KR100465829B1 - Production method of Bacterial cellulose using enzymatic saccharification solution of Food wastes - Google Patents

Abstract

The present invention relates to a method for producing bacterial cellulose using food waste enzyme saccharification liquid, and more specifically, to a fibrinolytic enzyme production step (ⓐ) in a bioreactor; A glycation solution production step (ⓑ) in the saccharification reactor in which food waste is injected into the fibrinolytic enzyme produced by the fibrinase production process and hydrolyzed in the saccharification reactor (ⓑ) to convert to reducing sugars; Bacterial cellulose production process (ⓒ) in which the saccharified liquid from which the saccharified residue was removed is cultured to produce bacterial cellulose in a bioreactor (ⓒ); It comprises a solidification process (ⓓ) for converting the saccharified residue into a functional material, can be mass-produced high value-added bacterial cellulose inexpensively using food waste enzyme saccharification liquid without the addition of other nutrients Is expected to be greatly converted, and the saccharified residue can be converted into a functional material through a solidification process, and the waste generated by using the culture residue generated in the production process of the bacterial cellulose (BC) as a nutrient source for the poor load and wastewater treatment plant is almost eliminated. Has no effect.

Description

Production method of Bacterial cellulose using enzymatic saccharification solution of Food wastes

The present invention relates to a method for producing bacterial cellulose using food waste enzyme saccharification liquid, and more specifically, fibrinase production process (ⓐ); Saccharification solution production process (ⓑ); Bacterial cellulose (BC) production process (©) for culturing to produce bacterial cellulose; Including the solidification process (ⓓ), it is possible to produce a large amount of high value-added bacterial cellulose (BC) inexpensively using food waste enzyme saccharification liquid without adding other nutrients. It is highly expected, and the saccharified residue can be converted into a functional material through the solidification process, and the culture residue generated in the bacterial cellulose (BC) production process is used as a nutrient source for poor load and wastewater treatment plant, so that there is little waste. It relates to a method for producing bacterial cellulose using food waste enzyme saccharification liquid.

In general, cellulose is one of the most abundant natural polymers on earth, and it is estimated that approximately 10 ¹¹ tonnes of cellulose are biosynthesized, and most of them synthesize cell wall fiber components of higher plants.

The molecules of cellulose are composed of 14,000 units of glucose units, and form a twisted rope bundle in which they are maintained by hydrogen bonds in chains or microfibers. Due to the rich amount of cellulose and other physical properties different from other natural polymers, a lot of research has been conducted for various industrial applications as renewable resources.

On the other hand, since the fact that acetic acid produced cellulose by AJ Brown in 1886 was reported, cellulose produced by microorganisms (Bacterial Cellulose, hereinafter referred to as BC) has been a subject of constant research as a new material. . 1 and 2 are SEM images of plant-derived cellulose and bacterial cellulose (BC), respectively, showing a large structural difference. That is, microbial-derived bacterial cellulose (BC) is composed of ribbon-like bundles, while plant-derived cellulose is composed of bundles of microfibrils.

Bacterial cellulose (BC) derived from microorganisms composed of the ribbon-like bundles does not contain any lignin or hemicellulose, unlike cellulose derived from plants composed of bundles of microfibrils. It is produced in a pure state, and has been developed for various uses in various industries because of its unique advantages such as high mechanical strength, high moisture retention, high crystallinity, and biodegradability.

For example, in Japan, the young's modules of bacterial cellulose (BC) are large, so they are used as vibrators for high-end audio headphones or speakers using the advantages of fast sound speed, excellent sound reproduction, and low noise. Because of its high density, long breaking length and high degree of polymerization, it is used as an additive of high-grade and high-strength paper.It has a three-dimensional network structure and is used for burn treatment because of its high water content. It is also used as a dietary fiber.

However, until now, the production of bacterial cellulose using commercial materials such as glucose has limited the cost of bacterial cellulose (BC) at about 100,000 yen per kilogram (100,000 Yen / kg). There was a problem and there was no report on the production of bacterial cellulose using enzyme saccharification of food waste.

