WO2012008766A9 - Bacillus thermophilic strain, and method for preparing fish meal containing soybean meal by using same - Google Patents

Abstract

The present invention relates to a Bacillus thermophilic strain, Bacillus pumilus OYR 0108(SARC_BSS) of accession number KCCM 11066P, and a method for preparing fish meal containing soybean meal by using a Bacillus thermophilic strain, Bacillus pumilus OYR 0108(SARC_BSS), comprising the following steps: mixing fish meal and soybean meal; adding a Bacillus thermophilic strain, Bacillus pumilus OYR 0108(SARC_BSS) of accession number KCCM 11066P, to the mixture; and fermenting the same.

Description

Bacillus genus pyrogenic strain and preparation method of fish meal containing soybean meal

The present invention relates to a Bacillus pumilus OYR 0108 (SARC_BSS) and a method for producing a fish meal containing soybean meal using the same Bacillus pumilus OYR 0108 (SARC_BSS) capable of growing at a high temperature and excellent decomposition ability of the material.

Fish meal can be largely divided into white fish meal, brown fish meal, by-product fish meal and fermented fish meal according to the raw materials produced.

White fishmeal and brown fishmeal have high protein content and low ash content, so they are classified as high-quality fishmeal.

By-product fishmeal is a fishmeal made of the remaining parts except the edible part of fish species used as food, and is classified as a low fishmeal with low protein content and high ash content. Fermented and used as fertilizer.

As a criterion for evaluating the quality of the fish meal, the content of protein and fat, freshness (protein: TVN, Histamine, fat: acid value (AV), etc.), and bioavailability (protein denaturation and digestibility) are used.

In general, fish meal is produced using high heat during the manufacturing process, and other fish species (fish) obtained during the fishing process are collected and pressurized to remove oil (fish oil), followed by high heat to evaporate moisture, crush and pack In circulation.

The protein contained in the fishmeal produced in this way is denatured by high heat and is changed into a form that is difficult to digest, and fatty acids are oxidized to cause an increase in acid value, which is a measure of rancidity.

In addition, since the raw material of fishmeal itself uses by-products except the edible part, there is a problem of low protein and fat content.

In addition, a number of methods for producing fish meal using fermentation have been developed, but in general, since the microorganisms are grown at room temperature (below 37 ° C.), there is a problem in that the production period takes up to several months to increase production costs.

On the other hand, efforts are being made in Europe and Japan to replace fish meals with severe price fluctuations and supply and demand. In particular, research on vegetable protein sources is being actively conducted.

Among these vegetable protein sources, skim soybean meal is receiving the most attention because it can supply raw materials of a certain quality throughout the year, and because the content of protein and amino acids is higher than other vegetable protein sources, it is easy to use as a raw material of formulated feed.

However, soybean meal contains a large amount of carbohydrates and fiber, so the availability of the protein is low compared to animal protein sources (fish meal), so there is a problem that it is difficult to use as a fish meal.

The present invention has been made to solve the above problems, the problem to be solved of the present invention, it is possible to grow at a high temperature and excellent decomposition ability of the material, and in particular in a short time to decompose carbohydrates and fiber which is a vegetable protein source relative protein content To provide a Bacillus pumilus OYR 0108 (SARC_BSS) to increase the temperature.

Soybean meal prepared to make fishmeal using high-temperature strain Bacillus pumilus OYR 0108 (SARC_BSS) that can increase the protein and fat content of by-product fishmeal and increase the protein availability in soybean meal by removing carbohydrates and fiber, which are vegetable protein sources This is to provide a method for producing the fish meal containing this.

In order to achieve the above object, the strain of the present invention is characterized in that the Bacillus genus high temperature strain Bacillus pumilus OYR 0108 (SARC_BSS) of Accession No. KCCM 11066P.

And the present invention is mixed with soybean meal in fish meal, Bacillus pumilus OYR 0108 (SARC_BSS) Bacillus genus high temperature strain Bacillus pumilus OYR 0108 (SARC_BSS) characterized in that the fermentation by adding a Bacillus pumilus OYR 0108 (SARC_BSS) of Accession No. KCCM 11066P. Characterized in that the manufacturing method of the fish meal containing the soybean meal used.

According to the present invention by the above problem solving means, the following effects can be expected.

First, Bacillus pumilus OYR 0108 (SARC_BSS) is a high-temperature bacterium strain capable of growing at high temperatures, having excellent decomposition ability of materials, and decomposing carbohydrates and fibers in soybean meal in a short time.

 The fish meal containing soybean meal prepared using Bacillus pumilus OYR 0108 (SARC_BSS) has a high protein and fat content in fish meal and has an effect of increasing digestibility of the fish ingesting it.

1 is a phylogenetic lineage of Bacillus pumilus OYR 0108 (SARC_BSS).

Figure 2 shows the base sequence of Bacillus pumilus OYR 0108 (SARC_BSS) genus Bacillus.

Figure 3 is a photograph confirming the enzyme properties of the SARC_BSS strain using the APIzyme kit.

Figure 4 is a photograph confirming the enzyme properties of the SARC_BSS strain using the APIzyme kit.

