KR20240024426A - Culture Medium for Thermophylic Bacterium Strain of Bacillus sp.strains and Fed-batch Culture Method using Thereof for Treating Food Waste - Google Patents

Abstract

The present invention provides an optimal culture medium for thermophilic bacterial strains of the genus Bacillus and B. subtilis subsp. The optimal cultivation method and use of B. subtilis LSK 0604 (SARC-BSL) and B. pumilus OYR 0108 (SARC-BSS) strains are disclosed.

Description

Culture medium for thermophylic Bacterium Strain of Bacillus sp.strains and Fed-batch Culture Method using Thereof for Treating Food Waste}

The present invention relates to a culture medium for a thermophilic bacterial strain of the genus Bacillus, a method for cultivating the same strain using the same, and its use, and more specifically, to the thermophilic bacterium B. subtilis subsp. Culture medium for B. subtilis LSK 0604 (SARC-BSL) and B. pumilus OYR 0108 (SARC-BSS) strains. Fed-batch culture method of the thermophilic bacterial strain using the same culture medium, probiotic composition containing the same culture strain, and the same probiotic. It is about the food waste disposal method used.

Animal LB medium (Luria-Bertani broth) is usually used as a specialized base medium for culturing genetically modified microorganisms in biotechnology or genetic engineering technology, and vegetable TS medium (Tryptic Soy broth) is used for liquid culture of general microorganisms. In addition, LA medium, NA medium, PDA medium, or TSA medium are used as agar medium as a basic culture medium in microbiology.

For laboratory cultivation of microorganisms, it is common to batch culture at the universal bottle or conical flask level using solid medium or liquid medium. Fed-batch culture, unlike batch culture in which nutrients necessary for the production of specific substances such as amino acids, organic acids, enzymes, and antibiotics are temporarily added, is a biotechnology that uses a limited amount of a specific substrate. Fermentation products are not withdrawn until they are put into a reactor, such as a bench-top or jar-fermenter, to induce and control metabolites such as wet cell paste or target chemicals, so the batch culture and continuous culture are used. This is a technique that falls in the middle. In biotechnology, once the metabolic characteristics of a microbial strain are generally confirmed through flask-level batch culture, the cultural characteristics of the microorganism according to the metabolic characteristics of the target microorganism are identified through fed-batch culture, and further, the metabolites of the specific target are identified. Biosynthesis is possible.

On the other hand, thermophilic microorganisms or bacteria (hereinafter referred to interchangeably as thermophilic microorganisms or bacteria in the present invention) survive in extreme conditions such as high temperature, strong acidity, or strong alkalinity, so their cultivation, isolation, and identification are relatively difficult. Therefore, much research has been conducted due to the potentialities of producing useful biologically active substances such as antibiotics, anticancer substances, proteins, or enzymes from thermophilic microorganisms. In particular, the solid agar medium used for the isolation and culture identification of thermophilic microorganisms has weak gel strength and causes moisture loss due to syneresis, so research on an appropriate solid medium was necessary.

In addition, since thermophilic microorganisms have grown naturally in extreme conditions, if the culture conditions suitable for the extreme environment during artificial culture, that is, the culture medium composition, especially the energy source (carbon source), pH, temperature, and ventilation, are not properly adjusted, their mass cultivation and specific metabolism may be impaired. Since the biosynthesis of the product or its isolation and purification was difficult, research on mass cultivation of these strains was necessary.

Republic of Korea Patent Registration No. 10-2197224 discloses a botulinum toxin-producing strain (Clostridium botulinum, type A) that is not an extremophile microorganism but contains peptone, yeast extract, and glucose as a replacement for regular TPYG medium. A culture medium composition is disclosed, and a culture medium composition deficient in sugars, especially lactose, which is an energy source, as a seed medium composition for the food poisoning bacterium E. coli 0157:H7, and a method for detecting food poisoning bacteria using the same are disclosed (Republic of Korea) Patent No. 10-1882090).

Meanwhile, Republic of Korea Patent Registration No. 10-2175256 discloses a culture composition for cryopreservation of microbial cells, especially natural killing, for the purpose of not reducing the recovery rate, survival rate, and cell activity of immune cells, etc., when thawing after cryopreservation of animal cells. As a cryopreservation culture medium for cells, a special culture medium containing DMSO and Dextran as a carbon source and albumin as a nitrogen source is known.

In addition, Korean Patent Registration No. 10-1591584 discloses bone marrow-derived stem cell culture medium compositions, which are all related to the survival and activity of immune cells or differentiation and formation of animal dermal fibroblasts after cryopreservation of animal cells. .

Korean Patent Registration No. 10-1875564 and No. 10-1875502 respectively disclose fed-batch culture technology, the former of which involves producing glucose and glycerol from E. coli. The biosynthetic conversion of the target compound, omega-hydroxy fatty acid, is depicted using a fed-batch culture medium containing limited carbon source, and in the latter case, the optimal growth temperature in the medium temperature range is 35 to 39°C, and the optimal pH is 7.0. A method for bioconversion of a specific compound, vitamin K2, is known from the Bacillus subtilis KCTC 1021 strain through fed-batch culture.

Meanwhile, the thermophilic bacteria Bacillus subtilis subsp. A method for manufacturing fishmeal containing subtilis LSK 0604 (SARC - BSL) strain (hereinafter referred to as Bacillus 'A strain') and soybean meal is described in Korean Patent Publication No. 10-2012-06601 and a thermophilic bacterial strain of the same Bacillus genus. A different species of B. pumilus OYR 0103 (SARC - BSS) strain (hereinafter referred to as Bacillus 'B strain') and a method for manufacturing fishmeal containing soybean meal using the same are disclosed in Domestic Patent Publication No. 10-2012. Each is disclosed in No. -06606.

The growth temperature of the thermophilic bacterial strains is generally in the high temperature range of 37℃, i.e., 35 to 39℃ or higher, but in water (H ₂ O) or liquid medium at 40 to 50℃ or above the growth temperature of the Bacillus genus spawn. Although self-growth and growth are possible, it has been confirmed that growth is completely impossible at high temperatures above 60°C (see specifications in the above-mentioned patent publications).

The thermophilic bacterial strains of the genus Bacillus (referred to as strains A and B) were deposited with the International Microorganism Depository as KCCM 11067P and KCCM 11066P, respectively, on February 19, 2010, and have been effectively preserved to this day.

However, so far, the deposited thermophilic bacterial strains have been cultured in the solid medium agar medium and liquid medium (NA broth) at a temperature range of 40 to 50°C, but the solid medium has been cultured in the high temperature range of 60°C to 80°C. Characteristics and cultural characteristics Furthermore, mass culture in liquid media in bottles and flasks or biosynthesis of specific enzyme proteins such as lipase and alkaline phosphatase enzymes for detergent or food waste treatment, and furthermore, the enzyme characteristics or strains. Cell growth characteristics have not been studied at all.

