KR20110002512A - Geobacillus sp. hw1 strain producing thermophilic lipase and usage thereof - Google Patents

Abstract

PURPOSE: A Geobacillus sp. HW1 strain which produces thermophilic lipase is provided to quickly decompose lipid ingredient in a high temperature and rapid decomposition process. CONSTITUTION: A Geobacillus sp. HW1 strain producing lipase is alive at pH 6.0-9.0 and 30-70°C. The pH concentration and temperature which is able to produce lipase is pH 5.5-7.0 and 45-60°C. The strain has 16s rRNA having a base sequence of sequence number 1. The lipase activity of the strain is enhanced by culturing the strain in a medium containing carbon source of olive oil, fish oil, sesame oil, soybean oil or canola oil. A microorganism formulation for composting waste food contains Geobacillus sp. HW1 strain.

Description

H1 strain of genus Geobacilli, which produces pyrolipase, and its use {Geobacillus sp. HW1 strain producing thermophilic lipase and usage

The present invention is a method of increasing the lipase activity by culturing the strain under a specific condition of the genus Geobacillus HW1 producing thermophilic lipase, the microbial agent for composting food waste comprising the strain and treating the strain to food waste It relates to a food waste composting method comprising the step.

As of 2004, the amount of food waste generated in Korea was 11,463 tons per day, accounting for 23% of total domestic waste, and the amount of treatment amounted to 15 trillion won. Since 2005, a law prohibiting direct landfilling of food waste has come into force, and it is urgently needed to improve the recycling rate of waste generated. Food waste consists of about 80% moisture, 17% flammable content, and 3% ash content. Excluding moisture, odor and leachate are generated due to high biodegradability. These odors and leachate are generated in all processes, such as collection, transfer, treatment, and the like, causing infestation of flies, insects, and pathogens. Food waste is expected to have high water content, low compression efficiency, low calorific value when incinerated, and over-consumption of fuel (83l diesel fuel is consumed per 1 ton), and high water content lowers the incineration temperature to reduce pollution such as dioxin. It is a cause of release of materials.

In 2002, 52% of food waste was recycled, 43.5% of compost, 4.4% of methanation and others. Feeding has declined sharply since the outbreak of foot-and-mouth disease, and there is a lack of methanation-related technologies, and the rate of composting is expected to increase significantly. When using food waste for composting, the first problem is that it is highly likely to be decayed by bacteria, and secondly, it is difficult to compost acidified food waste in relation to domestic special eating habits. As a solution to these problems, a high temperature and high speed fermentation process using high temperature and acid resistant microorganisms has attracted attention as a method for the rapid treatment of food waste. By using high temperature and acid resistant microorganisms, composting is carried out at high temperature of 50 ~ 75 ℃ and weakly acidic condition between pH 5 and 6, which prevents the breeding of mesophilic bacteria and improves the speed of composting by the heat of oxidization generated from microorganisms. Can be. Microorganisms involved in composting are aerobic microorganisms that can live only in the presence of oxygen and break down food into water and carbon dioxide, leaving the compost called compost. At this time, the heat of oxidation is generated at the same time as decomposition, and the temperature rises to 60-70 ° C. without applying a temperature from the outside. In addition, at this time, the water contained in the food is evaporated by the fermentation heat and decomposes about 85 to 90%, thereby obtaining a decomposition rate of 95% or more by composting. Decomposition process of high temperature microorganism organic matters firstly, sugars, amino acids, etc. are rapidly decomposed, and after 1 ~ 2 days, the temperature of compost pile starts to rise, followed by decomposition of cellulose pectin, which usually lasts for several weeks. do. However, it is pointed out that in this process, fat components such as animal and vegetable oils in food waste are not decomposed and accumulated on the bottom during fermentation.

