1 week Feed conversion 6 weeks 8 weeks pen 102.0 106.0 107.0 1.59 2.46 2.82 number 2 hours 103.0 3 weeks 106.2 106.8 1.59 3 weeks 2.40 2.72 1 4th hour 97.0 105.1 101.5 101.8 1.57 1.89 2.42 2.79 2 6th hour 95.0 105.3 94.6 97.1 1.61 .1.81 2.45 2.80 3 10th hour 94.7 102.0 96.5 99.3 1.56 '1.83 2.38 2.75. 4th 16th hour 100 95.3 100 100 1.66 1.84 2.59 2.98 5 control 95.5 1.82 . 6th (without addition) 100 1.89

Note: 100 chicks per group were tested.
Feed conversion: expressed by feed consumption / weight gain.

Example 4

A Marburg Nr.l60 strain of Bacillus subtilis was grown for 4 hours in the manner described in Example 1, then the culture broth was heated at a pH of 7.0 at 70 ⁰ C and held for 2 hours at this temperature. This heat-treated broth had very little amylase and protease activity. It was dried to a powder in a spray dryer. 0.05% by weight of the powder was added to the forage used in Example 3 and an experiment was conducted in which 100 1-day-old Rockhorn Fj-i $ ) chicks were raised in each of the experimental groups. The results are shown in Table 4.

table

Weeks groups Average weight
(G) Weight gain
(%) Feed conversion Beginning Experimental group 34.4 - - Control group 34.4 - - 2 Experimental group 136.2 102.9 1.84 Control group 133.2 100 1.87 4th Experimental group 341.4 104.8 .1.90 Control group 327.1 100 2.00 6th Experimental group 616.8 106.1 2.50 Control group 583.0 100 2.57 8th Experimental group 989.4 107.3 2.75 Control group 924.6 100 2.81

Example5

An N strain of Bacillus subtilis was grown for 4 hours in the manner described in Example 1, then the culture broth was heated to 60 ° C. for 2 hours. The heat treated broth was then dried to a powder in a spray dryer. 0.1, 0.05 and 0.025 percent by weight of the powder added to the base feed. Day old chicks were in each group Raised for 8 weeks.

1-day-old White Cornish- _Fr (Ä) and White Rock- _Fr ($) chicks were used for the experiments. 1000 chicks, of which $ 500 and $ 500 were divided into three experimental groups and a control group so that each group consisted of 250 chicks with equal numbers of males and females. The basic forage had the main components content given below. The basic forage I was from 0 to

4th week administered and basic forage II given from 5th to 8th week.

Raw
protein fish
flour Other
animal '
Products alfalfa
flour Basic forage I ...
Basic forage II .. 23%
21% 5%
4% 0%
0% .3%
3%

55 Each feed also contained 0.01% furazolidone, 30 g / t of a tetracycline antibiotic and 11 mg / t vitamin B ₁₂ and thus formed an adequate nutritional basis. It just became this feed

and water administered. -

The experiments were carried out until the end of the 8th week. Every two weeks, percentages of body weight and total feed conversion were taken definitely. The results are shown in Table 5.

109E85 / 88

Table 5 Feeding experiments on broiler chickens

10

Added supplementary feed 0 2 Weeks 6th 8th Group number 0.1% body weight (g) 38 147 4th 980 1430 1 Feed conversion - 1.68 440 1.95 2.15 0.05% body weight (g) 38 146 1.80 978 1425 2 Feed conversion - 1.68 435 1.96 2.16 0.025% body weight (g) 38 146 1.81 965 1400 ■ 3 Feed conversion - 1.69 430 2.00 2.19 0% body weight (g) 38 138 1.82 920 1360 control Feed conversion - 1.72 410 2.10 2.32 1.86

Examples

0.04 percent by weight of a powdered additional feed, which had been prepared in the manner described in Example 5 were added to a basic forage and egg production and feed conversion of 10 month old laying hens for 6 months definitely. Laying hens of the White Leghorn variety, the balanced one, were used as test hens Had laying performances. 1000 animals were divided into an experimental group and a control group with 500 hens each. The main forage contained the following main components.