1: Patent No. 2197224 Registered Patent Gazette, 2: Patent No. 1882090 Registered Patent Gazette, 3: Patent No. 2175256 Registered Patent Gazette, 4: Patent No. 1875564 Registered Patent Gazette, 5: Patent No. 1875502 Registered Patent Gazette, 6: Patent Publication No. 2012-0006601, 7: Patent Publication No. 2012-0006606.

Therefore, the purpose of the present invention is to provide cultural characteristics, that is, an optimal culture medium composition, of thermophilic bacterial strains including thermophilic microorganisms of the genus Bacillus derived from hot spring water (FIGS. 1, 2, and 5).

Another object of the present invention is to use a liquid medium to which 3% or more Maltose, 0.5% Tryptone, and 0.5% Yeast extract are added as carbon sources at the beginning of fermentation using a jar-fermenter of 2.5L or more, and then when the carbon source is used up. From the 16th hour of fermentation onwards, glucose stock soln. prepared by sterilizing it in a separate autoclave (121°C/15 minutes) is continuously supplied to perform a fed-batch culture method for mass thermophilic bacterial strain cells reaching a CDW of 16.0 g/L or more. To provide (Figure 2).

Another object of the present invention is to achieve a maximum Alkaline Phosphatase (AP) activity of 11.6 U/g wet cell weight within 16 hours of fermentation in the fermentation medium under the above conditions at the beginning of fermentation, with a lipase enzyme activity of up to 2,000 U/g wet cell weight. The purpose is to provide an optimal culture method of thermophilic bacterial cells with a high level (Figure 3).

Another object of the present invention is to provide a probiotic preparation containing a thermophilic bacterial strain having the above-mentioned lipolytic enzyme activity and its use, that is, a method of treating food waste using the probiotic.

The above object of the present invention is achieved through various examples and experimental results.

Identifying the optimal culture medium and culture conditions through universal bottle and conical flask level cultivation of thermophilic bacterial strains (A, B) according to the present invention; Initial medium concentration, that is, carbon source (carbon source), is a fed-batch culture condition using a bench-top fermenter for mass production of cells of the thermophilic bacterial strains according to the optimal medium composition and culture conditions obtained in the above step. ), maltose, and the optimal nitrogen source are added, and after that, when maltose, which is the energy source (carbon source) in the early stages of growth, is exhausted, pH, ventilation speed (vvm), and stirring speed are adjusted. adjusting the (rpm) and supplying glucose instead of maltose as the energy source (carbon source) at a feed rate of 5 to 7 g/L/h to achieve maximum activity of lipase and alkaline phosphatase (AP) enzymes; centrifuging the microbial cells obtained in the above step to obtain a wet cell paste, and adding the wet cell paste to food waste, especially food waste, to prepare a microbial probiotic suitable for rapid decomposition and treatment; This is achieved through providing and evaluating a food waste treatment method using the microbial probiotics obtained in the above step.

The present invention has the effect of providing a basal medium composition (M) containing maltose as a carbon source for optimizing the growth of the thermophilic bacterial strains of the present invention, and also provides the basal medium composition (M) at the beginning of the growth of the strains. The most preferred fed-batch culture method uses a glucose solution prepared by sterilizing it in an autoclave separately as a carbon source when the medium (M) is used and the maltose is used up, and mass culture of the thermophilic bacterial strain cells using the same. and has the effect of providing a method for lipase enzyme biosynthesis. In addition, the present invention has the excellent effect of providing a probiotic composition for food waste waste treatment and a food waste treatment method using the probiotic.

Figures 1a to 1j show that when thermophilic bacterial strains according to the present invention are batch cultured using universal bottles or flasks, glucose at a concentration of 0.3% or more is mixed with Tryptone and Yeast Extract 2. A graph showing that it cannot be used after sterilization in an autoclave at 121°C/15 minutes by adding it simultaneously as a culture medium component along with the nitrogen source of the species,
Figure 2 shows Medium G ₂ (5% maltose, 15% yeast extract and 15% yeast extract) in which the thermophilic bacterial strains according to the present invention changed the contents of the components of the basic medium (M) to optimized values in the bench-top fermenter. After starting fermentation by autoclaving at 121℃/15 minutes using Tryptone), when maltose is used up, glucose is added at OD (580nm) 36.7 and CDW (g) under the conditions of feed rate 6.0g/L/h, 1,500 rpm, 1.33 vvm. /L) Graph showing the maximum cell growth of 15.8,
Figure 3 shows AP enzyme activity (u/g) and cells in oil-free and phosphate-free medium (Medium G ₂ ), which is the substrate of Lipase and AP enzymes secreted by thermophilic bacterial strains according to the present invention during culture. Graph showing cellular growth,
Figure 4 shows thermophilic bacterial strains according to the present invention being plated on a conventional agar medium and a maltose-added maltose medium for probiotic preparation of the present invention, respectively, and incubated in an incubator at 60°C and 600 rpm for 4 h to 10 h. During culture, in the case of agar media (4a, 4b and 4c), hardening cracks appeared after syneresis occurred due to high heat, but in the maltose media (4d) of the present invention, this phenomenon was not observed and a photo showing normal cellular growth and colony growth was observed. do,
Figure 5a shows that when thermophilic bacterial strains according to the present invention are simultaneously autoclaved sterilized at 121°C/20 minutes using a mixture of glucose, Tryptone, and yeast extract, and then used as a culture medium for batch culture, glucose and amino acids are milled. In the process of metabolizing the substances produced as a result of the reaction, a large amount of organic acids and polysaccharides are biosynthesized and the polysaccharides are encapsulated on the extracellular surface, confirming evidence of a long lag phase of more than 30 hours. do,
Figure 5b is a micrograph showing the growth phase of the rod as a result of the thermophilic bacterial strains according to the present invention growing normally without polysaccharide biosynthesis in the basal medium (M) of the present invention, that is, a medium supplemented with mal tose;
Figure 6 is a graph showing the results of HPLC conversion of thermophilic bacterial strains according to the present invention from a carbon source to organic acid during fed-batch culture.

According to the present invention, the thermophilic bacterial strains (A and B) used as samples in the present invention exceed the medium temperature range (35 to 40 ℃), and the use of agar medium at a high temperature of 60 ℃ or higher is suitable for agar. It was confirmed that moisture was transferred due to the high heat, and if further progress was made, the surface of the medium hardened and cracked, making it unusable. Hardening and cracking of the agar medium above 60℃ are caused by the combination of the water (H ₂ O) and free sugar contained in the agar and the amino acids contained in the skim mill or yeast extract added during the production of the agar medium, millard for more than 4 hours. The progression of the reaction was determined to be the cause.

