KR101073986B1 - New Strain of Bacillus subtilis and Solid Phase Fermentation Process of a Arecaceae Kernel Cake Using the Same - Google Patents

Abstract

Disclosed is a Bacillus subtilis strain having a mannanase generating ability and a fermentation ability of a palm fruit seed and a solid state fermentation method of a palm fruit seed meal using the same. The novel Bacillus subtilis strain according to the present invention has the effect of producing a variety of enzymes to help digestion absorption of feed, and the fermented palm fruit seed meal produced by the solid phase fermentation method of the palm fruit seed meal according to the present invention has been improved digestibility In addition, nutrients such as proteins are increased, and it contains a large amount of microorganisms and enzymes beneficial to animals, which is useful as an animal feed or additives thereof.

Palm seed seed, poultry feed, met, met antase, solid fermentation, mixed fermentation

Description

New Strain of Bacillus subtilis and Solid Phase Fermentation Process of a Arecaceae Kernel Cake Using the Same}

The present invention relates to a novel Bacillus subtilis strain and a solid fermentation method of palm fruit seed meal using the same, and more particularly, Bacillus subtilis having a mannanase production ability and a palm fruit seed fermentation ability. The present invention relates to a strain and a solid fermentation method of palm fruit seed meal using the same.

In the palm oil industry, palm seed meal, palm oil sludge and palm extruded fiber are produced as by-products that can be used as animal feed. Among the by-products, palm kernel cake is a powder remaining after oiling of palm seed oil, and is most usefully used as an animal feed or an organic fertilizer.

Palm seed meal is an intermediate protein source that provides protein and energy, and the protein quality of palm seed meal is known to be slightly better than copra meal and lower than peanut meal or wheat. Palm seed meal also contains high fiber content, making it suitable for feed of ruminants. Therefore, palm seed meal is a beneficial feed for ruminants and has been used in other countries as beef and dairy feed additives along with other nutrients such as cornmeal and soybean meal.

However, palm seed meal is composed of fibrous mannan-based fiber, which is difficult to digest, relatively low in metabolic energy, and contains 15-17% of difficult-to-digest shells during crushing. It is not suitable as a unit animal feed.

The bark content of palm seed cake can be reduced to 7% using a method of removing the bark of palm seed cake using a sieve after crushing.

The fiber in palm seed cake is mainly met type of hemicellulose (Daud and Jarvis, Phytochemistry , 31 (2): 463-464, 1992), so that digestibility can be enhanced by the treatment of the metase, a metase. When hydrolyzed, the fiber met by palm seed meal produces various digestible sugars that can be digested and absorbed by unit animals, including mannose (Dusterhoft et. al ., Bioresource Technology , 44, 39-46, 1993; Daud et al ., Proc. of the 19th Malaysian society of animal Production Annual Conference. 8-10 september 1997, Johor Bahru, Johor, Malaysia). Mannosides, such as mannose and mannooligosaccharides, which are naturally present in palm seed meal or are produced during hydrolysis of palm seed meal, are known to have an effect of inhibiting the settlement of Salmonella in the intestinal tract of cattle (Oyofo et al. , Poultry Science). , 68: 1357, 1989; Allem et al . , British Poultry science , 38 (5): 485, 1997).

Copra meal is a powder remaining after oiling from copra of Cocos palm, which is the same as the palm seed, and is used as a feed for ruminants. Consists of mannan-based fiber, which is not suitable for unit animal feed.

US Patent No. 6,096,918 is a commercially available beta-mannanase, beta-mannanase, mannosidase, hemicellulase, such as met methalytic enzymes or acid-catalyzed met and fibrous high content foil A feed additive comprising mannose produced by degradation was disclosed. However, such a single enzyme reaction alone can not be expected to effectively decompose the insoluble powder palm seed cake, as well as enzyme price, liquid It is not economical considering the cost of enzyme reaction and drying. In addition, there is a problem of contaminating bacteria growth during the enzyme reaction.

Typically beta-met kinase has been isolated from fungi and higher plants, such as a Bacillus bacterium, Aspergillus (Aspergillus), such as (Bacillus).

Japanese Patent JP5176767 discloses a method for producing beta-1,4-mannanase for food by anaerobic culture of Clostridium tertium and Lactobacillus sp., Japanese Patent JP2001145485 Discloses a metnase to prevent precipitation of coffee beverages using Penicillium and Eupenicillium .

In addition, studies using fermentation of microorganisms have been disclosed to enhance the feed value of palm seed meal. Lyayi and Aderolu reported that palm seed cakes were fermented with Trichoderma viride for 14 days to increase protein, water-soluble sugars, energy and decrease fiber content (Lyayi and Aderolu). , African Journal of Biotechnology , 3 (3): 182, 2004). However, since there is no mention of enzymatic activity such as mannanase, which is necessary for effective poultry feeding of palm seeds, it is not known how effective the microorganisms used are for fermenting palm seeds, and the fermentation period is too long. There is a problem that safety is not secured as a feed microorganism.

Therefore, there is a need in the art for the development of an effective mannanase-producing microorganism having a fermentation ability of palm fruit seeds and the development of animal feed containing palm fruit seed meal fermented using the same.

Therefore, the present inventors have identified and identified the microorganisms having a mannanase generating ability and fermentation ability of palm fruit seed, and as a result of diligently trying to develop a solid fermentation method of palm fruit seed using the same, the novel Bacillus subtilis strain of the present invention It was confirmed that the ability to meet the metase, the present invention was completed.

After all, the main object of the present invention is to provide a novel Bacillus subtilis strain having the ability to produce mannanase and fermentation of palm seed.

It is another object of the present invention to provide a solid phase fermentation method of palm seed seeds using the strain.

Still another object of the present invention is to provide an animal feed or an additive containing a palm fruit seed meal fermented by the solid phase fermentation method of the palm fruit seed meal.

Still another object of the present invention is to provide a method for preparing a mannanase from palm seed seed cake fermented with the strain.

In order to achieve the above object, the present invention provides a novel Bacillus subtilis strain having a mannanase production ability.

The present invention also provides a solid phase fermentation method of palm seed seed cake, characterized in that using the Bacillus subtilis strain.

The present invention also provides an animal feed or an additive containing a palm fruit seed gourd fermented by the solid phase fermentation method of the palm fruit seed gourd.

The present invention also provides a method for producing a met kinase from fermented palm fruit seed using a novel Bacillus subtilis strain having a mannanase producing ability.

 According to the present invention, the novel Bacillus subtilis strain has the effect of producing a variety of enzymes to help the digestion absorption of the feed, in particular, it is excellent in the metinase activity to improve the digestibility of palm seed meal feed such as palm seed gourd It is effective to The fermented palm fruit seed meal produced by the solid fermentation method of the palm fruit seed meal according to the present invention not only has improved digestibility, but also has increased nutrients such as proteins, and contains a large amount of microorganisms and enzymes beneficial to animals. Useful as

In one aspect of the invention, the present invention relates to a novel Bacillus subtilis strain having a mannanase generating ability. The Bacillus subtilis strain is characterized in that Bacillus subtilis MBS1 (Accession No. KCTC 11003BP), and the separation and identification method is as follows.