Raw
protein Calcium phosphorus Fish meal alfalfa 16.5% 3.2% 0.65% 5% 3%

The feed also contained 0.01% furazolidone and 20 g / t of a tetracycline antibiotic and formed an adequate nutritional basis. Only this food and water were given. In the Table 6 is the percent egg production and feed conversion, per month for each group reproduced after the start of the experiments.

Table experiments with laying hens

1 2 Moi
3 late
4th 5 6th Egg production (%)
Experimental group
Control group
Feed conversion
Experimental group
Control group ... 88
83
2.23
2.37 86
81
2.28
2.43 83.
77
2.37
2.56 79
74
2.48
2.66 76
70
2.58
2.81 74
65
2.66
3.03

B e i s ρ i e 1 7

A powdery supplementary feed prepared in the manner described in Example 5 was added to a milk substitute feed for piglets. 15 day old piglets were reared in 65 day trials. 16 F ₁ -Ferkel a Landrace breed (<$) and a Yorkshire-middle white-breeding (?), 15 days old, with body weights as balanced as possible, one of which came every 8 piglets from a sow and $ 8 and $ 8 were divided into a test group and a control group so that sex and body weight were balanced.

A milk substitute feed I was given to the animals from the 15th to the 30th day after birth and a milk substitute feed II administered from the 31st to the 65th day after birth; In the experimental group were feed I 0.05 percent by weight of the additional feed described above and feed II 0.04 percent by weight of the same additional feed added.

The milk substitute feed contained the following main components:

Crude protein

total quantity
more digestible

nutrient

Fish meal

. Dairy by-products alfalfa

Antibiotics (g / t)

Procaine penicillin

Dehydro,
streptomycin

Tetracycline

acid
Protease *)

Milk substitute feed I.; .v,

Milk substitute feed II

22%

18%

78%

75%

6%

4%

0%

0%

10

40

20th

0.2

• 0.1

*) The acidic protease was a preparation with KXH) units / g.

11 12

. Both milk substitute feeds I and II contained 0.01% furazolidone, 40 g / t of an antibiotic and 20 mg / t of vitamin B ₁₂ and provided sufficient nutrition. Only this food and water were given to each group.

In Table 7, mean body weight and percent total feed conversion for each group is 10 days reproduced after the start of the experiment.

Table 7
Feeding experiments on piglets

15th 25th Days after of birth 55 65 groups 35 45 Experimental group 4.3 6.0 16.8 22.0 Body weight (kg) ■ - 1.23 8.8 13.2 1.58 1.70 Feed conversion 1.35 1.42 Control group 4.3 5.8 15.1 19.8 Body weight (kg) .... - 1.25. 8.2 12.0 * 1.66 1.82 Feed conversion - 1.38 1.49

In the control group, 4 piglets suffered from mild diarrhea on about day 35 and showed one low growth deficit. In the test group, on the other hand, the piglets showed no diarrhea at all and grew splendidly.

Example 8

A supplementary feed prepared in the manner described in Example 5 was mixed with a feed and given to dairy cattle. The amount of milk and the fat content of the milk were determined. There were two cows of a Holstein breed used, which were of the following age and body weight:

Cow a
Cow

body weight
(kg)

580
650

Age in
Years

4th 5

frequency
of calving

2 3

Months after calving

3 to 5

4 to 6

The trial period was divided into four sections of 2 weeks each. 0.2% of the above Additional feed was mixed with a feed and cows a and b in the second and fourth sections administered during the trial period, while none in the first and third sections. Additional feed given would.

The first week was used for preparation and the second week for measurement.

The sample feed was a compound feed for dairy cattle with 16% crude protein and 68% total TDN digestible nutrients. Each cow was given 8 kg of this feed per day. Both cows were fed outdoors with raw fodder made up of a total of 40 kg of grass and maize waste as well as rice straw per day existed.