According to the present invention, the thermophilic bacterial strains of the present invention are cultured in a universal bottle or small-capacity flask level by shaking culture at a larger-capacity flask level or by feeding-fed-culture in a bench-top fermentor. When cultivating batches, using it directly for inoculation was effective in maintaining homeostasis and uniformity of fermentation.

According to the present invention, the thermophilic strains (A and B) of the present invention are completely different from the usual mesophilic strains of the genus Bacillus, and at the beginning of cultivation, they contain maltose as a carbon source for 100% by weight of the total medium composition. ) 0.3% by weight (w/v), 0.3% by weight (w/v) of tryptone as a nitrogen source, 0.3% by weight (w/v) of yeast extract, and inorganic salt solution, such as, Castenholz inorganic component 10 × drainage stock soln. Most preferably, it should be cultured in maltose basal medium (M) consisting of 0.1% by volume (v/w) (Figure 1b). In addition, the present inventors confirmed the possibility of maximum growth up to the average OD (580nm) of 1.4 and 1.7, respectively, in three repeated batch cultures using bottles or flasks using a dilution medium (M/5) diluted to 1/5. (Figure 1a). The limiting factor preventing the thermophilic bacterial strains from growing according to the present invention to an OD of 1.4 or 1.7 or higher in the bottle or flask was confirmed to be lack of oxygen (O ₂ ).

According to the present invention, in fed-batch culture for mass production of cells of the thermophilic bacterial strain of the present invention, the stirring speed and oxygen supply rate of the stirrer are adjusted to maximize oxygen supply, and NaOH is supplied to correct the decrease in pH caused by organic acid production. This was definitely required. The preferred stirring speed was found to be 1,000 to 2,000 rpm and the oxygen supply rate was 1.00 to 1.55 vvm, with the most preferred being 1,500 rpm and 1.33 vvm.

In addition, according to the present invention, the growth rate of the thermophilic bacterial strain according to the present invention, that is, rapid culture in 1/5 diluted medium (M/5) compared to the maltose basal medium (M) of the present invention is higher in the high concentration basal medium (M ), it was confirmed that this was because these strains biosynthesized various organic acids more rapidly during the cell growth process and began to rapidly drop from the optimal growth pH of 7.0 to the acidic pH of 5.0, resulting in greater inhibition of growth.

To prevent such growth inhibition, automatic supply of alkaline solution (NaOH) was essential.

As a result of numerous experiments, the present inventors confirmed that the most desirable carbon source during batch culture using bottles or flasks is maltose as a disaccharide. On the other hand, when using monosaccharides such as glucose or its isomer, dcxtrose, tryptone and/or yeast extract derived as a nitrogen source are added together (simultaneously) during the sterilization process in an autoclave at 121°C/15 to 20 minutes. It was confirmed that the amino sugars produced by the amino acid and Milard reaction caused severe growth retardation of the thermophilic bacterial strains according to the present invention, making it impossible to obtain large quantities of cells. When glucose and the nitrogen source were sterilized by adding simultaneously (○) at a concentration of 0.5% or more, it was impossible to obtain the thermophilic bacterial strain of the present invention (FIGS. 1C to 1J).

Meanwhile, the thermophilic bacteria of the present invention obtain the energy necessary for cell activity in the decomposition stage of glucose during the cell proliferation process under high temperature and high temperature conditions of 60°C or higher and actively biosynthesize organic acids, causing the pH of the culture medium to rapidly decrease to below 5.0. At this time, a large amount of alkaline substances are required, and if NaOH, etc. are not supplied in an appropriate timely manner, the thermophilic bacterial strains of the present invention produce a large amount of mucous polysaccharides and are encapsulated, causing the strain's growth to quickly stop. confirmed (Figure 5a). The photograph in Figure 5a shows that the thermophilic bacterial strain of the present invention becomes fat (halo) and that organic acids and polysaccharides are biosynthesized in the culture medium, resulting in a yellow color.

In addition, the present inventors confirmed that the optimal growth pH was 7.0 and the optimal growth temperature was 60°C through batch culture using bottles or flasks of the thermophilic strains of the present invention, and the optimal inoculm size was 0.1% (w/v), which was 0.1 In cases where it was less than % and in cases where it was larger than 0.3%, it was confirmed that the lag phase lasted for more than 24 hours.

According to the present invention, through the examples and experiments disclosed in the optimal pH, optimal growth temperature, and optimal inoculum range of the thermophilic bacterial strains of the present invention, the present inventors maximized cell production of the bacterial strain of the present invention, that is, for mass attribute production. During batch culture, maltose must be used as the carbon source, and during fed-batch culture, maltose must be used even in the early stages. However, from the carbon source exhaustion stage to subsequent stages, the carbon source must be sterilized separately so that the carbon source can be monitored through a sugar assay. Even if the prepared glucose solution (stock soln.) is supplied at a constant supply rate, the strains of the present invention rapidly decompose glucose and use it to rapidly lower the pH, causing NaOH to be automatically pumped into the fermenter, thereby maintaining the pH in the optimal growth range of 7.0. It was also found for the first time that mass production of thermophilic bacterial cells was possible without the accumulation of various organic acids and biosynthesis of polysaccharides by continuously maintaining the temperature at ~ 7.5 (Figure 3).

On the other hand, when fed-batch culture is performed in a bench-top fermentor to mass-produce the thermophilic bacterial strain cells according to the present invention and simultaneously maximize lipolytic enzyme biosynthesis, 5% of maltose of the total weight of the medium composition is added. After autoclove at 121°C/20 minutes (in situ) in a medium containing 15% of Yeast extract and 15% of Tryptone (maltose), the culture was started, and at the stage when the maltose was exhausted, 0.3% (w/v) was added. It was most desirable to feed and culture a glucose stock solution that had been separately sterilized and prepared at a feed rate of 5 to 6 g/L per hour. In this case, no growth lag or polysaccharide biosynthesis was found in the thermophilic bacterial strain according to the present invention (FIG. 4).

Figure 5b is an optical microscope photograph taken by the present inventors of a sample obtained by random sampling during the fed-batch culture, and shows the bacillus cell shape at the point when cell growth is maximized without organic acid accumulation or polysaccharide biosynthesis and the activity of Lipase or AP enzyme is localized. has been confirmed.

The most preferred fed-batch culture method of the thermophilic bacterial strain according to the present invention uses maltose as a carbon source to suppress the lag phase, and at the stage when the maltose begins to be used up, a separately sterilized and prepared glucose solution is added. The optimal pH for growth is maintained at 7.0 by automatically supplying NaOH using an alkali feed pump.

