KR100499871B1 - Method for preparing enzyme by employing fermentation product from food wastes - Google Patents

Abstract

The present invention relates to a method for culturing mold in fermentation products obtained by fermenting food waste by bacteria, in particular by using solids as a medium, and according to the present invention, an enzyme having a high added value can be produced.

Description

Production method of enzyme using food waste fermentation {METHOD FOR PREPARING ENZYME BY EMPLOYING FERMENTATION PRODUCT FROM FOOD WASTES}

The present invention relates to a method for producing an enzyme using a food waste fermentation product, and more particularly, to a method for producing an enzyme by culturing mold using a solid dehydrated from a food waste fermentation product as a medium.

Currently, food waste, which is a representative organic waste generated in Korea, is reported to occupy about 30% of total household waste. However, due to its high perishability, it is difficult to recycle due to bad smell, sewage leakage, etc. However, landfill treatment is not only wasteful and takes a long time to stabilize, but also causes secondary environmental pollution of soil and groundwater by leachate and the like.

Against this backdrop, various recycling methods have been tried to feed or compost food waste. However, most of the products are food wastes that are simply dried foods or fermentation products that use bacteria, and thus produce feed products having a relatively lower commodity value than conventional feed products.

Bacteria fermentation of food waste has been reported (Ref .: Vol. 9, No. 2, pp. 16-20), but it can replace some of the excipients added to formulated feed due to its high salt content and insufficient nutrient composition. The added value is relatively lower than the additives (probiotics, enzymes, etc.) added to the existing compound feed with a high degree of quality.

Accordingly, there is a need for an effective method for developing high value-added products by efficiently treating food waste in response to the shortage of landfill facilities and the growing demand for organic waste disposal.

It is an object of the present invention to provide a method for culturing mold using food waste and commercially useful enzymes produced thereby.

In order to achieve the above object, the present invention provides a method for producing the enzyme by culturing the mold using a fermented cake of solid content dehydrated the fermentation product obtained by fermenting food waste by bacteria as a medium.

The food waste fermentation product may be prepared by fermenting the crushed food waste by its own bacteria or after fermenting hot bacteria. Fermentation by self bacteria can be carried out at a high temperature, preferably 50 to 80 ° C., in the presence of oxygen, using, for example, aerobic mold inside the food waste. In addition, the fermentation by inoculation of the bacteria is carried out in the food waste from the outside thermophile, for example, Thermopolyspora species, Thermomonospora species, Aspergillus species, Peregrine indeed, my process species (Paecilomyces species), Cellulofine Pseudomonas species (Cellulomonas species), Agrobacterium species (Agrobacterium species), Pseudomonas species (Pseudomonas species), Flavobacterium (Flavobacterium species), jaento Pseudomonas species (Xanthomonas species ), Micrococcus species, thermophilic species of Bacillus subtilis , Bacillus stearotheromphilus , Bacillus acidocaldarius , and the like, inoculated at 50 to 80 ° C. It can be carried out by fermentation in.

In particular, the inoculated food wastes were inoculated with Bacillus stearothermophilus (TS-LDF 905), Bacillus axidocalcarius (TS-LDF SP2) or mixtures thereof, which were obtained by continuous fermentation at 50 to 65 ° C. It is more preferred to use food waste fermentation products. New strains selected by the present inventors Bacillus steareromophilus TS-LDF 905 and Bacillus axido caldarius TS-LDF SP2, respectively, dated August 17, 2001 to the Korea Microorganism Conservation Center (KCCM) Accession No. KCCM Deposited as -10310 and KCCM-10309.

As a medium for culturing the mold used in the present invention, it is preferable to use a solid fermented cake obtained by dehydrating the food waste fermentation product in a conventional manner.

The fungus used in the present invention includes Aspergillus niger , Aspergillus oryzae or Aspergillus awamori , among which Aspergillus niger , Aspergillus duck Ash is preferred, particularly Aspergillus Niger.

Cultivation of the mold using food waste fermentation cake may be carried out at 25 ℃ to 30 ℃, 25 ℃ is most preferred.

In addition to the fermented cake, a carbon source, a nitrogen source, a trace element, an excipient, and the like may be further added as a medium used in the culture process of the mold. These carbon sources, nitrogen sources or trace elements multiply the number of cells of the fungus and thus increase the production of enzymes that are fungal culture products.