The present invention has been made to solve the above problems, an object of the present invention is to produce a large amount of high value-added bacterial cellulose (BC) inexpensively using food waste enzyme saccharification without the addition of other nutrients. The industrialization is expected to be greatly expected, and the saccharified residue can be converted into a functional material through a solidification process, and the waste generated by using the culture residue generated in the bacterial cellulose (BC) production process as a nutrient source for poor load and wastewater treatment plants. The present invention provides a method for producing bacterial cellulose using almost no food waste enzyme saccharification solution.

In order to achieve the above object, the present invention is to crush rice waste straw, waste paper, and then pulverized liquor, rice straw, soybean crushed liquid culture or fiber waste using finely pulverized fiber waste waste, and then finely FPase, CMCase, xylanase, and β-glucosidase in a bioreactor (ⓐ) using a solid state culture vessel using ground fiber waste as a substrate. Fibrinase production process (ⓐ) in a bioreactor for producing fibrinase of avicelase, amylase; Fpiaase (FPase), CMCase (CMCase), xylanase, beta-glucosidase, β-glucosidase, avicelase, amylase produced by the fibrinase production process In the saccharification reactor converting the food waste, which has been selected and pulverized into the complex of fibrinolytic enzymes of 10 to 20% by weight based on the dry weight of the fibrinolytic enzyme, saccharifies food waste in the saccharification reactor (ⓑ) and converts it into monosaccharides and disaccharides. Saccharification solution production process (ⓑ); The saccharified liquid converted in the saccharified liquid production process is centrifuged to remove saccharified residue, and the saccharified liquid from which the saccharified residue is removed is cultured to produce bacterial cellulose in a bioreactor ⓒ (BC). ) Production process (ⓒ); The saccharified liquid generated in the saccharified liquid production process is centrifuged before being introduced into the bacterial cellulose production process, and comprises a solidification process (ⓓ) for converting the remaining saccharified residue after the centrifugation into a functional material. It is done.

1 is a SEM photograph of plant-derived cellulose,

2 is a SEM photograph of the bacterial cellulose (BC),

3 is a view schematically showing the entire production method of the present invention,

4 is a diagram showing the fibrinase productivity according to the mixing conditions of fiber waste in liquid culture,

FIG. 5 is a diagram showing the fibrinolytic production characteristics in a medium of 2% of rice straw and waste paper and 0.1% peptone, which is an optimal fiber waste waste combination (liquid culture),

Figure 6 is a view showing the fibrinase production characteristics in solid culture using fiber waste to increase enzyme productivity,

7 is a view showing the enzyme activity according to the mixing ratio of alcoholic beverage, rice straw, soybeans (solid culture),

8 is a diagram showing reducing sugar concentration with time in enzymatic hydrolysis of food waste,

9 is a view showing the amount of reducing sugar, glucose and bacterial cellulose (BC) with the incubation time in the production of bacterial cellulose (BC) using the enzyme saccharification solution.

※ Explanation of main parts of drawing ※

Ⓐ: Bioreactor (enzyme production process)

Ⓑ: saccharification reactor (glycosylation process)

Ⓒ: Bioreactor (Bacterial Cellulose (BC) Production Process)

Ⓓ: solidification process

①: Selection process ②: Grinding process

③: Steam boiler ④: Centrifugal separation process

⑤: Filtration process ⑥: Cleaning process

⑦: pH adjusting device ⑧: Fresh water supply device

Hereinafter, with reference to the accompanying drawings will be described in more detail the production method according to the present invention.

Figure 3 is a schematic representation of the overall method of the present invention.

As shown, the production method of the present invention is generally a fibrinolytic production process (ⓐ) in a bioreactor; Saccharification liquid production process (ⓑ) in the saccharification reactor in which food waste is injected and hydrolyzed in the saccharification reactor (ⓑ) to be converted into reducing sugars; Bacterial cellulose (BC) production process (©) in which the saccharified liquid from which the saccharified residue was removed is cultured to produce bacterial cellulose in a bioreactor (ⓒ); It comprises a solidification process (ⓓ) for converting the saccharified residue into a functional material.