Glucose or its isomer, di-glucose, is added and used as a culture medium at a high temperature of 60°C along with nutrients such as other nitrogen sources, Tripton or/and yeast extract. In this case, the bacterial strains of the present invention use the sugars as an energy source to biosynthesize and release a large amount of organic acids and polysaccharides in the fermenter, which reduces the cell growth rate and promotes a long lag phase. It was confirmed that it causes (Figure 6).

According to the micrograph, it was confirmed that a large amount of organic acids and polysaccharides were formed and accumulated in the culture broth, giving off a yellow color (Figure 5a), and the results of the fed-batch culture of the present invention compared to HPLC of the standard solution. The types of organic acids produced were confirmed to be lactic acid (2), acetic acid (3), and propionic acid (4) (Figure 6).

Thermophilic bacterial strains according to the present invention easily decompose glucose into organic acids above 60°C during fed-batch cultivation to lower the pH. At this time, the NaOH pump is activated to maintain the optimal growth pH of 7.0 (neutral). At this time, yeast extract or Tryptone-derived It is believed that the so-called amilo reaction, which easily combines with the amino acid, does not occur, and thus the formation of yellow-brown amino sugars is suppressed, thereby preventing growth inhibition of the thermophilic bacterial strain of the present invention and maintaining normal growth.

When the thermophilic bacterial strains according to the present invention are fed-batch cultured, lactic acid, acetic acid, propionic acid, and other organic acids produced from glucose-derived saccharides (FIG. 6) are not adjusted to pH 7.0 and are maintained below pH 5.0, polysaccharide (polysaccharide) ) It is thought that the accumulation and the cells covering it cause long-term growth delay (lag phase).

As a result of performing fed-batch at a glucose feed rate of 6 g/L/h in the basic culture medium (M) according to the present invention, the present inventors found that cells were The dry weight (CDW) was at the level of 5g/L and 10g/L, respectively, but it was confirmed that the maximum CDW of 16g/L and OD (580mm) could reach 36.4 at an agitator stirring speed of 1,500rpm and an oxygen supply rate of 1.33vvm (Figure 2 ).

The thermophilic bacterial strains (A, B) according to the present invention biosynthesize lipase and alkaline phosphatase enzymes involved in the decomposition of lipid components contained in soybean waste, and the biosynthetic characteristics of these enzymes form a linear curve with each other. In relation to this point, it has already been shown that maximum growth is observed at 8 hours (h) at 45°C, 100 rpm, and 0.3 vvm in TBS liquid medium, and that enzyme activity is 200 U/g wet cells and 2 U/g wet cells, respectively, at 8 h from the start of culture. It has been confirmed (see Patent Publication No. 10-2012-6601 and Patent Publication No. 10-2012-6606). The enzyme properties were confirmed using an API zyme kit, and in addition, the present inventors tested the enzyme activity using paranitrophenyl caprylate and paranitrophenyl phospate as substrates, respectively. Lipase and AP enzymes were randomly sampled during fed-batch culture, and the hydrolyzed P-nitrophenol was confirmed by measuring the absorbance on a spectrometer (450 nm). As a result, the fed-batch culture conditions of the present invention, that is, the modified medium of the basic medium (M), were confirmed. In culture using (G) (Run 24), the maximum enzyme activity was 2,000U/G and 11.6%, respectively, 16h after the start of mass culture under the conditions of 60℃, pH 7.0, 1,500rpm, 1.33vvm, and glucose feed rate of 6.0g/L/h. It was confirmed that U/g wet cells were up to about 200 times higher than the known conventional patent (Figure 3, Table 4).

The present inventors found that the thermophilic bacterial strains of the present invention, unlike microbial strains that grow well in the normal mesophilic (20 to 35°C) or high temperature (30 to 45°C) ranges, have a specific activity of the enzymes in the Mg It is more effective against Ca ²⁺ ions than ²⁺ ions, and is also very effective against divalent cations such as Ba ²⁺ , Mn ²⁺ , Cu ²⁺ , Fe ²⁺ , and Co ²⁺ ions ( Table 7) When preparing the basic medium composition (M) and modified medium composition (G) for culturing thermophilic bacterial strains of the present invention (Table 7), the types and addition amounts of inorganic salts and trace elements were optimized (Table 1).

The present inventors confirmed that the use of oils such as olive oil, palm oil, and sesame oil as a carbon source for the culture medium of thermophilic bacterial strains according to the present invention was not desirable because it interfered with cellular growth. . In addition, the thermophilic bacterial strains according to the present invention rapidly decompose oils and other maintenance substances derived from food waste only after normal growth begins in the basic medium composition (M) to which maltose, a disaccharide, is added and colonies become dominant. It was judged that it could be magnetized and used as an energy source.

The present inventors also confirmed that the thermophilic bacterial strains according to the present invention maximize the activity of alkaline phosphatase, which is an indicator of lipase enzyme activity, in phosphate-free and oil-free media (FIG. 3).

Below, preferred embodiments and experimental examples of the present invention are described, but it is clear to those skilled in the art that the present invention is not limited thereto.

[Example 1] Basal medium composition (M) for culturing thermophilic bacterial strains according to the present invention

The composition and content of the solid medium for smearing the strains of the present invention on a Petri-dish plate and the basal medium (M) for liquid culture using a bottle or flask are shown in Table 1 below.

Solid medium for petri-dish smear or basal medium composition (M) for container culture of thermophilic bacterial strains (A, B) according to the present invention ingredient Content (weight or volume)/L percentage maltose 3g 0.3wt% Tryptone 3g 0.3wt% Yeast extract 3g 0.3wt% Inorganic salt solution (10 ×) 100mL 0.01% _[ Note _] Inorganic salt (10
MgSO ₄ ㆍ7H ₂ O 1.0g, NaCl 0.08g, KNO ₃ 1.03g, NaNO ₃ , 6.81g, NaHPO ₄ 1.11g, FecL ₃ soln
0.28g/L, 10mL, 10mL trace element solution. The trace element solution is 0.5mL of H ₂ SO ₄ (98%) per L,
MnSO ₄ ㆍH ₂ O 2.2g ZnSO ₄ ㆍ7H ₂ O 0.5g, H ₃ BO ₄ 0.5g, CuSO ₄ 0.016g, Na ₂ MoO ₄ ㆍ2H ₂ O,
CoCl ₂ ㆍ6H ₂ O is 0.046g.

The thermophilic bacterial strains of the present invention were grown in a medium diluted 1/5 of the basic medium (M) (M/5) and compared to this, 3g of glucose per L, 3g of Yeast extract, 15g of peptone, 0.5g of L-cystine, 2.5g of NaCl, Using TSB (Tryptic soy broth, MBT1053) medium (pH 7.0 each) consisting of 0.5 g of sodium thioglycolate, culture was performed in an incubator at 60°C with shaking at 150 rpm using a universal bottle, and then cultured in a 2L side-cell in a sterile room. It was transferred to an arm shake flask and a comparative culture experiment was performed again in the incubator at the same temperature, pH, and stirring speed.