As the carbon source, it is advantageous to add one or more selected from the group consisting of molasses, glucose, sucrose, galactose, mannitol and maltose in an amount of 1 to 2% by weight, preferably 2% by weight.

The nitrogen source is one selected from the group consisting of ammonium sulfate, yeast extract, peptone, soyton, tryptone, malt extract, casamino acid, corn steep liquor, monosodium glutamate fermentation by-product (MSG) and nucleic acid It is advantageous to add the above in an amount of 1 to 2% by weight, preferably 2% by weight.

As the trace element, it is advantageous to add one or more selected from the group consisting of zinc sulfate, calcium sulfate, ferrous sulfate (FeSO ₄ ) and magnesium chloride in an amount of 1 to 3 mM, preferably 1 mM.

In addition, in the fungal culture of the present invention, an excipient may be further added to the food waste fermentation product, and such excipient may increase breathability and provide additional nutrients. As the excipient, soybean meal, rye, cottonseed meal, corn fine grains, wheat flour or a mixture thereof may be used, and it is preferable to add so that the water content of the whole medium used in the present invention is 45 to 65%.

By the method of the present invention, the enzyme is produced as the mold is cultured from the food waste fermentation product. Examples of the generated enzyme include amylase, cellulase, xylanase and the like. The produced enzyme can be separated from the culture by conventional methods, for example, precipitation, gel filtration, various chromatography methods, and the like.

By obtaining the enzyme according to the method of the present invention, the added value is very high compared to the conventional method of simply drying the raw food waste to use as feed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Preparation of Medium

(1) Characteristics of food waste used

The food waste generated in public housing in Anseong-si, Gyeonggi-do was collected and provided by a special vehicle for food waste collection as a raw material for producing badges.

ingredient moisture Crude protein Crude fat Crude fiber View minutes calcium sign NaCl NPN % 85 3.52 2.07 1.09 2.70 0.68 0.14 0.71 0.018

(2) medium production method

The collected food waste was first crushed using a paper pulper (sail machine) without additional water supply or additive injection, and light foreign matter and crushing was not completely removed at the top of the pulper during the first crushing. . Heavy debris (bones, bottle caps, cutlery, etc.) was removed by centrifugal force from the Junk Box at the bottom of the pulp. The particle size was controlled by passing through an 8 mm diameter sieve at the bottom of the pulper when discharged from the pulper. Prior to transfer to the fermenter, an in-line grinder was used for secondary crushing to a maximum particle size of 4 mm or less, with an average particle diameter of 50 μm.

Subsequently, the prepared raw food pulverized product was placed in a fermenter, and then inoculated with Bacillus axido caldarius (KCCM 10309) in an amount of 0.5% (v / v), followed by 60 ° C., pH 5.8, and dissolved oxygen of 1-5%. The fermentation was continued for about 8 hours. After fermentation, the particle size of the solids decreased to 38 μm due to degradation by mold.

The food fermentation slurry fermented as described above was introduced into a decanter (decanter type) by high-speed centrifugation and dehydrated to separate the cake and the desorption solution from the solids.

Soybean meal as an excipient was added to the obtained food fermentation cake in the same ratio as the cake (1: 1) to prepare a medium, and the medium was wet sterilized at 121 ° C. for 15 minutes without adjusting the pH.

Example 2: Screening of Mold

In order to find the fungus strain showing the best growth in the medium prepared in Example 1, 11 Aspergillus Niger standard strains (KFCC 32316, KCCM 11724, KCCM 11240, KCCM 32005, KCCM 60317, KCTC 6985, KCTC 6906, KCTC) 6971, KCTC 6986, KCTC 7278, KACC 40280) and 20 Aspergillus duck standard strains (KCCM 11371, KCCM 11896, KCCM 12089, KCCM 12698, KCCM 32319, KCCM 60166, KCCM 60241, KCCM 60345, KCCM 60355 6377, KCTC 6095, KCTC 6983, KCTC 6909, KACC 40232, KACC 40233, KACC 40234, KACC 40242, KACC 40244 and KACC 40247).

40 g of the medium prepared in Example 1 was placed in a 250 ml bottle of baffle, in which each mold was inoculated with 1.9 × 10 ⁷ spores in suspension and shaken once every 24 hours while standing incubated at 25 ° C. After a period of time, a sample was taken and the titers of the resulting amylase, cellulase and xylanase were measured. The results are shown in FIG.