First, the samples collected from Meju, Cheonggukjang, Doenjang, etc. are properly diluted, then smeared on a medium, cultured to form colonies, and microorganisms are formed that form a decomposition ring that has become transparent by mannalysis. Isolated strains with high mannanase activity were identified by performing molecular phylogenetic methods based on morphological and 16S rDNA sequences. At this time, in order to facilitate the separation of microorganisms in Bacillus, a sample pretreated for 30 minutes at 70 ℃ can be used.

The medium includes LBG plate medium, Tryptic soy plate medium, Nutrient plate medium, Luria-Butani (Lung bean gum (LBG)), the main component of which met with the main carbon source. Luria-bertani) can be used.

The kinase activity of the microorganisms forming the degradation ring was enzymatically reacted in a constant pH buffer solution using locust bean gum as a substrate, and then the reducing sugars liberated by mannose decomposition were developed using DNS (dinitrosalicylic acid) reagent and the absorbance thereof was measured. It can measure by measuring.

The Bacillus subtilis strain according to the present invention is characterized in that it further has the ability to produce protease, starch enzyme, lipase, cellulosease and xylanase.

 Palm seeds are made of hemicellulose, protein, and fat, which are hard-tolerant, mainly composed of met. Therefore, in order to effectively decompose it, enzymes such as proteases (proteases), cellulose (celluloses) and xylanases (xylases), lipolytic enzymes (lipases), and starches (amylases) in addition to the metases It must work in combination.

The present invention relates to a solid-phase fermentation method of palm fruit seed palm, characterized in that it uses a Bacillus subtilis strain having a met kinase production capacity and a fermentation capacity of palm fruit seed.

In the present invention, the Bacillus subtilis strain can be used without limitation, as long as it has the ability to produce nanase and the fermentation ability of palm seed seed, and may include Bacillus subtilis MBS1 strain and the like.

In the present invention, the palm seed cake may be a palm seed cake (palm kernel cake), copra meal (copra meal) and the like.

Solid fermentation of the palm seed seed is preferably carried out at aeration conditions for 1 to 5 days at 25 to 45 ℃. The solid phase fermentation method of the palm seed oil is (a) heat treatment of palm fruit seed at 70 to 135 ℃; (b) 10-200 kGy irradiation of palm seed seeds; And (c) pre-treating the palm seed meal with at least one selected from the group consisting of treating the palm seed meal with 0.125 to 0.5% organic acid.

The heat treatment is to prevent the growth of contaminants present in the palm fruit seed, to relax the rigid structure of the palm fruit seed, to facilitate the action of microorganisms and enzymes. The heat treatment temperature may be performed by steaming, autoclave, dry heat at 70 to 135 ° C, and preferably at 100 to 121 ° C. When the heat treatment temperature is less than 70 ℃ is not satisfactory growth growth and enzymatic activity of the contaminating bacteria, there is a problem that the production cost increases when it exceeds 135 ℃. The heat treatment depends on the temperature, but is preferably carried out for 15 to 90 minutes.

The irradiation or organic acid treatment is a non-thermal treatment method, in place of the heat treatment, to prevent the growth of contaminants present in the palm fruit seed meal, and to relax the rigid structure of the palm fruit seed meal, more smoothly the action of microorganisms and enzymes It is to.

As the radiation, gamma rays such as cobalt-60 (Co-60) are preferably used.

It is preferable that the radiation absorption dose of the said palm seed seed foil is 10-200 kGy. When the radiation absorbed dose is less than 10kGy, the growth prevention and enzymatic activity increase effect of the contaminating bacteria is not satisfactory, and if the radiation absorption time exceeds 200kGy, there is a problem that the productivity decreases as the radiation treatment time becomes longer. Here, one gray (Gray, Gy) means that one joule of radiation energy is absorbed for 1 kg of material.

In addition, the organic acid is acetic acid, lactic acid, citric acid. Propionic acid and these may be mixed and used, preferably acetic acid.

The concentration of the organic acid is preferably 0.125 to 0.5% (w / w) of the weight of the palm seed seed substrate. When the concentration of the organic acid is less than 0.125%, the effect of preventing growth and enzymatic activity of contaminating bacteria is not satisfactory. If the concentration of the organic acid exceeds 0.5%, there is a problem of preventing the growth of the Bacillus subtilis strain of the present invention.

Compared with heat treatment, the non-thermal treatment method has an advantage that the equipment for fermentation is simple, low in cost, and economically advantageous.

The solid phase fermentation method of the palm fruit seed gourd according to the present invention is characterized by adjusting the moisture content of the palm fruit seed gourd to 35-65% (w / w). The water content of the palm seed meal is to control the activity and production of the met kinase, it is possible to control the water content by adding water to the powdered palm seed seed. When the moisture content of the palm seed meal is less than 35%, there is a problem of the growth of the strain and the reduction of enzyme production, and if it exceeds 65%, there is a problem of the decrease in air supply and the growth of contaminating bacteria.

In addition, the solid fermentation method of the palm fruit seed gourd according to the present invention is characterized in that the pH of the palm fruit seed gourd is adjusted to 6.4 to 7.5 before fermentation. The pH control of the palm seed meal is to promote the growth of Bacillus subtilis strains, the pH regulators are sodium hydroxide (sodium hydroxide), potassium hydroxide (potassium hydroxide), calcium hydroxide (calcium hydroxide), calcium oxide (calcium) basic substances such as oxides, ammonium hydroxide, uric acid, animal and plant ashes, and pH buffering substances such as phosphate and sodium phosphate. If the pH is less than 6.4, there is a problem that the growth of the Bacillus subtilis strain is not properly made, and if the pH exceeds 7.5 there is a problem that the production cost according to the use of the pH regulator increases.

When the basic substance is treated with palm seed meal, it is possible not only to promote microbial growth but also to obtain an additional effect of expanding and partially dissolving the palm seed meal substrate.

The solid fermentation method of the palm seed seed according to the present invention is characterized in that the fermentation by the addition of a fungal strain.

The fungal strain Aspergillus duck material (Aspergillus oryzae), Aspergillus material (Aspergillus sojae), Aspergillus and this (Aspergillus niger), Aspergillus Usami (Aspergillus usamii), Aspergillus Usami Shiro Usami (Aspergillus usamii mut. shirousamii), Aspergillus awamori (Aspergillus awamorii), rayijo crispus (Rhizopus), rayijo mu core (Rhizomucor), Trichoderma can be exemplified such as rijeyi (Trichoderma reesei), and Aspergillus duck material, Aspergillus It is preferable to use the material, Aspergillus Niger.

As such, when Bacillus subtilis strains and fungal strains are fermented to fertilize palm seeds, the synergistic effect of the mixed strains causes the growth rate of Bacillus subtilis, production of metase, and metase. There is an advantage that the activity is increased. In addition, when the mixed fermentation is performed, various kinds of enzymes having different characteristics may be produced from each strain, and these enzymes may be helpful for fermentation of palm seed seeds and digestion of animals.