The flask shaking culture comparison experiment was performed 3 times each, and the cell growth rate was checked by OD (580 nm) and the average value was calculated. The results are shown in [Table 2].

division cellular growth OD (580 nm) strain 2h 4h 6h 8h 10h 12h Invention M/5
dilution medium A 0.7 1.6 1.7 1.8 1.6 1.5 B 0.8 1.7 1.7 1.7 1.6 1.4
TSB badge A 0.3 0.5 0.7 0.9 0.8 0.7 B 0.4 0.6 0.8 0.8 0.8 0.7

As a result of the experiment, the thermophilic bacterial strains (A, B) according to the present invention were confirmed to have a cell growth rate of about twice that of the conventional TSB broth within 4 hours from the start of cultivation in the M/5 diluted medium of the present invention. The reason why the cell growth rate of thermophilic bacterial strains according to the present invention is delayed in TSB medium is that when sterilized in an autoclave (high pressure sterilizer) at 121°C/15 to 20 minutes, glucose added to the medium is derived from yeast extract and peptone. It is believed that it easily combines with amino acids, especially L-Cystine, to form amino sugars, which limits its use as an energy source, resulting in delayed cell growth.

[Example 2] Optimal pH and temperature (T) of the culture medium for thermophilic bacterial strains (A, B) according to the present invention

The present inventors additionally performed shaking culture experiments using bottles or flasks several times to confirm the optimal culture pH and temperature (T) of the thermophilic bacterial strains (A, B) according to the present invention. As a result of the experiment, when using glucose at a concentration of 0.3% or more as a carbon source simultaneously with Tryptone and/or yeast extract as a nitrogen source and sterilizing it in an autoclave (121℃/15 minutes), it was incubated at 60℃ and pH 7.0. Growth delay (Lag Phase) of more than 36 hours (h) was confirmed (Table 3). This is because the thermophilic bacteria of the present invention cannot decompose and utilize the glucose, yeast extract, or tryptone-derived amino acids or peptides that make up the medium and the amino sugars produced due to the browning reaction during the autoclaving process at 121°C/15 minutes or more and produce polysaccharides. It was confirmed to be biosynthesized. This same growth delay of bacterial strain cells was also confirmed in sucrose, lactose, and celobios. However, when glucose and maltose, which is an α1→4 bond of glucose among disaccharides, were added and used, the Millard reaction did not occur after 4 h of culture, and normal growth was achieved without growth delay, reaching an OD (580 nm) of 1.6 (Table 3).

That is, the bacterial strains according to the present invention were confirmed to grow normally even at a high temperature of 60°C when batch cultured using bottles and flasks only when maltose was used as a carbon source. However, when the carbon source is glucose and the two nitrogen sources are mixed and used after sterilization, if the glucose concentration is less than 0.3%, they are added simultaneously and sterilized (○), or sterilized separately from the nitrogen sources (autoclaved separately) and then added ( ●) Regardless of whether it is used, all grow normally (1a ~ 1b), but when adding glucose concentration of 0.5% or more, glucose must be sterilized separately from the nitrogen sources and then added (1c ~ 1f). On the other hand, when glcose is used at a high concentration of 1% or more, the growth of the thermophilic bacteria (A, B) of the present invention is maintained regardless of whether glucose and nitrogen source are sterilized separately and added (●) or simultaneously sterilized and added (○). There was a delay and few cells could be obtained (1g to 1j).

(pH 7.0, T60℃)
Carbon Source Cellular growth OD (580nm) 2h 4h 6h 8h 10h 12h (glucose)
α1→2gloucose
-
-
-
-
-
0.4 (sugar)
α1→2Sucrose
-
-
-
-
-
0.5 (lactose)
β1→4Lactose
-
-
-
-
0.4
0.5 (cellobiose)
β1→4celobios
-
-
-
-
-
0.5 (maltose)
α1→4maltose
0.4
1.6
1.8
2.0
2.0
1.8 [Note] The remaining added inorganic salts and trace elements are the same as the basic medium in [Table 1].

As a result of the experiment, the thermophilic bacterial strain according to the present invention reached the highest growth rate OD 2.0 (580 nm) in 8 hours of cultivation at 60°C.

The present inventors confirmed the growth rate of the bacterial strains according to the present invention after 8 h of incubation time at a culture temperature of 65°C or higher and 70°C, and found that there was no significant difference from that at 60°C, so they can be used for subsequent bottle or flask culture and even bench-top culture. During fed-batch culture, use basic medium (M) containing maltose as a carbon source and Tryptone and yeast extract as a nitrogen source at pH 7.0 and T 60°C. During fed-batch culture, when the carbon source begins to run out, it is sterilized separately in an autoclave (121°C/121°C). 15 minutes) prepared glucose stock soln. It was finally decided to adopt the feeding method.

The glucose solution prepared in advance will not induce the so-called milder reaction, which chemically combines glucose with amino acids or peptides, when a glucose feed rate of 5 to 7 g/L/h is adopted during fed-batch cultivation of the present invention. In addition, it was possible to obtain a large amount of wet cell paste after 8 h of culture without biosynthesis of polysaccharides and growth delay by the bacterial strains of the present invention. In particular, maximum cell growth was achieved under the fermentation conditions shown in Table 4 below. Could.

Fed-batch culture conditions (glucose feed rate, stirring speed, and oxygen supply rate) for maximizing cell growth of bacterial strains according to the present invention.
Fermentation conditions basic badge
× 5 glucose feed
rate(L/h) stirrer speed
(rpm) aeration rate
(vvm) CDW
(g/L) wet cell weight (g/L) Experimental Example 1 M × 5 4.0g 1,500 1.33 8.5 17.5 Experimental Example 2 M × 5 5.0g 1,500 1.33 9.4 21.4 Experimental Example 3 M × 5 6.0g 1,500 1.33 10.1 26.5 Experimental Example 4 M × 5 7.0g 1,500 1.33 9.8 25.2 [Note] 1. Initial medium (G) × 5 times that of basal medium (M) contains 0.5% maltose and 1.5% tryptone. 1.5% Y east extract is used, and after 8 hours of initial incubation, the pumping of glucose feed and NaOH solution is started, and the incubation is completed after 8 hours of additional incubation.
2. pH 7.0/T 60℃
3. CDW: cell weight (g/L)

As a result of the above experiment, the bacterial strain of the present invention was confirmed to have an optimal glucose feed-rate of 5.0 to 7.0 g/L/h, most preferably 6.0 g/L/h, in fed-batch culture at pH 7.0/T 60°C. . The present inventors were able to obtain wet cell weight of up to 26.5 g/L at the above glucose feed rate (Table 4).