Aspergillus Niger species showed higher enzyme expression titer than Aspergillus duck species. Among them, Aspergillus Niger KCCM 11724, which had the highest titer of xylanase, was selected for further experiments. .

Example 3: Enzyme Titer Comparison According to Culture pH

As a medium, the fermented cake (water content: 72.5%) prepared as in Example 1 was lyophilized (water content: 51.1%), and sodium hydroxide (NaOH) and hydrochloric acid (HCl) were further added to pH. The range was 1.97 to 6.63. 40 g of the prepared medium was placed in a 250 ml baffle bottle, and Aspergillus Niger KCCM 11724 selected in Example 2 was inoculated in the same amount as in Example 2, and then cultured at 25 ° C. for 5 days. To 1 g of the culture thus obtained, 4 ml of distilled water was added, vortexed for 3 minutes, and centrifuged at 10000 rpm for 5 minutes to extract enzymes (amylase, cellulase and xylanase) from the supernatant. The titer of the enzyme according to the culture pH was measured and shown in FIG. 2. As shown in FIG. 2, the enzyme titer was shown only at pH 6.05 or higher and the highest enzyme titer at pH 6.38.

Example 4 Comparison of Enzyme Titers According to Incubation Time

40 g of the medium prepared in Example 1 (pH 6.05) was placed in a 250 ml bottle of baffle, and the Aspergillus Niger KCCM 11724 selected in Example 2 was inoculated in the same amount as in Example 2, followed by 8 at a temperature of 25 ° C. Incubated for days. Enzymes (cellulase and xylanase) were extracted from the culture thus obtained in the same manner as in Example 3. The titer of enzyme with incubation time was measured and shown in FIG. 3. As shown in Figure 3, both cellulase and xylanase continued to increase in titer for 5 days after incubation, but remained unchanged from 5 to 8 days later.

Example 5: Comparison of Enzyme Titers According to Culture Temperature

40 g of the medium prepared in Example 1 (pH 6.05) was placed in a 250 ml bottle of baffle, and then inoculated with Aspergillus Niger KCCM 11724 selected in Example 2 in the same amount as in Example 2, at 15 to 25 ° C. Incubated for 8 days. Enzymes (cellulase, amylase and xylanase) were extracted from the culture thus obtained in the same manner as in Example 3. The titer of enzyme according to the culture temperature was measured and shown in FIG. 4. As shown in Figure 4, cellulase showed the highest enzyme titer when fermented at 25-30 ^° C and xylanase and amylase showed the highest enzyme titer at 25 ^° C.

Example 6 Comparison of Enzyme Titers According to Carbon Source Added to the Medium

As a medium, 2% by weight of glucose, galactose, maltose, sucrose, mannitol and molasses, which may be carbon sources, were added to the medium prepared in Example 1, and Aspergillus Niger KCCM 11724 was prepared in Example 2 and After inoculating the same amount and incubated for 8 days at 25 ℃, pH 6.05, the titer of each enzyme is measured and shown in Figure 5. As can be seen in Figure 5, it was observed that the enzyme was produced in all experimental groups except the control group ((-) control) not inoculated with the fungus at all, especially in the case of the addition of galactose in xylanase The highest enzyme titer was found.

Example 7 Comparison of Enzyme Titers According to Nitrogen Source Added to the Medium

As a medium, yeast extract, malt extract, peptone, soyton, corn steep liquor, monosodium glutamate fermentation by-product (MSG; fermentation waste solution of monosodium glutamate production process) and nucleic acid which may be nitrogen sources in the medium prepared in Example 1 2 wt% of each was inoculated with Aspergillus Niger KCCM 11724 in the same amount as in Example 2, and then incubated at 25 ° C., pH 6.05 for 8 days, and the titers of the respective enzymes were measured. Indicated. As can be seen in Figure 6, it can be observed that the enzyme production in all the experimental zones and the highest titer of xylanase in all nitrogen sources, especially the highest in the malt extract.

Example 8 Comparison of Enzyme Titers According to Trace Elements Added to the Medium

As a medium, 1 mM (w / w) of zinc sulfate, calcium sulfate, ferric sulfate, and magnesium chloride were respectively added to the medium prepared in Example 1 as a trace element, and Aspergillus Niger KCCM 11724 was prepared as in Example 2. After inoculation with the amount was incubated for 8 days at 25 ℃, pH 6.05, the titer of each enzyme was measured and the results are shown in Figure 7. As can be seen in Figure 7, it was observed that the enzyme was produced in all the experiments, the addition of calcium sulfate showed the highest xylanase titer.