Lipase (glycerol ester hydrolases, EC 3.1.1.3) hydrolyzes triglycerides in the suspension of lipids and water to form monoglycerides, glycerol and free fatty acids, as well as alcohols and fatty acids. It may also catalyze the synthesis or exchange reaction of esters. Since the enzyme was first found in animal pancreatic fluid, it has been found to exist in animal organs and plant seeds, and microorganisms derived from microorganisms such as fungi and bacteria have been found and widely distributed throughout the biological system. Among them, the exocrine lipase produced by the microorganism is attracting much attention as an industrial enzyme because it is more stable to heat than lipases derived from animals and plants.

Therefore, the present invention selects the microbial material for producing high-temperature lipase for the high-speed composting of hardly decomposable fat component that degrades degradation during high temperature and high speed fermentation of food waste, and the production of lipase enzyme targeting the final selected strains The purpose of the present invention is to improve the lipolytic activity of selected strains by optimizing the culture conditions to increase.

In an effort to find a microorganism strain that satisfies the above conditions, the inventor of the present invention, HW1 strain of genus Bacillus, which produces pyrolytic lipase isolated from Japanese hot spring, decomposes fat components in a short time during high temperature and fast composting of food waste. It was confirmed that this can be done, and the present invention has been completed.

One object of the present invention is to provide a strain of genus HW1 of the genus Geobacilli that produces pyrogenic lipases.

Another object of the present invention is to provide a method of increasing the lipase activity by culturing the strain under specific conditions.

Another object of the present invention to provide a microbial preparation for food waste compost comprising the strain.

Another object of the present invention to provide a food waste composting method comprising the step of treating the strain to food waste.

The present invention relates to a strain of genus HW1 of the genus Geobacillus, which produces pyrogenic lipases.

The present inventors isolated strains excellent in lipolytic activity from Japanese hot springs. The strains of the present invention are bacilli, are gram positive and have no flagella. In addition, the growthable pH is 6.0-9.0 (optimum pH 8.0) and the growthable temperature is 30-70 ° C (optimum temperature 55 ° C). Physiological characteristics are similar to the strain of genus Geobacilli, as shown in Table 1. In addition, 16s rRNA sequence analysis and homology results, showed a high homology with the strain of genus Geobacillus. As described above, the strains of the present invention were classified as physiological and biochemical properties as soft bacteria of the genus Geobacillus, and the present strain was identified as genus HW1 of the genus Geobacillus (see Example 2-1).

The present invention also relates to a method of increasing the lipase activity of the strain by culturing the strain under specific conditions. As the activity of the lipase increases, the fat component resolution of the strain may be further improved.

In order to increase the lipase activity of the strain of genus HW1 according to the present invention, the strain is cultured in a medium containing a carbon source selected from the group consisting of olive oil, lard oil, fish oil, sesame oil, soybean oil and canola oil. It is preferable. It is more preferable to use a carbon source selected from olive oil, fish oil and sesame oil. It is even more preferable to culture in a medium containing 0.5 to 1.0% of olive oil (see Example 2-2).

In order to increase the lipase activity of the genus HW1 strain of the genus Geobacillus, the pH is preferably 5.5-7.0, the temperature is cultured at 45 ~ 60 ℃. More preferably, the culture is carried out under the condition that pH is 6.0 and the temperature is 50 ° C (see Example 2-3). As described above, the strain of the present invention exhibiting higher lipase activity under mildly acidic conditions is a food waste of Korea near pH (pH: 4.0-6.5, rice, kimchi, soy sauce, miso, tofu, beef soup, boiled potatoes, etc.). Is particularly useful for composting

In order to increase the lipase activity of the genus Geobacilli HW1 strain according to the present invention, it is preferable to culture in a medium containing Mg ²⁺ or Mn ²⁺ with metal ions. Mg ²⁺ is more preferred. On the other hand, it is preferable not to include Co ²⁺ , Fe ²⁺ , Ni ²⁺ , Cu ²⁺ (see Example 2-4).

In addition, the present invention relates to a microbial preparation for composting food waste comprising the strain of genus HW1.

When the strain of the present invention is used as a microbial preparation for food waste composting, it is preferable to use the strain of the present invention by adsorbing it to a decomposition medium (inorganic carrier), for example, zeolite, activated diatomaceous earth, vermiculite and white carbon and Among the same inorganic carriers: 1) excellent moisture control ability, 2) can act as a carrier by microbial adsorption, 3) pH buffering ability, 4) stable to mechanical wear, 5) excellent adsorption of odorous substances You can choose.