[Example 3] Bottle or flask culture of the bacterial strain of the present invention and sterilization conditions and cell growth of carbon source and nitrgen source for fermentation in bench-top fermenter

When preparing a culture medium for the thermophilic bacterial strain of the present invention, the growth results of glucose as a carbon source and Tryptone and yeast extract as a nitrogen source are shown in Figures 1A to 1A under separate (●) or simultaneous (○) sterilization (autoclave) conditions. As already shown in 1f.

That is, when batch culturing using a bottle or flask of the thermophilic bacterial strain of the present invention, glucose and tryptone or/and yeast extract are simultaneously added at a concentration of 0.5% or more and sterilized at 121°C/15 to 20 minutes. As shown in Figures 1c to 1f, cell growth was delayed, and as seen in Figures 1g to 1j, when the glucose concentration was added at a concentration of 1% or more, uptake was not achieved due to the Millard reaction with the amino acid derived from the nitrogen source, and the polysaccharide was not taken up. Because of biosynthesis, cell growth of the strain was extremely delayed (lag phase). Therefore, it was confirmed that when mass producing thermophilic bacterial cells or biosynthesizing specific target substances through batch culture or fed-batch culture, glucose at a concentration of 0.3% or more cannot be used by simultaneous sterilization with trytone or yeast extract. .

According to the above experimental results, the addition and use of glucose as a carbon source during batch culture of the bacterial strain of the present invention cannot be used up to a concentration of 0.3% or higher, but it cannot be used at a concentration higher than that in fed-batch culture. It was confirmed that the above concentration, that is, most preferably, the glucose feed rate of 5.0 to 7.0 g/L/h (pH 7.0, 1,500 rpm, 1.33 vvm) could be used.

According to the present invention, glucose as a carbon source is simultaneously added at a concentration of 0.5% or more along with other nitrogen sources and sterilized (121°C/15 minutes) in a bottle or flask in an incubator at pH 7.0 and T 60°C. The results of the culture showed that the bacterial strains of the present invention biosynthesized polysaccharides in the medium due to the amino sugars produced, delaying growth for a long time (Figure 5a), and maltose instead of glucose as a carbon source was simultaneously added at a concentration of 0.5% with other nitrogen sources and sterilized. When used, there was no amino sugar produced in the medium, so there was no polysaccharide production and no delay in cell growth (Figure 5b).

[Example 4] Optimal fermentation conditions (maximizing cell growth and enzyme biosynthesis) during fed-batch culture using the modified medium composition (G1 ~ G2) of bacterial strains according to the present invention

division Inventive bacterial strain A Inventive bacterial strain B Modified badge type G1 G2 G1 G2 Initial culture medium
(Ratio)
5:10:10
5:15:15
5:10:10
5:15:15 Maximum O.D.
(580nm)
26.3
[36.4]
26.5
[36.7] maximum time
O.D.(h)
18
[35]
18
[34] MaxCDW
(g/L)
10.4
[15.9]
10.5
[15.6] Total
CDW(g)
26.0
[39.8]
26.2
[40.1] GFR
(g/L/h)
6.0
6.0
6.0
6.0 glucose
Consumption (g)
72.5
[93.5]
66.3
[85.7] NaOH
Neutralization amount (g)
21.6
[26.2]
21.6
[27.1] yeild
g cells/g GF
0.36
[0.43]
0.40
[0.47] Max AP Enzyme
activity
U/g wet cell
9.5
[11.6]
9.3
[11.2] Max AP Enzyme
Active time (h)
19.5
[16.5]
19.0
[16.5] Remarks [Note 1] The ratio of the components of the initial culture medium (G1, G2) is that of maltose, tryptone, and yeast extract.
Preloaded weight beam.
[Note 2] GFR is glucose feed rate (g/L/h)

The cell growth rate (g cell/g glucose feed; GF) of the thermophilic bacterial strains A and B of the present invention according to the above example was calculated according to the following formula.

The cell growth rate of thermophilic bacterial strains according to the present invention is a function of pH, temperature (T), stirring speed, and substrate (s) glucose feed rate (GFR).

In the bacterial strains (A, B) of the present invention, CDW (g/L) increased with an increase in wet cell weight (WCW), and the glucose feed rate increased in the range of 5 to 7 g/L/h, which was a significant difference. There was no.

According to the present invention, when the pH in the fermentor decreases, the production of organic acids rapidly increases and the pumping amount of NaOH rapidly increases.

In addition, the optical density (OD 580 nm) of the bacterial strains according to the present invention increased as the glucose feed rate increased, and as the glucose addition rate increased, various organic acids and polysaccharides that inhibit cell growth were also biosynthesized. The former was believed to be caused by a decrease in pH, and the latter was caused by oxygen limitation.

In order to alleviate oxygen deficiency, in fed-batch culture of the bacterial strains of the present invention, a glucose feed rate of 5 to 7 g (L/h), an aeration rate of 1.33 (vvm), and a stirring speed of 1,500 rpm were most preferable.

The bacterial strains of the present invention reached a maximum OD (580 nm) of 36.4 to 36.7 at 34 to 35 h after the start of fermentation and a maximum CDW (g/L) of 15.0 or more (Figure 2).

Meanwhile, in the bacterial strains (A, B) according to the present invention, a large amount of alkaline phosphase (AP enzyme), including lipase, was biosynthesized in the glucose feed rate medium for fed-batch culture of the present invention. The standard AP enzyme had a fermentation time of 16.5 h. After passage, the maximum activity was 11.6 U/g and 11.2 U/g wet cell weight, respectively, in G2 medium (Figure 3, Run 24).

The above experimental results provided important clues for maximizing cell growth of thermophilic bacterial strains according to the present invention and for increasing lipase biosynthesis and maximizing activity when developing probiotics for processing food waste such as detergents for high-temperature machine cleaning and food waste.

[Example 5] Lipase and AP enzyme biosynthesis characteristics of thermophilic bacterial strains according to the present invention

The results of investigating the biosynthetic characteristics of lipase and AP enzymes of thermophilic bacterial strains (A, B) according to the present invention are shown in [Table 6] below.

After culturing for 8 hours (h) in a 5-fold (M Experimental results were observed by randomly sampling samples at 1-hour intervals and measuring absorbance (OD 405nm) in a spectrophotometer while culturing the sample for an additional 8 hours (h) at a rate of /h. is shown in Table 6 below.