In still another aspect, the present invention relates to an animal feed or an additive containing a palm fruit seed meal fermented by the solid phase fermentation method of the palm fruit seed meal.

Coconut seed meal fermented by the solid phase fermentation method according to the present invention has a higher water-soluble protein, free reducing sugars, amino acids, and vitamins, and lower fiber and hemicellulose content than conventional coconut seed meal. Do. In addition, palm and seed seed fermented by the solid-phase fermentation method contains various enzymes such as mannanase, protease, cellulase, xylanase, lipase, amylase and can be used as animal feed or additives thereof. It can be used as feed or feed additive.

In addition, Bacillus subtilis is a probiotic strain known to have a beneficial effect on animal health (Hong et al ., FEMS Microbiology Reviews , 29 (4): 813-835, 2005), so Bacillus subtilis MBS1 (deposited) When the fermented palm fruit seed meal according to the present invention including the number KCTC 11103BP) is used as an animal feed or an additive, a more excellent effect can be obtained.

In another aspect, the present invention relates to a method for producing a mannase, characterized in that after extracting the fermented palm fruit seed meal by the solid phase fermentation method of the palm fruit seed meal, the extract is purified.

In the method for preparing a mannase according to the present invention, a solvent such as distilled water or a buffer solution is added to a palm fruit seed seed fermented by the solid phase fermentation method, and then the fermented palm fruit seed seed is suspended, and solids are removed by precipitation and centrifugation. After extracting the crude enzyme solution, the step of purifying the metase through a conventional enzyme separation purification process.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Isolation and Identification of Palm Seed Fermented Microorganisms

end. Primary Isolation of Palm Seed Fermented Microorganisms

In order to isolate the microbial strain having the ability to meet the enzyme, samples were taken from Meju, Cheonggukjang and Doenjang. After diluting the sample, LBG plate medium (ammonium sulfate 0.5g; yeast extract 2g; K ₂ HPO ₄ 2g; MgSO ₄ 7H ₂ O 0.2g) with pH of 5, 6, 7, 8, 9, 10, respectively. 0.01 g of FeSO ₄ · 7H ₂ O; 0.05 g of CaCl ₂ · 2H ₂ O; 5 g of locust bean gum; 15 g of agar; DW 1 L) and 2 at 30, 40, 50, 55, 60 ° C. Each day was incubated. Here, LBG plate medium has locust bean gum, which is mainly composed of met, as the only carbon source. Also, tryptic soy plate with 0.5% locust bean gum, Nutrient plate, and Luria-bertani plate were used for the same LBG plate. It was cultured by the method.

Next, after separating the colony forming a decomposition ring in the surrounding medium from the medium, it was passaged twice in tryptic soy liquid medium. Subsequently, subcultured strains were again inoculated into 2 μl of LBG plate medium, incubated at 30 ° C. and 50 ° C. for 20 hours, and first, strains having a large degradation ring size were selected.

The size of the degradation ring was measured in parallel with the following measurement method.

a) A method of directly measuring the ring of lysis that has become transparent due to the decomposition of locust bean gum.

b) staining the surface of the medium with Congo red staining solution (0.1% Congo red) for 30 minutes, then removing the non-absorbed Conco red staining medium, and again applying 1 mol (1M) NaCl solution to the surface of the medium, How to measure the transparent ring generated when shaken slowly for 15 minutes

c) Method for measuring the clear ring that is not stained when the medium is stained with Iodine staining solution (Iodine 1g, Potassium Iodide 2g, D.W. 300ml)

I. Secondary Separation of Palm Seed Fermented Microorganisms

Sodium hydroxide and distilled water were treated to sterilize the palm seed cake adjusted to pH 6.8, water content 55% at 121 ° C. for 15 minutes. After inoculating the culture medium of the strain first selected in 500 g of sterile palm seed gourd so that the initial number of bacteria to about 1 × 10 ⁷ level per 1 g of the dry weight of palm seed gourd, it was incubated at 30 ° C. Next, the bacterium MBS1 was secondarily selected as a strain having excellent microbial growth and metase activity.

Microbial metase activity was determined by quantifying the reducing sugars released after the enzymatic reaction using locust bean gum as a substrate. The quantitative measurement of the free reducing sugar is as follows.

After shaking 9 ml of sterile distilled water and 1 g of palm seed cake fermented sample, centrifuged at 12,000 rpm for 15 minutes, the supernatant was separated and used as an enzyme solution. Next, 0.5 ml of enzyme solution, 1 ml of 1% (w / v) locust bean gum solution suspended in distilled water, and 0.5 ml of 200 mM sodium citrate buffer (pH 5.8) were mixed and mixed at 50 ° C. for 10 minutes. Reacted. After the completion of the reaction, 1 ml of reaction solution and 1 ml of 3, 5-dinitro salicylic acid (DNS) reagent (Miller et al . Analytical Biochemistry , 2: 127-132, 1960) were added, Color reaction was carried out for a minute. Next, 10 ml of distilled water was added to the reaction, and then the absorbance was measured at 540 nm. Here, mannose was used as a reference material, and 1 unit of enzyme activity was calculated for the amount of the enzyme that produces reducing sugar corresponding to 1λmol of mannose from locust bean gum for 1 minute under the above conditions.

Through the above process, the maximum metase activity during fermentation of palm seed cake of MBS1 strain selected as an excellent strain is shown in Table 1 compared with Bacillus specimen.

Strain Metase activity (unit / g) MBS1 1,220 Bacillus subtilis ATCC 6633 455 Bacillus subtilis 168 6 B. licheniformis ATCC 14580 One

In Table 1, 1 unit of enzyme activity refers to the amount of enzyme that produces a reducing sugar corresponding to 1λmol of mannose from locust bean gum for 1 minute under the above conditions.

All. Identification of Palm Seed Fermented Microorganisms

MBS1 with high mannase activity and excellent palm seed fermentation ability was identified by molecular phylogenetic method based on morphological characteristics and 16S rDNA (16S rRNA coding gene) sequence. To analyze 16S rDNA sequences, DNA extracted from each strain was PCR amplified using universal primers (5'-GAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 1), 5'-ACGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2)). It was. The amplified 16S rDNA was purified using a PCR product purification kit (Qiagen), followed by sequencing. Sequencing was performed by CLUSTAL W (Thompson et. al ., Nucleic Acids Research , 22: 4673-4680, 1994) and the PHYLIP program (Felsenstein, PHYLIP: Phylogenetic Inference Package, version 3.5.Seattle: University of Washington, 1993).

16S rDNA sequence homology analysis of each strain (SEQ ID NO: 3), the isolated microorganism was identified as Bacillus subtilis ( Bacillus subtilis ). Bacillus subtilis isolated from the present invention was deposited on March 30, 2007 to the Korea Institute of Biotechnology Gene Bank (Accession Number: KCTC 11103BP).