The microbial preparation for food waste composting of the present invention can be carried out using conventional techniques known in the art. In one embodiment, a method of culturing the genus Geobacillus HW1 strain under certain conditions capable of increasing the lipase activity described above; Adsorbing the culture to an inorganic carrier and drying to prepare a dried product of the culture; And mixing nutrients of excipients and strains in the dry matter of the culture.

The excipient may be selected from elite, activated carbon and elvan, and the like, and preferably 0.1 to 5 times by weight based on the dry matter of the culture. In addition, the nutrient of the strain is preferably a carbon source capable of promoting the growth and lipase activity of the strain, it is preferably 0.5 to 5% by weight relative to the excipient.

In addition, the present invention relates to a food waste composting method comprising the step of treating the strain to food waste. Here, not only the strain itself but also a culture of the strain is available, and the culture of the strain is preferably a culture obtained by the method of increasing the lipase activity described above. The strain of the present invention can decompose fat components in a short time in a high temperature and high speed composting device (see Example 2-5).

HW1 strain of the genus Geobacillus of the present invention can decompose fat components in a short time during the high temperature and high speed composting of food waste.

In addition, since the strain of the present invention exhibits higher lipase activity under mildly acidic conditions, food wastes in Korea close to acidic pH (pH: 4.0-6.5, rice, kimchi, soy sauce, soybean paste, tofu, beef soup, boiled potatoes, etc.) It is especially useful for composting.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1-1: Strain Isolation and Identification

In this experiment, tributyrin was used as a substrate for the screening of lipolytic bacteria among microorganisms isolated from soil collected at Arima hot spring in Japan, and plate assay method (Kouker, G. et al. 1987. Appl. Environ. Microbiol. 53, 211-213) was selected as a strain with high lipase activity. Lipid-degrading bacteria selection medium was inoculated with bacteria to form a zona pellucida around the colonies as lipase-activated positive bacteria, and these were cultured in 1% olive oil nutrient broth to finally select the strain with the highest enzyme activity. The morphology of the isolated strain HW1 was observed by a negative stain using a transmission electron microscope (JEM 1200 EX-II electron microscope, JEOL). Morphological and physiological characteristics were investigated using the method of Romano et al. (Romano. I, A. et al. 2005. J. Gen. Appl. Microbiol . 51, 183-189) and 16s rDNA sequence analysis was performed by Kim et al. , DJ et al. 1995. J. Ferment. Bioeng. 79, 87-94).

Example 1-2 Enzyme Production

Geobacillus sp. For enzyme production. After incubating the strain of HW1 for 24 hours at 50 ° C. in LB medium, the culture medium was minimally grown [0.7% NaNO ₃ , 0.2% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.01% KCl, 0.05% MgSO ₄ ㆍ 7H ₂ O, 0.001% CaCl ₂ , 0.0012% FeSO ₄ ㆍ 7H ₂ O, 0.01% (v / v) trace element solution (0.026% H ₃ BO ₃ , 0.05% CuCl ₂ ㆍ 2H ₂ O, 0.05% MnCl ₂ ㆍ 4H ₂ O, 0.006% Na ₂ MoO ₄ ㆍ 2H ₂ O, 0.07% ZnSO ₄ ㆍ 7H ₂ O), 0.1% yeast extract] inoculated at 1% concentration in a medium containing 0.5% olive oil at 50 ℃, 36 hours incubation Then, the supernatant was recovered by centrifugation (10,000 rpm, 10 minutes). Here, 70% of ammonium sulfate was saturated and left at 4 ° C. for 12 hours, and the precipitate obtained by centrifugation (10,000 rpm, 20 minutes) was dialyzed with 50 mM sodium phosphate (pH 6.0) as a crude enzyme solution.