AP enzyme and Lipase enzyme activity of thermophilic bacterial strains (A, B) according to the present invention Incubation time
(h) AP enzyme activity (unit: U/g wet cells) Lipase enzyme activity (unit: U/g wet cells) A strain B strain A strain B strain 8 2.2 2.1 100 95 9 2.4 2.3 104 106 10 2.3 2.4 108 100 11 2.5 2.6 110 109 12 2.7 2.8 117 115 13 4.8 4.5 225 220 14 5.7 5.6 472 470 15 8.4 8.7 1,520 1,687 16 11.5 11.0 2,000 1,998 17 11.0 10.8 1,850 1,810 18 9.2 9.4 1,720 1,700 19 8.4 8.3 1,500 1,505 20 5.5 5.2 398 462

As a result of the Fed-batch culture experiment according to the present invention, the thermophilic bacterial strains of the present invention have high Lipase and AP enzyme activities, and are used as probiotics for food waste treatment such as cleaning agents for mechanical equipment or food waste used at high temperatures in the range of 40 to 70 ℃. It was confirmed that it would be very useful to use.

[Example 6] Enzyme characteristics of thermophilic bacterial strains (A, B) according to the present invention

The lipase and AP enzymes secreted by the thermophilic bacterial strains according to the present invention showed their activity even at 65°C, and the enzyme activity gradually decreased above 70°C.

These enzyme characteristics were in contrast to the fact that mesophilic bacterial strains of the genus Bacillus typically show optimal activity at 40 to 45°C, and their enzyme activity rapidly decreases above 50°C.

The enzyme characteristics of the thermophilic bacterial strains (A, B) according to the present invention are similar to those of lipase or AP enzymes derived from the representative commercially available Cordida cylondraacea yeast strain, which are widely used in the processing and pharmaceutical industries, losing their enzyme activity above 50°C. It was a contrast.

In addition, regarding the enzyme activity of the thermophilic bacterial strains (A, B) according to the present invention, the results of experiments using crude enzyme sonicated with the addition of Triton It was released and released rather quickly at 65°C.

Additionally, the optimal pH of the thermophilic bacterial strains (A, B) according to the present invention was pH 7.5 at 45°C and pH 8.0 at 60°C, respectively.

Meanwhile, the effect of monovalent and divalent cations on the enzyme activity of the thermophilic bacterial strains (A, B) according to the present invention was as shown in [Table 7] below.

Ion characteristics of Lipase and AP enzymes of bacterial strains (A, B) of the present invention
division Lipase activity AP active Strain A crude enzyme solution Strain B crude enzyme solution Strain A crude enzyme solution Strain B crude enzyme solution

monovalent ion K ⁺ 100 100 100 100 ^NH ₄₊ 100 100 100 100 Na ⁺ 100 100 100 100 Tris/HCl 0 0 0 0


divalent ion Mg ²⁺ 100 100 100 100 Ca2 ⁺ 176 172 172 175 Ba ²⁺ 121 123 120 123 Mn2 ⁺ 0 0 0 0 Cu2 ⁺ 0 0 0 0 Fe2 ⁺ 0 0 0 0 [Note] Standard assay; 100 Standard assay is performed for 2 minutes at an optimal temperature of 65℃

In [Table 7], Lipase and AP enzyme activities in the crude enzyme solution from the two strains (A, B) were generally highly stable with monovalent cations. However, Na ⁺ ion was the best, and among divalent cations, Ca ²⁺ ion was the best, confirming that it is useful as a cofactor in enzyme stability and activity.

[Example 7] Preparation of probiotics for waste treatment such as food waste using the thermophilic bacterial strain of the present invention

As confirmed through Examples 1 to 4 of the present invention, wet cell paste was obtained by centrifuging the strain culture while the specific activity of AP enzyme and Lipase enzyme was maximized.

Cells cultured in a 2.5L Bench-Top fermenter are centrifuged at 8,000rpm for 30 minutes using a J10 Roter.

The wet cell paste obtained as a result of the centrifugation is formulated as a probiotic using the most desirable biopolymer and can then be usefully used as a probiotic for processing food waste such as food waste or wastewater.

According to the present invention, cells that biosynthesize AP and lipase enzymes with high activity are recovered by centrifugation, and the wet cell paste recovered through centrifugation contains some of the free water that exists between microbial strains. Despite these free water, the inventors primarily selected pectin as a gelling agent that is advantageous for forming probiotic gels of thermophilic microorganisms. However, it is desirable that the appropriate amount of pectin used does not exceed 20% by weight of the total composition content.

Gellan gum (gelrite) is advantageous for gel formation of live thermophilic bacteria that can grow above 70°C, but it is expensive and has a drawback in that it has weak viscoelasticity. In consideration of this, it was confirmed that in the present invention, it is preferable to use 10% by weight or less of the total probiotic composition secondarily.

The present inventors selected pullurans as a biopolymer that is stable in a wide pH range of 2.5 to 8.5, has excellent viscoelasticity, and has little synergy. It was preferable to use 10 to 20% by weight of flurane based on the total probiotic composition. However, at more than 20% by weight, hardening of the gel composition was accelerated and synergy occurred, which was not desirable.

The present inventors tried carrageenan and agar as gelling agent ingredients derived from seaweed, but the thermophilic strains according to the present invention lowered pH due to biosynthesis of large amounts of organic acids during the growth stage at high temperatures above 60°C. It was confirmed that its use was undesirable due to the severity of the condition.

According to the present invention, as the most preferred embodiment of the probiotic composition for waste treatment such as food waste, the final components and contents are shown in [Table 8].

Components and added amounts of thermophilic bacterial probiotic composition according to the present invention
division
ingredient content
(g/L) ratio
(weight%)
note


(1)Basic badge
(M)Ingredients
maltose (required)
30.0
3.0 [Note] Glucose of more than 0.3% by weight cannot be used because it delays growth by more than 30h when used simultaneously with the nitrogen source below.
Tryptone (required)

30.0
3.0 [Note] When glucose is used together with Tryptone, it cannot be used because growth is delayed for more than 30h.
Yeast Extract (required)
30.0
3.0 [Note] When glucose is used together with yeast extract, it cannot be used because growth is delayed for more than 30h. Inorganic salt solution (required)
10X stock soln.
100mL
10.0 [Note] Most preferably, after preparing the inorganic salt stock solution, use a 1/10 times diluted solution.
(2)Gelling agent
Pectin
150.0
15.0
It is desirable to use less than 20% by weight.
gelrite
150.0
15.0
It is desirable to use less than 20% by weight.
pullurans
150.0
15.0
It is desirable to use less than 20% by weight.
(3)Breathable material Kalolinite 120.0 12.0 [Note] Domestic products are preferred (to improve breathability) Buckwheat Husk Powder
(10 to 50 mesh)
120.0
12.0
[Note] Not burned (to improve breathability)
(4) Gel stabilizer β-cyclo
Textrin (required)
120.0
12.0
[Note] To prevent liquid synergy and sedimentation of gelling agent sum 100.0 thermophilic bacteria A or B strain 1.0 0.1 ^1.0 [Note] The components of the basal medium (M) for thermophilic bacterial strains according to the present invention were placed in a 2.5L Flas container and dissolved in 1L of water, and the pH of the solution was adjusted to pH 7.0 by adding 0.1M NaOH solution. After adding the remaining components, heat sterilization at 121°C/15 minutes in an autoclave, then move to a clean bench, open the Cotton Flask cap cooled to 60°C, and inoculate the microorganism strain A or B of the present invention. Disposal of food waste according to the present invention by shaking and culturing in an incubator for 4 to 6 hours.
The probiotic composition for treatment is solidified and manufactured into a sterile sealed product.