Example 2 Bacillus according to the culture temperature Subtilis Mannanase Production Characteristics

Two microliters of each strain (Bacillus subtilis MBS1 and Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) incubated in Tryptic Soy liquid medium for 16 hours were plated with LBG (ammonium sulfate 0.5). g; yeast extract 2g; K ₂ HPO ₄ 2g; MgSO ₄ · 7H ₂ O 0.2g; FeSO ₄ · 7H ₂ O 0.01g; CaCl ₂ · 2H ₂ O 0.05g; 5 g locust bean gum; ag 15g inoculated in 1 liter of distilled water (pH 7.0) and incubated at 30, 35, 40, 45 and 50 ° C. for 20 hours, and then the size of the degraded ring was measured. The measurement results are shown in Table 2 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 35 ℃ 40 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 21.0 24.0 27.8 27.0 23.5 B. subtilis ATCC 6633 19.0 22.5 24.5 26.5 24.5 B. subtilis 168 17.0 19.5 22.0 24.0 21.0 B. licheniformis ATCC 14580 15.0 19.0 22.0 23.0 23.5

Example 3 Enzyme Production Characteristics of Bacillus subtilis

end. Starch Enzyme Production Characteristics

The production characteristics of amylase of the fermented bacteria from isolated palm seed cake were investigated. 2 μl of each strain (Bacillus subtilis MBS1, Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) cultured for 16 hours in tryptic Soy liquid medium was added with 1% of starch as a substrate. Inoculated in a Nutrient plate medium (pH 7.0), incubated at 30, 37, 45 and 50 ℃ 20 hours, respectively, and the size of the degradation ring was measured. The measurement results are shown in Table 3 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 37 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 14.8 17.9 19.5 18.5 B. subtilis ATCC 6633 12.5 16.2 21.6 20.0 B. subtilis 168 12.5 14.2 17.0 18.5 B. licheniformis ATCC 14580 7.5 11.0 13.5 13.5

I. Protease Production Characteristics

The protease production characteristics of the isolated palm seed cake fermented bacteria were investigated. 2 μl of each strain (Bacillus subtilis MBS1, Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) cultured for 16 hours in a tryptic soy liquid medium was used as a substrate. Inoculated in% added Nutrient plate medium (pH 7.0), and incubated at 30, 37, 45 and 50 ℃ 20 hours, respectively, the size of the degradation ring was measured. The measurement results are shown in Table 4 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 37 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 21.5 24.8 27.3 19.0 B. subtilis ATCC 6633 21.0 24.0 26.5 23.5 B. subtilis 168 19.2 21.0 24.0 21.0 B. licheniformis ATCC 14580 11.0 12.0 16.8 18.0

All. Lipolytic Production Characteristics

Lipase production characteristics of the isolated palm seed cake fermented bacteria were investigated. Tributyrin (tributyrin) was used as a substrate for 2 μl of each strain (Bacillus subtilis MBS1, Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) culture medium incubated for 16 hours in tryptic soy liquid medium. ) Was inoculated in Nutrient plate medium (pH 7.0) to which 0.5% (w / v) was added, and incubated at 30, 37, 45, and 50 ° C for 20 hours, respectively, and the size of the degraded ring was measured. The measurement results are shown in Table 5 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 37 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 12.0 14.0 14.0 10.0 B. subtilis ATCC 6633 12.8 14.5 14.0 11.5 B. subtilis 168 11.0 12.0 10.2 10.2 B. licheniformis ATCC 14580 6.0 7.5 8.2 8.8

la. Fibrinase Production Characteristics

The cellulase production characteristics of the isolated palm seed cake fermented bacteria were investigated. Two microliters of each strain (Bacillus subtilis MBS1, Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) cultured for 16 hours in a Tryptic Soy liquid medium were used as a substrate for carboxymethyl cellulose ( Carboxymethyl cellulose) was inoculated in Nutrient plate medium (pH 7.0) to which 0.5% (w / v) was added and incubated at 30, 37, 45, and 50 ° C for 24 hours, and then the size of the degradation ring was measured. The measurement results are shown in Table 6 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 37 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 18.5 21.5 23.5 23.3 B. subtilis ATCC 6633 19.5 23.5 24.5 25.0 B. subtilis 168 13.0 16.0 19.2 18.8 B. licheniformis ATCC 14580 11.0 12.0 14.0 13.0

hemp. Xylanase Production Characteristics

The production characteristics of xylanase of isolated palm seed cake fermented bacteria were investigated. Oat-derived xylan was used as a substrate using 2 μl of each strain (Bacillus subtilis MBS1, Bacillus subtilis ATCC 6633, Bacillus subtilis 168, and Bacillus rickenformis DSM13) incubated for 16 hours in Tryptic Soy liquid medium. Inoculated in% added Nutrient plate medium (pH 7.0), incubated at 30, 37, 45 and 50 ℃ for 24 hours, respectively, the size of the degradation ring was measured. The measurement results are shown in Table 7 below.

Strain Disintegration Ring Diameter (mm) 30 ℃ 37 ℃ 45 ℃ 50 ℃ Bacillus subtilis MBS1 20.8 23.0 25.7 19.2 B. subtilis ATCC 6633 18.0 21.5 24.0 23.5 B. subtilis 168 16.5 20.0 21.5 22.8 B. licheniformis ATCC 14580 13.8 19.0 22.0 22.8

Example 4 Measurement of Palm Seed Fermentation Conditions of Bacillus subtilis

end. Effect of pH on Palm Seed Fermentation

Sodium hydroxide was added to the palm seed meal to adjust the pH of the palm seed meal to 6.0, 6.4, 6.6 and 6.8, respectively. At this time, the moisture content was adjusted to 55%. After sterilizing 500 g of each palm seed foil substrate having a pH adjusted at 121 ° C. for 15 minutes, the Bacillus subtilis MBS1 was inoculated to 1 × 10 ⁷ CFU level per 1 g of palm seed leaf dry weight and then incubated at 30 ° C. for 3 days. Incubated. Bacillus subtilis MBS1 according to the pH and the production capacity of the met kinase was measured and shown in Figures 1 and 2.

1 is a graph showing growth of Bacillus subtilis MBS1 according to pH. From Figure 1, it can be seen that Bacillus subtilis MBS1 increased or decreased in the case of pH of 5.2 and 6.0, while Bacillus subtilis MBS1 increased in the case of pH 6.4, 6.6, 6.8 and 7.5. At pH 6.8 and pH 7.5, the increase in Bacillus subtilis MBS1 was found to be the most active.

2 is a graph showing the met kinase activity of Bacillus subtilis MBS1 according to pH. 2, it was found that the pH of 5.2 and 6.0 of the palm seed meal was low, while the activity of the Bacillus subtilis MBS1 strain was low, whereas the pH of 6.4, 6.6, 6.8, and 7.5 was increased. . In particular, the pH of the 6.8 and 7.5 was the highest activity met by the first day, and the pH 6.4 and 6.6 was found to be the highest activity met by 1.5 to 2 days.

I. Effect of Water Content on Palm Seed Fermentation

In order to examine the effect of water content on palm seed meal fermentation, the water content of palm seed meal was added to 35, 40, 45, 50, 55, 60 and 65% (w / w), respectively. The pH was adjusted to 6.8 and autoclaved at 121 ° C. for 15 minutes. Next, Bacillus subtilis MBS1 was inoculated (1 × 10 ⁷ CFU per 1 g of palm seed cake dried) to palm seed cake, and cultured at 30 ° C. The production capacity of the met kinase of Bacillus subtilis MBS1 according to water content was measured on the first day of culture, and is shown in Table 8 below.