Example 1-3: Lipase Activity Measurement

Enzyme activity was measured by the colorimetric method (Puerto W, K. et al. 1986. J. Bacteriol. 168, 1070-1074) using PNPB (p-nitrophenyl butyrate) as a standard. Mix 30 mg of PNPB in 10 ml of 2-propanol and 0.207 g of sodium deoxycholate (Na-DOC) and 0.1 g of gum arabic in 90 ml of 50 mM sodium phosphate buffer (pH 6.0). Then, 0.1 ml of enzyme solution was added to 2 ml of the mixed solution and reacted at 50 ° C. for 10 minutes, and then 0.9 ml of 2M Na ₂ CO ₃ solution was added to stop the reaction. Lipase activity was measured at 410 nm to determine the amount of p-nitrophenol produced.

As another lipase activity assay, Kwon et al. (Kwon, DY et al. 1986. JAOCS 63, 89-92) were used. The substrate was added to emulsion stock solution (50 mM sodium phosphate buffer (pH 6.0), 20 mM CaCl ₂ , 1 mM deoxycholate, 5% gum arabic), followed by homoge It was used by emulsifying with a homogenizer. Enzyme activity was measured by adding 0.2 ml of the enzyme solution to 2 ml of the substrate and reacting at 50 ° C. for 1 hour while shaking at 200 rpm in a shaker bath. 0.5 ml of 6 N HCl was added as an enzyme stopping solution, and 5 ml of isooctane was added to dissolve free fatty acids, followed by mixing at 100 ° C. for 3 minutes. The separated isooctane layer was vortexed for 90 seconds after adding 1 ml of a colorant copper reagent (prepared with 5% cupric acetate, filtered through Whatman filter paper, adjusted to pH 6.1 with pyridine), and measured at absorbance at 715 nm. One unit of lipase was expressed as the amount of enzyme that produced 1 μM free fatty acid in one minute.

Example 2-1: Isolation and Identification of Thermophilic Lipase Production Strains

For the isolation of thermophilic lipase-producing bacteria, 8 kinds of strains were cultured in a separation medium containing tributyrin for 24 hours using a soil sample obtained from a hot spring zone and forming a zona pellucida around colonies. The isolates were cultured in a liquid medium, and finally the strains with the highest enzyme activity were selected and named HW1. This strain is a bacterium having a length of 2-3 μm and a diameter of 0.7 μm, as shown in the electron micrograph of FIG. 1, without Gram-positive, flagella. Growth potential of the isolated HW1 is 6.0 ~ 9.0 (optimum pH 8.0) and the growth possible temperature is 30 ~ 70 ℃ (optimum temperature 55 ℃) (Fig. 4, 5), the physiological characteristics are gelatin, starch as shown in Table 1 , Hydrolyzed aesculin (aesculin) showed similar characteristics to the strains of the genus Geobacillus . In order to identify strain HW1, 16s rDNA sequence (SEQ ID NO: 1) was analyzed and homology was analyzed using NCBI's BLAST program. Geobacillus thermoparaffinivorans and 99.9%, Geobacillus sp. The homology between A27 and 99.7%, Geobacillus thermoleovorans strain hs-1 and 99.3%, and Geobacillus stearothermophilus strain ARM 1 was 98.9%. As described above, strain HW1 has been classified as a flexible bacterium of the genus Geobacillus because its physiological and biochemical properties are classified as Geobacillus sp. Identifies Geobacillus sp. Named HW1. The named strain was deposited on May 26, 2009 with the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and assigned accession number: KACC91476P.

Example 2-2: Influence of Carbon Sources for Enzyme Production

The influence of carbon sources was investigated to select the optimal conditions for lipase production. As a carbon source, various natural oils such as olive oil, lard oil, fish oil, sesame oil, soybean oil, grapeseed oil and canola oil are added to a minimum medium so that the final concentration is 0.5% and incubated for 36 hours at 50 ° C. After taking the crude enzyme solution was measured lipase activity (Fig. 2). As a result of lipase activity, olive oil showed the highest activity, fish oil and sesame oil showed more than 95% activity, and soybean oil and canola oil showed about 50% activity. In contrast, lard and grapeseed oil showed relatively low activity. Olive oil showed the highest activity in a minimum medium culture at various concentrations of 0.1%, 0.5%, 1.0%, 1.5% 2.0% and measured lipase activity by time zone (Fig. 3). As a result, the lipase activity showed the highest activity at the olive oil concentration of 0.5% and maintained high activity from 18 hours to 36 hours, which is the growth stop period of the bacteria.