Hereinafter, look at in detail.

Ⓐ process for producing fibrinase in bioreactor (ⓐ)

The process of producing fibrinase (FPase, CMCase, xylanase, Avicelase, β-glucosidase, amylase) in a bioreactor (ⓐ) using pulverized fiber waste, which is a raw material, It takes about 5 days.

The fibrinase production process may be made of process ㉮ or process 과 as shown in Table 1 below, depending on the enzyme production (culture form and substrate used).

-Process ㉮: Fibrinase production process through liquid culture

-Process ㉯: Fibrinase production process through solid state culture

Enzyme productivity according to each enzyme production Use strain Culture type Substrate Enzyme Concentration (U / ml) Trichoderma sp. Liquid Culture Process ㉮ Rice straw, waste paper FPase: 0.25-1.0 Amylase: 1.0-5.0 Solid State Culture Process Berth, rice straw, bean curd FPase: 0.2 or more Amylase: above 1.0

Ⓑ Saccharification liquid production process in the saccharification reactor

It is a process of hydrolyzing food waste into reducing sugars (monosaccharides and disaccharides) in the saccharification reactor (ⓑ) with fibrinase produced by the fibrinase production process of ⓐ, and the residence time is about 1 day. .

Food waste to be added to the saccharification reactor (ⓑ) is a food waste that is already removed and finely broken food waste prior to addition by going through the sorting and grinding process described below, food waste introduced into the saccharification process (water content: 80 to 85% by weight) Is injected into the dry weight of 10 to 20% by weight.

The enzyme concentrations of FPase and amylase required for the saccharification solution production process are FPase (0.25U / ml) and amylase (1.0U / ml). This bioreactor (ⓐ) is introduced into the saccharification reactor (ⓑ) is a saccharification liquid production process. However, when the concentration of the enzyme solution produced in the enzyme production process is higher than the concentration required in the saccharification solution production process, it is possible to dilute the enzyme solution.

Ⓒ Bacterial Cellulose Production Process

The saccharified liquid converted in the saccharified liquid production process is centrifuged to remove saccharified residue, and the saccharified liquid from which the saccharified residue is removed is a biological process for producing bacterial cellulose (BC) in a bioreactor (ⓒ), The residence time is about 3 to 5 days depending on the culture type. (The BC production time in political culture is about 5 days, BC production time in stirred culture is about 3 days.) Bioreactor in this process (Ⓒ) is separate from the bioreactor of ⓐ for enzyme solution production.

Bacterial cellulose (BC) produced in the step ⓒ can not be used immediately, but must be subjected to the filtration and cleaning process described later.

Ⓓ solidification process

The saccharified liquid generated in the saccharified liquid production process is centrifuged before being introduced into the ⓒ bacterial cellulose production process, and the remaining saccharified residue after the centrifugal separation is separated into functional material (humidant, microbial carrier, environmental biotope planting material). ) Process.

Hereinafter, an additional apparatus and process for performing the above process will be described in detail.

① Sorting process: The sorting process indicated by ① in FIG. 3 is a process for protecting the mechanism of the grinding process described later by removing metals that may be mixed in food waste in advance, and basically removing metals containing iron by a magnet or the like. Of course, metals that do not contain iron are removed as much as possible using weight differences and density differences.

② Grinding: The grinding process indicated by ② in Fig. 3 is a source of cellulose enzyme production medium according to the size of the food waste required for the grinding of the fiber waste (straw straw, waste paper, ethanol, soybeans) and enzyme saccharification liquid production. It is a fine grinding process.

③ Steam boiler: Steam boiler shown in ③ in FIG. 3 is supplied with a high temperature steam to the bioreactor (ⓐ) and the bioreactor (ⓒ) of bacterial cellulose (BC) production for fibrinase production, medium It is a device for preventing the contamination of other microorganisms through sterilization.

④ Centrifugation: The centrifugation process indicated by ④ in FIG. 3 is a process for separating the solids contained in the saccharification solution produced from the saccharification reactor (ⓑ), in addition to increasing the reaction efficiency in the bioreactor (ⓒ). It is also necessary for the purpose of developing a functional material.