Moisture content (%) Metase activity (unit / g) 35 450 40 700 45 1,005 50 1,150 55 1,220 60 1,300 65 1,250

From the above Table 8, when the water content of the palm seed gourd was 60%, the rendezvous activity was the highest on the first day of cultivation, and showed a 75% or more activity of the highest value in a wide range of 45 to 65%, and the water content Even lower 40% showed more than 50% of the met kinase activity. That is, the Bacillus subtilis MBS1 according to the present invention not only exhibits met kinase activity even when it is higher than the water content conditions of a conventional solid phase fermentation process, but also has a low water content for reducing air supply, contaminant growth, and drying cost. It can be seen that even when fermented under the conditions, a considerable amount of met kinase activity is shown.

All. Effect of Heat Treatment on Palm Seed Fermentation

To investigate the effect of heat treatment on palm seed cake fermentation, the palm seed cake (55% water content, pH 6.8) was not heat treated and the cooked steam (100 ℃, 90 minutes) or autoclaved (121 ℃, 15 minutes or Fermentation characteristics in the case of heat treatment in 30 minutes) were investigated. The non-heat treated palm seed meal and each heat treated palm seed meal were inoculated with Bacillus subtilis MBS1 (1 × 10 ⁷ CFU per 1 g of palm seed meal dried) and incubated at 30 ° C. for 3 days. The strain amount of Bacillus subtilis MBS1 according to the heat treatment conditions and the production capacity of the met kinase were measured and shown in FIGS. 3 and 4.

3 is a graph showing growth of Bacillus subtilis MBS1 according to heat treatment. 3, the strain amount of the palm seed gourd without heat treatment (increase in steam or autoclave) was similar to the strain amount of the palm seed gourd subjected to heat treatment, but it decreased after 1 day of fermentation. It is believed that when palm seeds are not heat treated, growth of Bacillus subtilis MBS1 is inhibited due to the growth of contaminants.

4 is a graph showing the met kinase activity of Bacillus subtilis MBS1 according to the heat treatment. From FIG. 4, it can be seen that the normal fermentation proceeded when the heat treatment (a steam or autoclave) exhibited the best mannase activity on the first day of fermentation. On the other hand, fermentation without heat treatment showed a similar encounter with anneal activity until 16 hours of fermentation, but after that, activity was gradually decreased and continuous activity was not achieved.

la. Compositional Changes of Fermented Palm Seeds by Bacillus subtilis MBS1

Ingredients of fermented palm seed cake fermented by Bacillus subtilis MBS1 (crude protein, crude fiber, neutral detergent fiber (NDF), acidic detergent fiber (ADF), hemicellulose, reducing sugar, water-soluble) Protein, vitamin B ₂ ) was analyzed.

Analysis of crude protein, crude fiber, NDF and ADF was measured according to the method described in the feed standard analysis method of the feed process. In other words, crude protein was measured by the Kjeldahl method, crude fiber was measured by weighing the residual material after incineration at 600 ℃, NDF and ADF was added to the neutral detergent solution and acidic detergent solution, and then boiled and filtered to weigh the remaining insoluble material. Was calculated by measuring. Hemicellulose content is shown by calculating the difference in content of NDF and ADF.

Reducing sugar and water-soluble protein content was suspended in 9 times distilled water in a certain amount of palm seed cake fermentation, shaken for 5 minutes, centrifuged at 12,000 rpm for 15 minutes to separate the supernatant, and quantified the amount of reducing sugar and protein released in the separated supernatant It measured by.

The quantitative method of free reducing sugar was added 1 ml of the diluted supernatant to the effective range of the color reaction, and 1 ml of 3,5-dinitro salicylic acid (DNS) reagent, and the color was developed at 100 ° C. for 5 minutes. Reacted. Next, 10 ml of distilled water was added to the reaction, and then the absorbance was measured at 540 nm. Here, mannose was used as a standard.

Quantification of the free protein was determined by Lowry-Folin method. That is, 0.1 ml of 2N sodium hydroxide solution and 10 ml of copper sulfate solution (CuSO ₄ 20 g; NaCO ₃ 20 g; sodium potassium tartrate 10 g; distilled water; 1 L) were added to 0.1 ml of the sample (supernatant) extracted by the above method, followed by 10 minutes at room temperature. After leaving for a while, Folin reagent was added thereto and left for 30 minutes. Then absorbance was measured at 570 nm. Here, albumin (bovine serum albumin, BSA) was used as a standard.

And the quantification of vitamin B ₂ was analyzed using HPLC. Palm seed cake fermentation was extracted with 20% (v / v) methanol (methanol) solution containing 0.2M NaOH, neutralized, diluted to an appropriate concentration and filtered through a membrane filter (0.45 λ m) to prepare an extract. HPLC column used Metachem Monochrom (4.6mm × 250mm) and UV using 10mM phosphate buffer containing 12.5% (v / v) of acetonitrile as mobile phase solvent (flow rate, 1.2ml / min) The detector detected at 271 nm.

The change in composition after fermentation (after 2 days) by Bacillus subtilis MBS1 of palm seed cake is shown in Table 9 below.

Furtherance Palm seed
(No treatment) Fermented Palm Seeds Crude Protein (%, w / w) 16.5 17.5 Crude fiber (%, w / w) 23.5 21.5 NDF (%, w / w) 71.7 66.8 ADF (%, w / w) 43.8 46.8 Hemicellulose (%, w / w) 27.9 20.0 Reducing Sugar (mg / g) 11.5 29.1 Water Soluble Protein (mg / g) 19.5 132.1 Vitamin B ₂ (ppm) 1.67 4.34

From Table 9, the palm seed meal fermented by Bacillus subtilis MBS1 is increased in crude protein, water-soluble protein, reducing sugar, vitamin B ₂ , crude fiber, NDF, ADF and hemicellulose is reduced, digestibility is enhanced As a result, nutritional value was improved.

In addition, the amino acid content of the palm seed meal fermented by Bacillus subtilis MBS1 was analyzed. The amino acid content was analyzed by an amino acid autoanalyzer by treating the sample according to the ion exchange chromatography method (ninhydrin method) described in the feed standard analysis method.

Amino acid composition changes after fermentation by Bacillus subtilis MBS1 of palm seed cake are shown in Table 10 below.