Example 9 Comparison of Enzyme Titers According to Excipients Added to Fermented Cakes

As an excipient, a medium was prepared by the same method as Example 1 except for using soybean meal or adding rye, cottonseed meal, and corn fine grains instead of soybean meal, and aspergillus niger KCCM 11724 in Example 2 After inoculation with the same amount as incubated for 8 days at 25 ℃, pH 6.05, the titer of each enzyme was measured and the results are shown in Figure 8. It was observed from Figure 8 that the enzyme was produced in all the experiments, soybean meal was the most effective in the case of amylase and cellulase, the best effect of cottonseed foil in the case of xylanase.

Example 10 Comparison of Enzyme Titers According to Water Content in Fermented Cakes

As a medium, the fermented cake prepared in Example 1 (moisture content: 72.5%) was lyophilized and further moisture was added to adjust the initial moisture content of the culture to 25 to 70%. The titer of each enzyme obtained by inoculating 40 g of the prepared medium into a 250 ml bottle of baffle, inoculated with Aspergillus Niger KCCM 11724 selected in Example 2 in the same amount as in Example 2, and then incubating at 25 ° C. for 5 days. The results are shown in FIG. 9. As shown in FIG. 9, amylase and cellulase showed the highest enzyme titers in the water content of 50% of the medium and in the case of xylanase in the water content of 55% of the medium.

The present invention provides a method for producing an enzyme by culturing a mold using the fermented product produced by fermenting the food waste as a medium, accordingly, the product value is higher than the conventional method used by simply drying the food waste as a feed Since it is possible to produce excellent enzymes, it is economically useful, and furthermore, it is very useful for environmental industry because it can solve environmental problems such as secondary environmental pollution of soil and groundwater by leachate generated by simple landfill of food waste.

1 is a diagram showing the best growth strains by culturing various bacteria in food waste fermentation cake;

2 is a diagram showing the titer according to the culture pH of the enzyme produced according to the embodiment of the present invention;

3 is a diagram showing the titer of the enzyme according to the fermentation time of the food waste fermentation broth;

Figure 4 is a diagram showing the titer according to the fermentation fermentation temperature of the enzyme produced according to the embodiment of the present invention;

5 is a diagram showing the titers of enzymes according to various carbon sources added to the food waste fermentation cake;

6 is a diagram showing the titers of enzymes according to various nitrogen sources added to food waste fermentation cakes;

7 is a diagram showing the titers of enzymes according to various trace elements added to the food waste fermentation cake;

8 is a diagram showing the titers of enzymes according to various excipients added to food waste fermentation cakes;

9 is a diagram showing the activity of the enzyme according to the water content in the food waste fermentation cake.

Claims (11)

Food waste was selected from the group consisting of Bacillus stearotheromphilus TS-LDF 905 (KCCM-10310), Bacillus acidocaldarius TS-LDF SP2 (KCCM-10309), and mixtures thereof. Aspergillus niger , Aspergillus oryzae , Aspergillus awamori, Aspergillus awamori ) and a fungus selected from the group consisting of a fungus selected from the group consisting of amylase, cellulase, xylanase and mixtures thereof. delete The method of claim 1, Fermentation using bacteria is carried out at 50 to 65 ℃. delete The method of claim 1, Cultivation of the fungus is characterized in that the pH is carried out at 5.5 to 7.0, the temperature 25 to 30 ℃. delete The method of claim 1, To the medium further comprising at least one carbon source selected from the group consisting of molasses, glucose, sucrose, galactose, mannitol and maltose in an amount of 1 to 2% by weight. The method of claim 1, 1 to 2% by weight of one or more nitrogen sources selected from the group consisting of ammonium sulfate, yeast extract, peptone, soyton, tryptone, malt extract, casamino acid, corn steep liquor, monosodium glutamate fermentation by-product and nucleic acid It is characterized in that the addition further. The method of claim 1, The medium further comprises at least one excipient selected from the group consisting of soybean meal, rye, maize, cottonseed meal and wheat bran. The method of claim 1, The medium further comprises at least one trace element selected from the group consisting of zinc sulfate, calcium sulfate, ferric sulfate and magnesium chloride in an amount of 1 to 3 mM. The method according to any one of claims 1, 3, 5 and 7 to 10, characterized in that the water content in the medium is 45 to 65%.