⑤ Filtration: Filtration process indicated by ⑤ in Figure 3 is a process for filtering the culture solution to recover the bacterial cellulose (BC) produced in the bioreactor (ⓒ).

⑥ Cleaning: The cleaning process indicated by ⑥ in FIG. 3 is a process of cleaning the bacterial cellulose (BC) filtered during the filtration process of ⑤.

⑦ pH control device: pH control device shown in ⑦ in Figure 3 is a device for adjusting the pH of the enzyme production, saccharification solution production process and bacterial cellulose (BC) production process.

⑧ Fresh water supply device: Fresh water supply device indicated by ⑧ in Fig. 3 is a fresh water in the unit operation of the bioreactor (ⓐ), saccharification reactor (ⓑ), bioreactor (ⓒ), steam boiler (③) and washing process (⑥) It is a device for supplying.

Devices and processes such as the pH control device, fresh water supply device, steam boiler, etc. are pH, base mass, reaction temperature, stirring conditions, enzyme concentration in the efficiency of enzyme production, saccharification solution production process and bacterial cellulose (BC) production process. Is an important decision and is part of the device and process for enhancing this efficiency (see Examples based on Trichoderma strains described below).

Hereinafter, with reference to the accompanying drawings will be described in more detail the present invention through an embodiment of examining the production method of the present invention Trichoderma strain (Trichoderma sp.). However, the following examples should not be construed as limiting the scope of the present invention, and ordinary changes may be made by those skilled in the art within the scope of the present invention.

The Tricorderma strain can be easily purchased by those skilled in the art through the Korea Biotechnology Research Institute Genetic Center Genetic Bank (KCTC), located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea. Korean Culture Center of Microorganisms, KCCM, established in August 1989 as an affiliated organization of the Korean Federation of Culture Collections (KFCC), established by non-profit corporations. /www.kccm.or.kr) can be obtained easily.

The Gene Bank (Korean Collection for Type Cultures, KCTC) was officially approved by the government in 1985 and is a standard microbial strain. Animal and plant cell lines. Collect and preserve gene recombination resources such as gene libraries systematically and continuously from home and abroad, and distribute them to the general public, and directly produce chemical taxonomic information, phylogenetic information, molecular biological information, etc., and classify new useful resources. It is used for scientific research and exploration, is open to the public through databases, and it operates a bank system for efficient management of international strain depositing institutions for microbial patents. It is provided on the Internet through //kctc.kribb.re.kr, it is also possible to apply for strains through the computer network.

Example 1

Production of Fibrinase Using Trichoderma Strains

(1) enzyme activity analysis

① CMCase

CMCase enzyme activity was measured by reacting a reaction mixture of 0.5 ml of 2 wt% CMC (Sodium Carboxy methyl Cellulose; Aldrich, USA) solution with 0.5 ml of an enzyme solution appropriately diluted with a buffer at 50 ° C for 30 minutes. The reducing sugar produced by the measurement was measured by the DNS method. 50 mM citric acid (pH 4.8) was used as a buffer used to prepare a 2 wt% CMC solution and dilute the enzyme solution. Enzyme activity was defined as 1 unit of enzyme required to produce reducing sugars corresponding to 1 μmol min −1 glucose under standard reaction conditions.

② Xylanase

Xylanase enzyme activity was measured by mixing 0.5 ml of 2% by weight of Xylan (Sigma, USA) solution with 0.5 ml of an appropriately diluted enzyme solution and reacting the reactants at 50 ° C for 30 minutes. Measured. 2% by weight of xylan solution was dissolved in 50 mM citric acid (pH 4.8) by stirring for 24 hours, and then the solution was centrifuged (7,000 rpm, 30 minutes) to use a supernatant. Enzyme activity was defined as 1 unit of the amount of enzyme required to produce a reducing sugar corresponding to 1 μmol min-1 xylose under standard reaction conditions.