Amino acid composition
(g / 100g) Palm seed
(No treatment) Fermented Palm Seeds Aspartic acid 1.37 1.59 Throneine 0.53 0.55 Serine 0.76 0.70 Glutamic acid 3.34 3.50 Proline 0.6 0.75 Glycine 0.79 0.85 Alanine 0.69 0.71 Valine 0.82 0.77 Isoleucine 0.54 0.57 Leucine 1.57 1.46 Tyrosine 0.43 0.54 Phenylalnine 0.76 0.79 Histidine 0.45 0.54 Lysine 0.33 0.52 Arginine 1.94 1.52 Cystein 0.19 0.31 Methionine 0.25 0.19 Tryptophan 0.04 0.02

From Table 10, it was found that the palm seed meal fermented by Bacillus subtilis MBS1 increased the amino acid content, including lysine. It can be seen that the fermentation of palm seed meal by Bacillus subtilis MBS1 converts the constituents of palm seed meal and increases the amino acid content, thereby enhancing nutritional value.

hemp. Compositional Changes of Palm Seed Bak by Irradiation

Cobalt-60 was used to treat 20, 50, 100, 200 kGy of radiation to be absorbed into the palm seed cake. To investigate the change after irradiation of palm seed cake, 1 g of sample was suspended in 9 ml of distilled water, shaken for 5 minutes, centrifuged at 12,000 rpm for 15 minutes to separate the supernatant, and the amount of reducing sugar and protein liberated in the separated supernatant was quantified. It measured by. Quantification of free reducing sugars, free protein and NDF was measured in the same manner as in "D of Example 4" above. The compositional change of palm seed cake according to the irradiation is shown in Table 11 below.

Radiation Absorption Rate (kGy) of Palm Seeds 0 20 50 100 200 Reducing Sugar (mg / g) 11.50 16.56 17.67 19.33 21.22 Water Soluble Protein (mg / g) 19.50 25.56 27.67 27.80 29.56 NDF (%, w / w) 71.45 71.00 70.35 68.21 66.81

From Table 11, it can be seen that the content of reducing sugar and water soluble protein is increased and the content of NDF is decreased as the amount of radiation absorption of palm seed cake is increased. These results indicate that the fiber and protein of palm seed meal, including met, were partially degraded by radiation treatment.

bar. Effect of Radiation on Palm Seed Fermentation

Palm seed cake was irradiated with cobalt-60 to absorb 20, 50, 100, and 200 kGy of radiation, and distilled water and sodium hydroxide solution were added to obtain a water content of 55% and pH 6.8. Media was prepared without. Subsequently, Bacillus subtilis MBS1 was inoculated (1 × 10 ⁷ CFU per 1 g of palm seed cake dried) to the radiation-treated palm seed cake and incubated at 30 ° C. for 2 days. On the other hand, inoculated and cultured in the same manner for the non-heat-treated palm seed meal medium and autoclaved palm seed meal medium. Growth of Bacillus subtilis MBS1 and production capacity of the met kinase according to the radiation treatment conditions were measured and shown in FIGS. 5 and 6.

5 is a graph showing growth of Bacillus subtilis MBS1 following radiation treatment. From Figure 5, it can be seen that Bacillus subtilis MBS1 of palm seed foil treated with radiation shows a growth curve similar to Bacillus subtilis MBS1 of autoclaved palm seed foil.

6 is a graph showing the met kinase activity of Bacillus subtilis MBS1 following radiation treatment. From Figure 6, it can be seen that the Bacillus subtilis MBS1 of the radiation-treated palm seed cake had better mannase activity than the Bacillus subtilis MBS1 of the non-radiated or autoclaved palm seed cake. In particular, when the absorption of radiation was 100kGy at 24 hours of fermentation, it showed the highest kinase activity.

7 is a graph showing the change of reducing sugar during the fermentation process of palm seed cake according to the radiation treatment. From Fig. 7, it can be seen that the palm seed meal treated with radiation increased the reducing sugar more than the autoclaved palm seed meal. In particular, reducing sugars increased with increasing radiation absorption.

FIG. 8 is a graph showing changes in soluble protein during fermentation of palm seed meal according to radiation treatment. From Fig. 8, it was found that the palm seed meal treated with radiation increased more water-soluble protein than the autoclaved palm seed meal. In particular, as the amount of radiation absorption increased, the water-soluble protein increased.

four. Component Analysis of Irradiated Palm Seed Ferment

The components (fermented protein, crude fiber, neutral detergent insoluble fiber (NDF), acidic detergent insoluble fiber (ADF), reducing sugar and water soluble protein) of the fermentation of the radiation palm seed cake by the Bacillus subtilis MBS1 were analyzed.

Of Radiated Palm Seeds The compositional change after fermentation by Bacillus subtilis MBS1 is shown in Table 12 below.

Furtherance Palm seed
(No treatment) Fermented Palm Seeds (*)
Heat treatment Radiation dose (kGy) 20 50 100 200 Crude Protein (%, w / w) 16.5 17.5 19.1 18.8 18.7 19.6 Crude fiber (%, w / w) 23.5 21.5 17.7 16.2 15.2 16.7 NDF (%, w / w) 71.7 66.8 62.8 61.2 56.7 56.5 ADF (%, w / w) 43.8 46.8 42.4 39.7 36.8 43.4 Hemicellulose (%, w / w) 27.9 20.0 20.4 21.6 20.0 13.1 Reducing Sugar (mg / g) 11.5 29.1 35.0 42.0 50.9 51.2 Water Soluble Protein (mg / g) 19.5 132.1 107.1 121.9 130.6 130.6

^(*) Heat treatment: 121 ℃, 15min

From Table 12, it can be seen that the irradiated palm seed cake fermented product increased crude protein, reducing sugar and water-soluble protein, decreased crude fiber, NDF, ADF and hemicellulose, thereby improving digestibility and improved nutritional value. have.

Ah. Effect of Organic Acids on Palm Seed Fermentation

Acetic acid concentration was treated to palm seed cakes so that the acetic acid concentration was 0.125%, 0.25%, 0.38% and 0.5%, respectively, and the distilled water and sodium hydroxide solution were used so that the water content was 55% and pH 6.8. Next, Bacillus subtilis MBS1 was inoculated (1 × 10 ⁷ CFU per 1 g of palm seed cake dried) to the organic acid-treated palm seed cake, and incubated at 30 ° C. for 3 days. The results of measuring the number of Bacillus subtilis MBS1 and contaminating bacteria of palm seed meal treated with acetic acid by concentration according to fermentation time are shown in Table 13 below.

Fermentation time (days) Bacillus subtilis MBS1 can
(log cfu / g) Contaminating bacteria
(log cfu / g) Acetic acid concentration (%) Acetic acid concentration (%) 0 0.125 0.25 0.38 0.50 0 0.125 0.25 0.38 0.50 0 7.00 7.00 7.00 7.00 7.00 4.50 4.02 3.75 3.50 2.85 0.5 8.80 9.01 9.32 8.66 8.18 9.40 8.90 7.00 6.30 5.00 One 8.60 9.11 9.46 8.87 8.41 10.24 9.91 9.11 7.90 7.00 2 7.00 8.70 9.04 8.85 9.08 10.86 10.53 10.30 10.02 9.72 3 6.00
Below 8.00 7.65 7.60 7.45 11.20 11.14 10.79 10.41 10.17

From Table 13, it was found that when acetic acid was not treated, the growth rate of contaminated bacteria was high, and the growth of Bacillus subtilis MBS1 was slow. On the other hand, acetic acid treatment showed a slow growth rate of contaminating bacteria and a fast growth of Bacillus subtilis MBS1. That is, when acetic acid was not treated, it could be seen that the number of Bacillus subtilis MBS1 increased due to the growth of contaminants, but decreased after one day of fermentation. On the other hand, when 0.125, 0.25, 0.38 and 0.5% acetic acid was treated, it was found that the growth of Bacillus subtilis MBS1 was similar to that of heat-treated palm seed cake as the effect of inhibiting the growth of contaminants caused by acetic acid. Can be.