The strain in the present invention was identified as Bacillus, as a result of morphological characteristics, biochemical characteristics and sequencing of 16S rDNA, as Bacillus, Bacillus pumilus OYR 0108 (hereinafter referred to as SARC_BSS) of Bacillus Named and hereinafter referred to as SARC_BSS.

This was deposited on February 19, 2010, with the accession number KCCM 11066P to the Korea Center for Microbiological Conservation (KCCM).

Hereinafter, the present invention will be described in more detail.

The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Isolation of Thermophilic Strains.

5 g of the sample was added to 100 ml of physiological saline and shaken for 1 h in a shaking incubator at 30 ° C. 10 ml of the supernatant was added to 100 ml of PBS and shaken at 40 ° C. for 1 day. The culture solution was plated in NA (Nutrient Agar) medium and cultured for 1 day in an incubator at 40 ° C. The colony was named SARC_BSS and purely cultured in TSA (Trypticase soy Broth agar) medium.

1) medium condition,

       Pure cultured cells at 40 ° C. using NA medium, TSA medium and PDA medium showed good growth in TSA medium containing 5% NaCl. This resulted in SARC_BSS strains for carbon source and mineral (or salt). It was confirmed that the demand for) is high.

       2) Temperature conditions at the time of strain culture (lab scale).

① Stain SARC_BSS strain in TSA medium and incubated at 30 ℃, 40 ℃, 50 ℃ and 60 ℃ as a result the strain is good growth at 40 ℃ and 50 ℃ .

② Smear SARC_BSS strain in TSA medium and do not grow as a result of culturing at 60 ℃, 70 ℃ and 80 ℃

③ Smear SARC_BSS strain in TSA medium and left at 90 ° C. for 1 h, followed by incubation at 50 ° C., resulting in no growth.

④ Stain SARC_BSS strain on TSA medium and incubate at 45 ℃ for 1 day and store at room temperature and 4 ℃ for refrigeration and show shelf life over 6 months at room temperature and 4 ℃.

 3) Liquid culture conditions for strain culture

 ① Inoculated strains in TSB medium and cultured with static and shaking, showing good growth in shaking culture.

 ② Maximum growth after 8 h at TSB, 45 ℃, 100rpm, 0.3vvm as a result of mass culturing of JAR fermentor.

4) Confirmation of the characteristics of the strain (see FIGS. 3 and 4)

Enzyme characteristics of SARC_BSS strain were identified using APIzyme kit.

As a result, it was found to secrete a large amount of esterase and esterase lipase, and a small amount of alkaline phosphatase, acid phospatase and naphtol-AS-Bl-phospaohydrolase.

 In addition, b-glucuronidase, a carcinogenic enzyme, does not appear to be secreted.

5) Toxicity test of strain culture

Animal toxicity experiments were performed using strains cultured in commercially developed media.

No abnormal symptoms such as feeding mortality and weight loss for 3 weeks by oral administration of 1 × 10 ⁹ cfu / ml strain using experimental mice.

1 × 10 ⁷ cfu / ml of strains were mixed with the cultivated halibut feed for 4 weeks without abnormal symptoms such as mortality and weight loss.

1 × 10 ⁷ cfu / ml of strain is mixed with cultured eel feed for 4 weeks without abnormal symptoms such as mortality and weight loss.

 Looking at the results of the experiment, the BSS strain cultured at the laboratory scale grows well at a temperature of about 40 ~ 50 ℃, it is judged that the room temperature and refrigeration preservation is high. In addition, acute toxicity test results are considered to be harmless to the organism.

6) Soybean meal degradation capacity of SARC_BSS strain

Soybean meal degradation capacity of SARC_BSS strain cultured in TSB medium for 1 day was tested.

50g of raw soybean meal was mixed with 50ml of distilled water, and 0.1% of control or BSS was added thereto, fermented at 50 ° C inking incubator for 2 days, and then sampled at 1 and 2 day intervals for general analysis.

Table 1 below is an experimental result to determine the soybean meal decomposition ability of Bacillus pumilus OYR 0108 (SARC_BSS) strain.

Table 1

Experiment	1 day		2 days
	moisture	Crude protein (% by weight)	moisture	Crude protein (% by weight)
Control	54.18	48.43	56.28	50.46
BSS	57.08	51.55	60.10	55.76

Looking at Table 1, the protein and water content of SARC_BSS increased compared to the control, the thermophilic SARC_BSS strain was formed by using carbohydrates contained in soybean meal, and seems to increase the relative protein content.

That is, if SARC_BSS strain is added after mixing soybean meal with by-product fish meal, SARC_BSS strain will remove carbohydrates and fiber in soybean meal and increase the protein availability, and thus it is considered to supplement the low protein content of by-product fish meal.

Example 2 Field Application Experiments

1) Temperature condition experiment when manufacturing fish meal

(Experimental method)

-Tuna by-product: mix soybean meal in 8: 2 ratio

 -0.1% BSS culture of total raw materials

 -1st: drying after maintaining about 60 ℃ (90 ℃)

   Secondary: Maintain about 80 ℃

   Tertiary: Initial high temperature (about 2h, 80 ℃), low temperature (60 ℃) and drying (75 ℃)

   4th: Initial high temperature (about 2h, 80 ℃), low temperature (60 ℃) and then dried (75 ℃)

 -General ingredient analysis and microbial measurement after stirring and drying for 24h

Table 2 below is an experimental result for confirming the temperature conditions when manufacturing fish meal of the present invention.