Example 2-3 Optimal pH and Temperature for Enzyme Production

In order to determine the optimal pH for lipase production, enzyme activity was measured using different buffers in the range of pH 4-11. pH 4-5.5 at 0.05 M sodium acetate buffer, pH 6-7 at 0.05 M sodium phosphate buffer, pH 8-9 at 0.05 M Tris-HCl buffer, pH 10-11 at 0.05 M glycine-NaOH buffer The pH of the medium was adjusted and cultured at 50 ° C. for 36 hours to obtain enzyme activity by taking a crude enzyme solution (FIG. 4). As shown in FIG. 4, microbial growth was the highest at pH 8, but microbial growth was not good at low pH. On the other hand, lipase production showed a good effect in the pH range of 5.5-7.0 and the best enzyme production at pH 6.0. In order to determine the optimum temperature for the enzyme production, the enzyme activity was measured after incubation at 30 ℃, 37 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃ (Fig. 5). As a result, the microbial growth was the highest at 55 ℃ and the highest at 50 ℃ for enzyme production.

Example 2-4: Effect of Metal Ions on Enzyme Production

To determine the effect of metal ions on the lipase production of this strain, 2 mM metal ions were added to the medium and incubated at 50 ° C. for 36 hours, and then the lipase activity was measured by taking a crude enzyme solution (Table 2). As shown in Table 2, Mg ²⁺ and Mn ²⁺ in the metal ions showed a good effect on lipase activity, affecting enzyme production. In particular, Mg ²⁺ showed about 2.5 times higher activity than the control without the metal ion. On the other hand, Co ²⁺ , Fe ²⁺ , Ni ²⁺ , Cu ²⁺ have been shown to inhibit enzyme production.

Example 2-5: Lipolysis Characteristics in a Composting Apparatus

Using the isolated strain, the decomposition effect of olive oil was actually tested in the composting device. The apparatus used in this embodiment is a 10 L food waste composting apparatus (BK-01H, Oklin, Korea), which is 28 cm (L) x 32 cm (B) x 89 cm (H). The temperature inside the device was maintained at about 55 ~ 60 ℃ by a temperature sensor mounted on the bottom, and three stirring shafts were installed at right angles to the horizontally arranged rotating shaft. In the composting device, 500 ml of the strain culture medium incubated for 18 hours in a minimum medium to 1.0 kg of cedar sawdust serving as a microorganism carrier was evenly poured and stirred for 30 minutes, and then 50 ml of olive oil was added and stirred for another 30 minutes. Thereafter, 10 g of cedar sawdust was taken per hour for 36 hours, and the viable cell count and lipase activity in the sample were measured. As a result, the number of viable cells immediately after olive oil administration was 4.5 × 10 ⁸ CFU / ml, and increased rapidly to 13.2 × 10 ⁸ CFU / ml in 9 hours after the start of the reaction. In enzyme activity, the highest activity was found at 18 hours, after which the activity was slightly decreased, showing a relative activity of 52% at 36 hours (FIG. 6). As a result, Geobacillus sp. HW1 can be said to be very useful as a strain for decomposing fat in a short time in a high temperature and high speed composting device.

1 is Geobacillus sp. The electron micrograph of HW1 is shown (Bar, 0.5 micrometer).

2 is Geobacillus sp. The effect of carbon source on lipase production of HW1 is shown.

3 is Geobacillus sp. Concentrations of olive oil on growth of HW1 and lipase production (strain growth: -0.1%; ▼ -0.5%; ■ -1.0%; ◆ -1.5%; ▲ -2.0%), (lipase activity: ○ -0.1%; ▽ -0.5%; □ -1.0%; ◇ -1.5%; △ -2.0%).