③ beta-glucosidase (β-glucosidase)

Beta-glucosidase enzyme activity was measured in 1.8 ml of 0.1 M sodium acetate buffer (pH 4.8) and 1 ml of 5 mM PNPG ( p- nitrophenyl-β-D-glucoside; Sigma, USA) buffer. (0.2 ml of enzyme solution diluted appropriately with a buffer) were mixed and reacted for 30 minutes at 50 ° C. Then, 4 ml of 0.4M glycine buffer (pH 10.8) was added to terminate the reaction. The resulting р- nitrophenol was quantitated at 430 nm. It was. β-glucosidase 1unit was defined as the amount of enzyme required to produce 1μmol min-1 of р -nitrophenol under the above reaction conditions.

④ Avicelase

Avicelase activity was measured by reacting a 1% by weight of Avicel (Merck, Germany) with 1 ml of an appropriately diluted enzyme solution in buffer and 1 ml of enzyme solution at 50 ° C for 120 minutes to measure reducing sugars using the Somogyi-Nelson method. It was. Enzyme activity was defined as 1 unit of the amount of enzyme required to produce a reducing sugar corresponding to 1μmol min-1 cellobiose under standard reaction conditions.

⑤ FPase

Filter paper activity was expressed as FPase, which was produced by mixing 50 mg (1 × 5 cm) of filter paper (Watman No. 1) with 1.0 ml of enzyme solution diluted appropriately with buffer and reacting at 50 ° C for 60 minutes. The reduced sugar was measured by the DNS method. The amount of enzyme required to produce a reducing sugar corresponding to 1 μmol min −1 glucose was defined as 1 unit.

⑥ Total amylase

Amylase enzyme activity was measured by DNS method. In other words, first add 0.5 ml of enzyme solution properly diluted with buffer (50mM citric acid, pH 5.0) to the test tube, add 0.5 ml of 2% soluble starch solution in a 50 ℃ constant temperature water bath, and react for 30 minutes, and then add 3 ml of DNS reagent. The reaction was terminated. The reaction was heated in boiling water for 5 minutes, 20 ml of distilled water was added, and the absorbance was measured at 540 nm. Glucose was used as a standard, and the enzyme activity was defined as 1 uint of the enzyme producing 1 μmol of reducing sugar for 1 minute under the above reaction conditions.

(2) Enzyme Production Characteristics of Trichoderma sp.

① Culture condition

Yeast Malt Extract Agar (YMEA; malt extract 10.0g, yeast extract 4.0g, glucose 4.0g, agar 15g, distilled water 1.0L) was used as a medium for preservation and spore production. The cells incubated for 5 days were collected by scraping with platinum and suspended in sterile water, and then filtered by cotten wool plug.

Mandel's medium was used as a basic medium for enzyme production, and the medium composition is shown in Table 2 below. In the experiment of enzyme production, 100 ml medium was used in 500 ml flask, and 2% inoculation of spore suspension was incubated at 30 ° C., 100 rpm, for 5 days. Enzyme solution was centrifuged (10,000 rpm, 10 minutes) to the supernatant was used.

Composition of Mandel's Badge Trace element solution Avicel 5.0 g FeSO ₄ 7H ₂ O 5.0 g CMC 5.0 g ZnSO ₄ · 7H ₂ O 1.4 g KH ₂ PO ₄ 2.0 g MnSO ₄ 7H ₂ O 1.6 g (NH ₄ ) ₂ SO ₄ 1.4 g CoCl ₂ 2.0 g Eptone 1.0 g Distilled water 1.0ℓ MgSO _{4 7} H ₂ O 0.3 g CaCl ₂ · 7H ₂ O 0.3 g Urea 0.3 g Trace element solution 1.0 ml Distilled water 1.0ℓ

② Comparison of enzyme productivity

Optimal culture conditions for the enzyme production of Trichoderma sp. (Trichoderma sp.) Was 25-30 ℃, 100rpm, pH 6.0-7.0, incubation time 5 days. Fibrinase productivity in Mandel's medium and in modified Mandel's medium is shown in Table 3 below.