The results of Table 14 are shown in FIGS. 9 and 10. 9 is a graph showing growth of Bacillus subtilis MBS1 according to organic acid treatment. 10 is a graph showing the growth of contaminating bacteria according to organic acid treatment.

In addition, the results of measuring the activity of the met kinase according to the organic acid treatment 1.5 days are shown in Table 14 below.

Acetic acid concentration (%) Metase activity (unit) 0 125 0.13 280 0.25 545 0.38 420 0.50 385

From Table 14, as a result of comparing the activity of the met kinase at 1.5 days of fermentation, it was found that the treatment of acetic acid increased the met kinase activity than the treatment without acetic acid. In particular, 0.25% acetic acid treatment showed the highest activity. It is inferred that the growth of the Bacillus subtilis MBS1 and the production of mannase were possible by reducing the proliferation of contaminants through acetic acid treatment. Therefore, the method of fermenting palm seed seed by organic acid treatment is useful as an economical method that enables the production of a considerable amount of metase without heat treatment.

Example 5 Mixed Fermentation of Palm Seed Bak using Bacillus Subtilis MBS1 and Aspergillus Duck MFO1

end. Growth of Strains by Mixed Fermentation

After adding water to the water content of palm seed cake to 55% (w / w), the pH was adjusted to 6.2, 6.4, 6.6 and 6.8, respectively, and autoclaved at 121 ℃ for 15 minutes. Next, Bacillus subtilis MBS1 (1 × 10 ⁷ CFU per 1 g of palm seed cake dry) and Aspergillus duckwood MFO1 (strain No. KCTC 11105BP) (5 × 10 ⁵ spore per 1 g of palm seed cake dry) into palm seed cake ) Was inoculated by mixing and incubated at 30 ° C. for 5 days. In addition, Bacillus subtilis MBS1 was inoculated alone and cultured as a control.

11 is a graph showing the growth of Bacillus subtilis MBS1 according to the initial pH in mixed fermentation. 11, when mixed culture of Bacillus subtilis MBS1 and Aspergillus duck material MFO1, even if the fermentation is started at low pH conditions (pH 6.2, 6.4), it is adjusted to pH 6.8 to control the incubation of Bacillus subtilis MBS1 alone It was found that the growth of the strain was superior to that of the case. Therefore, when the mixed fermentation is performed, Bacillus subtilis MBS1 can be cultured in high yield while reducing the cost of adding the pH regulator.

I. Enzyme Activity by Mixed Fermentation

Water content of palm seed cake was 55% (w / w), pH was adjusted to 6.4, autoclaved at 121 ℃ for 15 minutes. Next, Bacillus subtilis MBS1 (1 × 10 ⁷ CFU per 1 g of palm seed cake dry) and Aspergillus duckwood MFO1 (strain No. KCTC 11105BP) (5 × 10 ⁵ spore per 1 g of palm seed cake dry) into palm seed cake ) Was inoculated by mixing and incubated at 30 ° C. for 5 days. Further, as a control, Bacillus subtilis MBS1 and Aspergillus duck MFO1 were inoculated and cultured, respectively.

12 is a graph showing the activity of the met antase according to the mixed fermentation of Bacillus subtilis MBS1 and Aspergillus duck material MFO1. From Fig. 12, it was found that when the Bacillus subtilis MBS1 and Aspergillus duck material MFO1 were mixed and cultured, the met kinase activity of the Bacillus subtilis MBS1 was superior to that of the single culture. In addition, when the Bacillus subtilis MBS1 alone was cultured, after 1 day of fermentation, the rendezvous activity gradually decreased, whereas in the mixed culture, the total nanase activity was increased by the production of the antagonist produced by the Aspergillus duck MFO1 strain. After days, the increase was found again.

In particular, on the 5th day of fermentation, in the case of the mixed culture, the kinase enzyme activity was greater than the sum of the individual cultures of each species, indicating that the synergy effect occurred in the production of the mannase in the mixed culture. Could. That is, in case of Bacillus subtilis MBS1 mainly produced the met kinase from the early fermentation, and after 2 days of fermentation, it was confirmed that the activity of the met kinase is gradually decreased while the active metabolism of the strain is stopped.

On the other hand, the Aspergillus duck MFO1 strain is active in microbial metabolism from the beginning of fermentation, but the activity of the metase starts after 1 day of fermentation and reaches its highest level after 3 days of fermentation. It can be seen that. Therefore, the fermentation of palm seed meal was made possible by the continuous action of metase and other enzymes from the first half to the second half of fermentation by mixed culture.

In addition, after mixing and fermenting Aspergillus Niger (KCCM 11239) in place of the Aspergillus duck MFO1, the met kinase activity is measured and shown in FIG.

All. Changes in Composition of Fermented Palm Seeds by Fermentation

Ingredients of palm seed cake fermentation product according to mixed fermentation of Bacillus subtilis MBS1 and Aspergillus duck MFO1 (crude protein, crude fiber, neutral detergent insoluble fiber (NDF), acidic detergent insoluble fiber (ADF), hemicellulose, Reducing sugars, water soluble proteins).

Of Palm Seeds by Mixed Fermentation The composition change after fermentation is shown in Table 15 below.

Furtherance Palm seed
(No treatment) Fermented Palm Seeds Single culture ^(*) Mixed Culture ^(**) 2 days 3 days 5 days Crude Protein (%, w / w) 16.5 17.5 17.8 18.0 Crude fiber (%, w / w) 23.5 21.5 20.5 23.2 NDF (%, w / w) 71.7 66.8 66.2 70.7 ADF (%, w / w) 43.8 46.8 48.1 58.6 Hemicellulose (%, w / w) 27.9 20.0 18.1 12.1 Reducing Sugar (mg / g) 11.5 29.1 35.1 72.0 Water Soluble Protein (mg / g) 19.5 132.1 92.7 155.0

^(*) Bacillus subtilis MBS1

^(**) Bacillus subtilis MBS1 vs Aspergillus duck MFO1

From the above Table 15, palm seed meal fermented by fermentation of Bacillus subtilis MBS1 and Aspergillus duck MFO1 fermented crude protein, reducing sugar and water soluble protein, crude fiber, NDF and hemicellulose are reduced, Digestion is enhanced and nutritional value is improved.

The amino acid content of palm seed gourd fermentation according to the mixed fermentation of the Bacillus subtilis MBS1 and Aspergillus duck material MFO1 was analyzed. Of Palm Seeds by Mixed Fermentation The amino acid content changes after fermentation are shown in Table 16 below.