TABLE 2

	Moisture after 24h	24h postprotein	Viable count after 24h
Primary production	37.8%	58.47	10 ^ 6
Secondary production	30.1%	63.85	10 ^ 7
Tertiary production	30%	62.76	10 ^ 9
4th production	25%	62.89	10 ^ 9

Looking at Table 2, the fermentation is judged to be good for the rest of the production except the primary production, the number of bacteria is significantly reduced when the temperature is maintained above 90 ℃, it seems that the fermentation is not made.

After the production of the first fishmeal, which produced a markedly high production temperature, the color appeared dark brown and the contents appeared to be carbonized. Thus, the fermentation and drying temperature of the fishmeal produced was 75 ° C. or less.

The SARC_BSS strain is almost killed at 50 ℃ or higher at strain culture (laboratory scale), but the fishmeal is grown at 75 ℃ when fermented (on site fermentation). This is because, on the site scale, growth is possible at higher temperatures by indirect heat in the air.

Through the above results, SARC_BSS strain can be confirmed that it is a high-temperature strain capable of growing well at a temperature of 40 ~ 50 ℃ at the laboratory scale and fermenting at 75 ℃ at the field scale.

 2) Experiment to confirm fishmeal production

In the preparation of fish meal, the internal temperature was set to confirm the maintenance and change, and the effect on the viable cell number was evaluated.

(Experimental method)

-3200kg of tuna by-product + 800kg of soybean meal in 10 ton fish meal production equipment

-20L of SARC_BSS and SARC_BSS culture medium prepared in 10 ^ 9 each

-Set the external temperature of the fishmeal preparation machine to 75 ℃, set the external temperature to 85 ℃ after 16h.

-Check temperature, moisture change and viable cell number over time.

Table 3 below shows the results of checking the temperature, moisture change and viable cell number according to the time of preparing the fish meal of the present invention.

TABLE 3

Hours (h)	Internal temperature (℃)	moisture(%)	Viable cell count (cfu / g)
0	41	53.4	3.4 × 10 ^ 6
3	60	50.4	_
4	61	50.4	_
6	62	47.2	_
8	60	50.2	4.8 × 10 ^ 9
10	61	50.0	2.1 × 10 ^ 9
12	63	48.6	1.1 × 10 ^ 9
14	60	45.7	4.2 × 10 ^ 8
16	60	45.1	4.5 × 10 ^ 8
18	70	43.5	2.6 × 10 ^ 8
24	77	24.7	5.7 × 10 ^ 8

Referring to Table 3, the 8h viable cell count was shown to be the maximum, and the growth of the microorganisms was almost completed during the initial 8h, and then the fermentation proceeded while maintaining the viable cell number.

In addition, the water content tends to increase after 8 h, which appears to generate a large amount of water as a soybean meal decomposition product by microorganisms.

When manufacturing fish meal, when the internal temperature is maintained at 60 ℃, the viable cell number of microorganisms is maintained at 10 ^ 8 ~ 10 ^ 9, and if the temperature is increased after 18h and dried, the moisture content is maintained at about 25% after 24h, so that the fermentation and drying are appropriate. It confirmed that it became.

3) Quality Evaluation of Fish Meal and Commercial Fish Meal of the Present Invention

To evaluate the quality by comparing the general components, acid value and pepsin digestion rate of the fish meal produced with the present invention and commercially available fish meal.

Table 4 below is a fishmeal quality evaluation results of the present invention.

Table 4

Item	Fermented Fish Meal	Domestic Tuna Fish
Crude Protein (%)	58	57
Ash content (%)	13	17
Crude fat (%)	11	9
Pepsin digestion rate (%)	95	78
Acid	7.6	32

Looking at Table 4, fermented fish meal of the present invention compared to the domestic tuna fish meal currently on the market is judged to have high quality of crude protein and crude fat and low ash content.

In addition, the pepsin digestion rate is very high, the utilization rate of the fish meal produced by the present invention is expected to be high, and the acid value is also very low value, it is judged that a very good product is produced even in freshness.

The present invention can be used in the production of Bacillus pumilus OYR 0108 (SARC_BSS) and soybean meal containing soybean meal using the same Bacillus pumilus OYR 0108 (SARC_BSS) capable of growing at a high temperature and excellent decomposition ability of the material.

Claims (2)

1. Bacillus pumilus OYR 0108 (SARC_BSS), genus Bacillus sp., Accession No. KCCM 11066P.
2. Soybean meal using Bacillus pumilus OYR 0108 (SARC_BSS), characterized by fermentation by adding soybean meal to fish meal and adding Bacillus pumilus OYR 0108 (SARC_BSS), Bacillus pumilus OYR 0108 (SARC_BSS), Accession No. KCCM 11066P. Method of preparing fishmeal.

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