Enzyme Production of Trichoderma Strains in Modified Mendel Medium badge Culture condition Enzyme Activity (U / ml) Mandel's Badge Incubation temperature: 30 ℃ Initial pH: 6.0 Stirring condition: 100rpm, Culture time: 5 days CMCase: 9.1Xylanase (4 days): 12.6β-glucosidase: 0.54 Avicelase: 0.21 Modified Mandel's Medium (Avicel + CMC: 2.5% Peptone: 0.5) Incubation temperature: 30 ℃ Initial pH: 6.0 Stirring condition: 100rpm, Culture time: 7 days CMCase: 41.2 Xylanase: 65.6β-glucosidase: 2.25 Avicelase: 4.72

(3) Production of Fibrinolytic Enzyme Using Fibrin Waste in Liquid Culture

① Culture condition

The enzyme production of the enzyme production characteristics of (2) Trichoderma sp. (Trichoderma sp.) Was examined by the method of culturing conditions, and the experiment was performed by replacing Avicel and CMC, which are carbon sources of Mandel's medium, with fibrin waste. The fiber waste used here is rice straw (Chunnam Jangseong; Rice Straw (RS)), sawdust (oak, Gwangju Mudeungsan; Sawdust (SD)), waste paper (box waste) (Paper Waste (PW)), which is 24 hours at 80 ℃. After drying, it was ground to 30mesh.

② Enzyme productivity review using fiber waste

Figure 4 is a diagram showing the fibrinolytic enzyme productivity according to the mixing conditions of the fiber waste in the liquid culture, Figure 5 is a fibrinolysis in the medium of 2% of rice straw and waste paper and 0.1% peptone of the optimal fiber waste combination It is a figure which shows the enzyme production characteristic (liquid culture). In Figure 4, MS means Mixture Substrate (straw straw + sawdust + waste paper).

As shown in the drawing, enzyme productivity was compared at a mixed concentration of 1% in combination with rice straw and sawdust, rice straw and waste paper, sawdust and waste paper, rice straw and sawdust and waste paper to produce fibrinolytic enzymes. In the experiment for enzyme production using fiber waste, a medium using 100 ml of fiber waste was used in a 500 ml flask, and 2% inoculation of spore suspension was incubated at 30 ° C., 100 rpm, for 5 days. As a result, the activity of fibrinase was high when rice straw and waste paper were used. At this time, the CMCase (CMCase), xylanase (xylanase), beta-glucosidase (β-glucosidase), avicelase (Avicelase) were 24.3, 38.7, 1.5, 0.6 U / ml, respectively (see Figure 4). Enzyme production characteristics in the media of 2% of rice straw and waste paper and 0.1% peptone, which is the optimal fiber waste combination, were obtained up to 0.75 U / ml on the fifth day of culture of FPase (see FIG. 5).

(4) Production of fibrinolytic enzymes using fiber waste in solid state culture

① Culture condition

Fibrous wastes used for solid state culture were made from alcoholic beverages (Bohae Brewery, Jeonnam Suncheon; Wine Waste, WW), rice straw (Chunnam Jangseong; Rice Straw, RS), sawdust (oak, Gwangju Mudeungsan; Sawdust; SD), waste paper (boxes). Paper Waste; PW), Soybean Flour (Nature and People, Jeonnam Damyang; Soybean Flour, SF) mesh).

Solid state culture for enzyme production was put into a 100ml Erlenmeyer flask with the appropriate amount of each substrate, followed by sterilization (121 ℃, 15min) was used in the experiment. Culture conditions were sterilized (121 ℃, 15 minutes) of the fibrous waste, and then added to the appropriate amount of 50mM citric acid buffer (pH 5.0) in order to adjust to 70% water content, the cells grown in YMEA medium to the above buffer solution 1 ml of the spore suspension prepared by suspension was inoculated and incubated at 30 ° C. for 5 days.

After the incubation, the enzyme solution was extracted, and 10 ml of buffer solution was added per 1 g of the substrate, stirred at 30 ° C. and 100 rpm for 1 hour, followed by centrifugation (10,000 rpm, 10 minutes) to prepare the supernatant as the enzyme solution.