Amino acid composition
(g / 100g) Palm seed
(No treatment) Fermented Palm Seeds Single culture ^(*) Mixed Culture ^(**) 2 days 3 days 5 days Aspartic acid 1.37 1.59 1.92 1.51 Throneine 0.53 0.55 0.66 0.62 Serine 0.76 0.70 0.89 0.96 Glutamic acid 3.34 3.50 4.04 3.70 Proline 0.6 0.75 1.08 0.74 Glycine 0.79 0.85 1.00 0.94 Alanine 0.69 0.71 1.13 0.83 Valine 0.82 0.77 1.15 0.70 Isoleucine 0.54 0.57 0.90 0.52 Leucine 1.57 1.46 1.89 1.54 Tyrosine 0.43 0.54 0.54 0.54 Phenylalnine 0.76 0.79 0.79 0.84 Histidine 0.45 0.54 0.50 0.51 Lysine 0.33 0.52 0.63 0.51 Arginine 1.94 1.52 1.73 2.45 Cystein 0.19 0.31 0.31 0.24 Methionine 0.25 0.19 0.21 0.14 Tryptophan 0.04 0.02 0.04 0.02

^(*) Bacillus subtilis MBS1

^(**) Bacillus subtilis MBS1 vs Aspergillus duck MFO1

From Table 16, it was found that the fermented palm seed meal fermented by the mixed fermentation of Bacillus subtilis MBS1 and Aspergillus duck material MFO1 increased the amino acid content. In particular, in the mixed culture it was found that the amino acid content was increased more than the case of culturing only Bacillus subtilis MBS1 alone. This can be seen that the fermentation by the mixed fermentation by Bacillus subtilis MBS1 and Aspergillus duck material MFO1 to increase the amino acid content as the constituents of palm seed gourd enhances nutritional value.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 and 2 are graphs showing growth and metase activity of Bacillus subtilis MBS1 according to pH.

3 and 4 are graphs showing growth and metase activity of Bacillus subtilis MBS1 according to heat treatment.

5 and 6 are graphs showing growth and metase activity of Bacillus subtilis MBS1 following radiation treatment.

7 and 8 are graphs showing changes in reducing sugars and soluble proteins of palm seed meal after radiation treatment.

9 is a graph showing growth of Bacillus subtilis MBS1 according to organic acid treatment.

10 is a graph showing the growth of contaminating bacteria according to organic acid treatment.

11 is a graph showing the growth of Bacillus subtilis MBS1 according to pH in mixed fermentation.

12 is a graph showing the activity of the met antase according to the mixed fermentation of Bacillus subtilis MBS1 and Aspergillus duck material MFO1.

Figure 13 is a graph showing the activity of the met kinase according to the mixed fermentation of Bacillus subtilis MBS1 and Aspergillus Niger (KCCM 11239).

<110> Korea Research Institute of Bioscience and Technology <120> New Strain of Bacillus subtilis and Solid Phase Fermentation          Process Using the Same <130> P07-B29 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gagtttgatc ctggctcag 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acggttacct tgttacgact t 21 <210> 3 <211> 1437 <212> DNA <213> Bacillus sp. <400> 3 cacttcggcg gctggctcct aaaggttacc tcaccgactt cgggtgttac aaactctcgt 60 ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc 120 gattactagc gattccagct tcacgcagtc gagttgcaga ctgcgatccg aactgagaac 180 agatttgtgg gattggctta acctcgcggt ttcgctgccc tttgttctgt ccattgtagc 240 acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg 300 gtttgtcacc ggcagtcacc ttagagtgcc caactgaatg ctggcaacta agatcaaggg 360 ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca 420 ccacctgtca ctctgccccc gaaggggacg tcctatctct aggattgtca gaggatgtca 480 agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540 ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt 600 aatgcgttag ctgcagcact aaggggcgga aaccccctaa cacttagcac tcatcgttta 660 cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt 720 cagttacaga ccagagagtc gccttcgcca ctggtgttcc tccacatctc tacgcatttc 780 accgctacac gtggaattcc actctcctct tctgcactca agttccccag tttccaatga 840 ccctccccgg gttgagccgg gggctttcac atcagactta agaaaccgcc tgcgagccct 900 ttacgcccaa taattccgga caacgcttgc cacctacgta ttaccgcggc tgctggcacg 960 tagttagccg tggctttctg gttaggtacc gtcaaggtgc cgccctattt gaacggcact 1020 tgttcttccc taacaacaga gctttacgat ccgaaaacct tcatcactca cgcggcgttg 1080 ctccgtcaga ctttcgtcca ttgcggaaga ttccctactg ctgcctcccg taggagtctg 1140 ggccgtgtct cagtcccagt gtggccgatc accctctcag gtcggctacg catcgtcgcc 1200 ttggtgagcc gttacctcac caactagcta atgcgccgcg ggtccatctg taagtggtag 1260 ccgaagccac cttttatgtc tgaaccatgc ggttcaaaca accatccggt attagccccg 1320 gtttcccgga gttatcccag tcttacaggc aggttaccca cgtgttactc acccgtccgc 1380 cgctaacatc agggagcaag ctcccatctg tccgctcgac ttgcatgtat agcacgc 1437  

Claims (14)

Novel Bacillus sub, characterized in that it has the ability to produce mannanase, protease, cellulose, xylanase, lipase and starch degrading enzyme based on coconut seed. Tilly's KCTC 11103BP. delete delete (a) heat treatment of palm seed meal at 70 to 135 ° C; (b) 10-200 kGy irradiation of palm seed seeds; And (c) pretreatment of the palm fruit seed meal by at least one method selected from the group consisting of treating the palm fruit seed meal with 0.125 to 0.5% of an organic acid, followed by Bacillus subtilis having the ability of producing met nanase and fermentation of palm seed seed. Solid fermentation method characterized in that the fermentation of palm seeds seed using KCTC 11103BP. delete The solid phase fermentation method according to claim 4, wherein the palm seed cake is a palm kernel cake or a copra meal. delete The solid phase fermentation method according to claim 4, wherein the organic acid is selected from the group consisting of acetic acid, lactic acid, citric acid, propionic acid and mixtures thereof. 5. The solid phase fermentation method according to claim 4, wherein the moisture content of palm seed seed meal is adjusted to 35 to 65% (v / v) before the fermentation. 5. The solid phase fermentation method according to claim 4, wherein the pH of the palm seed meal is adjusted to 6.4 to 7.5 before the fermentation. 11. The solid phase according to claim 10, wherein the pH of the palm seed meal is adjusted to a basic material selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, ammonia water, uric acid, animal and plant ash, and phosphate. Fermentation method. The solid phase fermentation method according to claim 4, wherein the solid fermentation of palm seed seed is further fermented by adding a mold strain. The method of claim 12, wherein the fungal strain Aspergillus duck material (Aspergillus oryzae ), Aspergillus sojae ), Aspergillus niger), Usami Aspergillus (Aspergillus usamii), Aspergillus Usami Shiro Usami (Aspergillus usamii mut . shirousamii), Aspergillus awamori (Aspergillus awamorii), rayijo crispus (Rhizopus), rayijo Mu cores (Rhizomucor), root recorders do rijeyi (Trichoderma reesei ) solid phase fermentation method characterized in that it is selected from the group consisting of. delete

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