The milk quantities and the fat content of the milk of both cows in the last half (the test period) of the section in which the supplementary feed was administered, and in the last half (the control period) of the section in which no supplementary feed was administered were measured for comparison. The results are in Table 8 reproduced.

table

Control period Trial period Increase over control
(%) Average quantities of milk produced (kg / day)
Cow a 17.5
18.2
3.70
3.38 19.0
19.5
3.81
3.51 + 8.6
+7.1
+ 3.0
+ 3.8 Cow b Fat content of milk
Cow a Kuhb .....

To the technical progress of the subject of the application in comparison to the product of the USA patent specification 2,906,622, the following comparative tests were carried out: The strains IAM 1069 (= ATCC-6633) and IAM 1186 (the designation IAM means one at the university Osaka deposited culture) were according to the present invention on the one hand and according to the United States patent 2 906 622 on the other hand, and the cultures then as supplementary feed for rockhorn chicks used.

The conditions and results used were as follows:

1. The IAM 1069 strain was grown with shaking at 37 ° C in a beet molasses medium of the following Bred composition:

Beet molasses 85.0 g / l

Potassium sulfate 2.0 g / l

Diammonium phosphate 8.5 g / l

Diammonium citrate 19.0 g / l

Yeast extract: 1.0 g / l

13 14

Zn (as chloride) 5 ppm dried. The product is in the following sample

Fe (as chloride) 5 ppm No. I named.

Mg (as chloride) 50 ppm Another 50 l of the culture broth were added

Mn (as chloride) 50 ppm taken from tank culture for 10 hours. This broth

Co (as chloride) 2 ppm 5 was immediately spray dried and without preheating

Ca (as chloride) 20 ppm is designated Sample No. II.

2 The IAM 1186 strain was among the same

The inoculum culture obtained (8 1) was used in 2001 to cultivate conditions as indicated above. From the same medium as indicated above, two samples were prepared in the same manner, which were inoculated in a culture tank as in Example 2, which is shown below as Sample No. IH (4-hour tank shown). The tank fermentation (cultivation, heat treatment and spray drying) was carried out at 37 ° C. with constant stirring and testing or sample No. IV (10 hour tank cultivation and ventilation at 200 l / min. The pH of the spray drying continued).
The medium during the culture was 6.3 to 3. Each of Sample Nos. I, II, III and IV was kept at 7.0 by adding ammonium hydroxide 15 in an amount of 0.05% by weight to the im as needed. The usual basic feed used in Example 1 was added to chicks for 4 hours after the start of breeding, and four different 50 liters of the broth in the tank were thus taken out and the types of feed prepared. The same feed without leftover was further bred. set was used as a control. This feed The extracted 50 1 were immediately sorten 2 hours 20 were heated for feeding tests on 100 long skirt to 6O ⁰ C and then spray-powder horn-Fi - used Ueken (S) -K

The results are summarized in the following table:

Lining 0 Average weight (g) 2 4th 6th Weight gain {%) 2 Weeks 6th barn
TsIr 35.1 Weeks 139.9 360.1 673.6 103.6 4th 106.5 1ΝΓ. Sample I. 35.1 135.8 348.4 644.8 99.5 106.0 101.7 1 Sample II 35.0 138.6 362.4 678.3 102.4 102.2 107.3 2 Sample III 34.9 134.2 349.2 651.2 98.1 106.8 102.8 3 Sample IV 34.9 136.1 341.5 634.4 100 102.5 100 4th control 100 5

Claims (2)

Patent claims: 1. A process for the production of supplementary feed by growing Bacillus subtilis or Bacillus natto in an aqueous nutrient medium containing carbon, nitrogen and inorganic nutrient sources with aeration and agitation at an initial pH of 6.0 to 8.0 and a temperature of 30 to 40 ⁰ C, characterized in that the cultivation see between 45 beginning and middle of the logarithmic Growth phase is terminated, the entire culture is immediately then subjected to a 1 to 3 hour heat treatment at a pH of 4.0 to 8.0 at a temperature of 50 to 80 ⁰ C and then dried to a powder or added directly to a feed . 2. The method according to claim 1, characterized in that the cultivation at a time stopped between 2 and 4 hours after inoculation of the nutrient medium.