② Enzyme Activity Analysis

Previously, (1) enzyme activity analysis was carried out according to the method of ⑤ FPase and ⑥ amylase, and the enzyme activity was divided by total dry enzyme (SDW, substrate dry weight) and FPase (FPase) ( FPA / g-SDW) and amylase (U / g-SDW).

③ Production of fibrinase

Fibrinase production is dependent on the use of carbon sources in the culture medium. Fibrinase productivity through solid-state culture was investigated using alcohol, rice straw, sawdust, waste paper, soybean waste, and food waste as substrates. In this experiment, the water content was selected as 70% (w / w), and then the substrate was investigated for the enzyme production through solid state culture through 5 days culture in 100ml Erlenmeyer flask. 50 mM citric acid buffer (pH 5.0) was used to control the water content and no other nutrients were added. As a result of measuring the fibrinase productivity after 5 days incubation, 1.1 FPA / g-SDW showed the highest enzyme production when the berth was used as a substrate. This suggests that berth is a suitable carbon source for solid state culture.

Figure 6 is a view showing the fibrinase production characteristics in solid culture using fiber waste to increase enzyme productivity. Experiments were carried out by mixing berths and other fibrous wastes to increase enzyme productivity. When each substrate is used alone, the components contained in the substrate may not be suitable for the growth of the strain and the enzyme production. Each fiber waste was incubated for 5 days on the basis of the best marinated enzyme. As a result, 1g of ethanol and rice straw were mixed and cultured, and the production of fibrinase was about 13 times higher than that of ethanol alone. The enzyme activity was 14.0 FPA / g- SDW (see FIG. 6).

7 is a view showing the enzyme activity according to the mixing ratio of ethanol, rice straw, soybeans (solid culture). The production of fibrinase was insignificant, but the soybeans, which had a favorable effect on cell growth, were mixed and cultivated in marinated rice and straw. As a result, the higher the mixing ratio of soybeans, the higher the enzyme production was confirmed. When marinated, rice straw and soybeans were mixed at a ratio of 1: 1: 1, the enzyme activity was 16.7 FPA / g-SDW (see FIG. 7).

Example 2

Enzymatic Saccharification of Food Waste

① Characteristics of Enzyme Produced by Trichoderma sp.

Optimum temperature, pH, and thermal stability were examined to characterize the enzymes produced by Trichoderma sp. Optimal temperature, pH and thermal stability of FPase, CMCase, Avicelase, Xylanase, β-glucosidase, amylase It is shown in Table 4.

Enzyme Properties Produced by Trichoderma Strains enzyme Optimum temperature (℃) Optimal pH Thermal safety FPase 60 5.0 95% CMCase 60 5.0 80% Xylanase 50 5.0 20% β-glucosidase 70 5.0 20% Avicelase 60 5.0 60% Amylase 60 4.5 90%

* Thermal stability expressed residual enzyme activity after 2 days at 50 ℃

* All enzymes had 100% residual enzyme activity after 2 days at 40 ℃

② Enzymatic hydrolysis of food waste

Enzymatic hydrolysis of food waste was performed by varying the food waste concentration by 5-20% (w / v) using a solid or liquid culture enzyme solution. Food waste (Chonnam National University cafeteria) was used after being dried at 80 ℃ for 24 hours and crushed into 30mesh. Each concentration was added to 50 ml of enzyme solution and 50 ml of 50 mM citric acid (pH 5.0), and the reducing sugars produced by reaction at 50 ° C. and 100 rpm were quantified.

8 is a view showing the reducing sugar concentration over time in the enzymatic hydrolysis of food waste. Enzymatic saccharification was greatly influenced by enzyme concentration, base mass, reaction temperature, pH, and agitation conditions.FFPase (0.25 U / ml) and amylase (20% (w / v)) of dried food waste amylase) (1.0 U / ml), at 67 ° C, 100 rpm, pH 5.0, 67g / L of reducing sugar was obtained at 24 hours, and 73g / L of reducing sugar was obtained at 48 hours.