[Comparative Manufacturing Examples 1 to 3]

Unlike Example 7 above, the present inventors prepared the probiotic composition according to the present invention according to the composition of the comparative preparation example below.

Comparative Preparation Example 1 contained equal amounts of pectin, gellan gum, and agar as gelling agents,

Comparative Preparation Example 2 contained equal amounts of gellan gum, carrageenan, and agar as gelling agents,

In Comparative Preparation Example 3, equal amounts of agar, pectin, and carrageenan were used as gelling agents, and the physicochemical properties were compared with the product in Example 7.

[Comparative Manufacturing Examples 4 to 6]

The present inventors did not use β-cyclotextrin as an anti-settling agent for the gelling agent used in Example 5, but instead used an equal amount of CaCl ₂ powder solution to prepare the gelling agent of the present invention and compared and evaluated its physicochemical properties.

As confirmed in Table 9 below, the experimental results showed that the gel strength and gel elasticity were poor.

Physicochemical properties of probiotics
division gel strength gel elasticity
note Hardness (N) Brittleness (N) Elasticity (mm) Cohesiveness (mm) Example 7 5.7 3.4 1.3 0.7 Comparative Example 1 3.4 1.6 1.1 0.3 Comparative Example 2 3.3 1.5 1.0 0.3 Comparative Example 3 3.5 1.7 0.9 0.3 Comparative Example 4 3.1 1.4 0.6 0.2 Comparative Example 5 3.0 1.5 0.5 0.2 Comparative Example 6 3.1 1.2 0.6 0.2 [Note] Gel strength and elasticity are measured using a physical property analyzer, the maximum load is 5N, and the solid medium thickness is 1mm.
Width × height = 2 cm × 2 cm.

[Example 8]

Growth experiment of thermophilic bacterial strain in solid gelling agent composition according to the present invention

Each sample of the probiotic composition according to the present invention prepared according to Example 7 and Comparative Examples 1 to 6 was dispensed onto a petri-dish on a clean bench, and then the strains of the present invention (A, B) (1.0 0.1 mL of a 10× dilution (10 ^{5 to 9} cfu/mL) was streaked, cultured in an incubator at 60°C for 2 days, and then the number of colonies was observed. The experiment was repeated three times, and the average number of colonies was calculated and listed in [Table 10].

Colony average value
division
colony number Is there anything wrong? cracks Synergy phenomenon Example 7 102 radish radish Comparative Example 1 53 you radish Comparative Example 2 57 you radish Comparative Example 3 47 you radish Comparative Example 4 51 radish you Comparative Example 5 57 radish you Comparative Example 6 61 radish you [Note] The flat pear Petri dishes on which the strain was smeared were placed in a plastic bag and cultured with shaking in an incubator for 4 hours to 10 hours, and then survived for 12 months when stored in a 4°C refrigerator.

As a result of the experiment, the probiotic composition of Example 7 according to the present invention not only had the best gel strength and gel elasticity, but also no cracks or syneresis of the plate medium was observed even at high temperatures of 60°C or higher. The growth condition of the thermophilic bacterial strain according to the present invention was good and viability was maintained even when refrigerated for more than 12 months (FIG. 4d).

Claims (5)

Batch culture in a solid or liquid container for Petri dishes characterized by 0.3% by weight maltose as a carbon source, 0.3% by weight tryptone, and 0.3% by weight yeast extract as a nitrogen source, with the remainder being 0.01% by volume of inorganic salt solution, based on the total weight of the medium composition. For thermophilic bacteria Bacillus subtilis subsp. Basal medium composition (M) of subtilis LSK 0604 (SARC-BSL) or Bacillus pumilus OYR 0108 strain. The thermophilic bacterium Bacillus subtils subsp. for batch culture in solid or liquid containers for Petri dishes, characterized in that the basal medium composition of claim 1 is diluted with 5x sterilized water. Dilution medium composition (M/5) of LSK 0604 (SARC-BSL) or Bacillus pumiuls OYR 0108 (SARC-BSS) strain. The thermophilic bacterium Bacillus subtilis subsp for batch culture in solid or liquid containers for Petri dishes according to claim 1, which contains glucose at a concentration of 0.3% by weight or less instead of the maltose. subtilis LSK 0604 (SARC-BSL) or Bacillus pumilus OYR 0108 strain medium composition. 0.5% by weight maltose based on the total weight of the entire culture medium. Thermophilic bacterium Bacillus subtilis subsp containing 1.5% by weight tryptone, 1.5% by weight yeast extract and 0.05% by volume of inorganic salt solution. Modified medium composition (G2) for fed-batch culture of subtilis LSK 0604 (SARC-BSL) or B. pumilus OYR 0108 (SARC-BSS) strains. (a) Introduce the modified medium composition (G2) for fed-batch culture of Paragraph 1 into a bench-top fementer and simmer at 121°C for 15 to 20 minutes. Autoclave and cool to a temperature below 70°C; (b) 0.1% by weight of the thermophilic bacterium Basubtilis subsp was added to the liquid culture obtained in the above step. subtilis LSK 0604 (SARC-BSL) or B. pumilus OYR 0108 (SARC-BSS) strain; (c) connecting an automatic acid-alkali pumping reservoir for adding HCL and NaOH to the fermenter inoculated with the thermophilic bacterial strain in the above step to adjust the pH to 7.0 and the temperature to 60°C; (d) In the above step, fermentation is started at a stirring speed of 1,000 rpm, and at 4 to 6 h when maltose is exhausted, the stirring speed is increased to 1,500 rpm, glucose is supplied at a glucose feed rate of 6 g/L/h, and the oxygen supply rate is 1.00 vvm. further culturing at a rate; (e) In the culture step, the oxygen supply rate (airation rate) was 1.33vvm and 200L/2.5L/60min until OD (580nm) was 36.4 and maximum CDW (g/L) was 15.9. A fed-batch culture method of thermophilic bacterial strains characterized by fermentation at a rate of 33.5 hours in total and then stopping.