4 is Geobacillus sp. PH effect on HW1 growth and lipase production [0.05 M sodium acetate buffer (pH 4-5.5), 0.05 M sodium phosphate buffer (pH 6-7), 0.05 M Tris-HCl buffer (pH 8 -9), 0.05 M glycine-NaOH buffer (pH 10-11)].

5 is Geobacillus sp. It shows the temperature effect on the growth of HW1 and lipase production (□ -strain growth; VIII-enzyme activity).

6 is Geobacillus sp. In the composting device. The number of viable cells (●) and lipase activity (○) of HW1 is shown.

<110> Dong-eui University Industry-Academic Cooperation Foundation <120> Geobacillus sp. HW1 strain producing thermophilic lipase and          usage <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1467 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA <400> 1 tcaggacgaa cgctggcggc gtgcctaata catgcaaggc gagcggacca aatcggagct 60 tgctctgatt tggtcagcgg cggacgggtg agtaacacgt gggcaacctg cccgcaagac 120 cgggataact ccgggaaacc ggagctaata ccggataaca ccgaagaccg catggtcttt 180 ggttgaaagg cggcctttgg ctgtcacttg cggatgggcc cgcggcgcat tagctagttg 240 gtgaggtaac ggctcaccaa ggcgacgatg cgtagccggc ctgagagggt gaccggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgaaag cctgacggag cgacgccgcg tgagcgaaga aggccttcgg gtcgtaaagc 420 tctgttgtga gggacgaagg agcgccgttc gaagagggcg gcgcggtgac ggtacctcac 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa tacgtagggg gcgagcgttg 540 tccggaatta ttgggcgtaa agcgcgcgca ggcggttcct taagtctgat gtgaaagccc 600 acggctcaac cgtggagggt cattggaaac tgggggactt gagtgcagga gaggagagcg 660 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 720 gctctctggc ctgcaactga cgctgaggcg cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaagtgt tagaggggtc acacccttta 840 gtgctgcagc taacgcgata agcactccgc ctggggagta cggccgcaag gctgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960 gaagaacctt accaggtctt gacatcccct gacaacccaa gagattgggc gttccccctt 1020 cggggggaca gggtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caaccctcgc ctctagttgc cagcacgaag gtgggcactc 1140 tagagggact gccggcgaca agtcggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tgggcggtac aaagggctgc gaacccgcga 1260 gggggagcga atcccaaaaa gccgctctca gttcggattg caggctgcaa ctcgcctgca 1320 tgaagccgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accacgagag cttgcaacac ccgaagtcgg tgaggtaacc 1440 cttacgggag ccagccgccg aaggtgg 1467  

Claims (10)

A genus HW1 strain of genus Bacillus, which has a viable pH of 6.0 to 9.0, a viable temperature of 30 to 70 ° C, and produces lipases. The method of claim 1, Lipase production possible pH is 5.5 ~ 7.0, the production temperature is a genus HW1 strain of the genus Bacillus, characterized in that 45 ~ 60 ℃. The method of claim 1, A genus HW1 strain characterized by having a 16s rRNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. The method of claim 1, HW1 strain of the genus Geobacillus, characterized in that the accession number is KACC91476P. The strain of genus HW1 according to any one of claims 1 to 4 is incubated in a medium containing a carbon source selected from the group consisting of olive oil, fish oil, sesame oil, soybean oil and canola oil. Methods of increasing lipase activity. The method of claim 5, If the carbon source is olive oil, characterized in that it comprises 0.5 to 1.0%. The method of claim 5, pH is 5.5 ~ 7.0 and the temperature is a method characterized in that the culture in 45 ~ 60 ℃. The method of claim 5, Method of culturing in a medium containing Mg ²⁺ or Mn ²⁺ with metal ions. Microbial preparation for food waste compost comprising a strain of genus HW1 according to any one of claims 1 to 4. Composting food waste comprising the step of treating the strain according to any one of claims 1 to 4 food waste.

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