Example 3

Production of Bacterial Cellulose (BC) Using Food Waste Saccharification Solution

(1) Characteristics of Acetobacter xylinum used to produce bacterial cellulose (BC)

① Bacterial Cellulose (BC) Analysis

The resulting bacterial cellulose membrane (BC pellicle) was centrifuged at 10,000 rpm for 30 minutes, then the supernatant was discarded and only the bacterial cellulose membrane (BC pellicle) was placed in 0.1 N sodium hydroxide (NaOH) solution for 20-30 minutes at 80 ° C. The cells were treated to dissolve the cells, and then filtered through a filter paper. Subsequently, the mixture was washed several times with distilled water to neutralize the bacterial cellulose (BC), followed by drying at 80 ° C. for 8 hours, and allowed to cool for 30 minutes in a tessitizer to measure the dry weight to determine the amount of the bacterial cellulose (BC).

② BC production in optimized GFC medium

The pre-culture for the production of bacterial cellulose (BC) was inoculated with 1% of cells in 100 ml of HS medium (see Table 5) in a 500 ml Erlenmeyer flask and used as an inoculum after incubation for 3 days. Stationary culture and stirred culture for BC production was used 100ml HS medium or 100ml GFC medium for 100ml BC production in 500ml slant baffled flask, incubated for 5 days at 30 ℃ by inoculating 1% pre-cultured strain culture. Condition of the stirring culture was adjusted to 150rpm stirring speed. The GFC medium composition is shown in Table 6 below.

BC production pre-culture in the optimal operation of the bioreactor was incubated for 3 days by inoculating cells into a 500 ml flask containing 100 ml of HS medium. In this culture, 5L of medium was added to a 10L jar fermentor (BioG, Hanil R & D Co., Korea), and 1% of the supernatant obtained from the stationary culture was inoculated with the inoculum, followed by incubation at 30 ° C for 80 hours. Culture conditions were adjusted to the carbon source concentration of 4%, CSL concentration of 6% in the above GFC medium, oxygen supply was 0.28atm (0.3vvm), agitation rate was adjusted to 460rpm. PH control of the culture was adjusted to 5.0 using 2N NaOH / HCl.

Table 7 below shows the results of examining the BC productivity using acetobacter xylinum (Acetobactor xylinum). In the culture culture, when the HS was used, 3.2 g / L of BC was produced, and in the stirred culture, the modified GFC medium produced 7.2 g / L of BC. Under optimum culture conditions of the bioreactor, 11.7 g / L of BC was produced.

Composition of HS (Hestrin & Schramm) Medium Glucose 20 g Bacto peptone 5 g Yeast extract 5 g Na ₂ HPO ₄ 2.7 g Citric acid monohydrate 1.15 g Distilled water 1.0L pH 5.25

Composition of GFC medium for BC production Glucose 5 g Fructose 15 g Corn steep liquor (CSL) 20 g Na ₂ HPO ₄ 2.7 g Citric acid monohydrate 1.15 g Distilled water 1.0L pH 5.25

BC productivity review using Acetobacter xylinum Culture Used badge BC concentration (g / L) BC yield (g / g) flask Political Culture HS medium (Table 5) 3.2 0.16 Stirring Culture GFC Badge (Table 6) 7.2 0.36 Bioreactor Modified GFC medium (carbon source: 4%, CSL: 6%) 11.7 0.29

(2) BC production using food waste enzyme saccharification liquid

① Types and Conditions of Used Food Waste

The enzymes used for enzymatic glycosylation were FPase, CMCase, xylanase, beta-glucosidase, avicelase and amylase, respectively. ) Were 0.38, 10.04, 17.92, 0.32, 0.06, 2.0 U / ml.

Food wastes were collected by source as shown in Table 8 below. The collected food waste was dehydrated in the liver under natural conditions, and then ground and used directly in a wet state, or after drying at 80 ° C. for 2 days in a drier.

Enzymatic saccharification conditions were adjusted to pH 5.0, 50 ℃, 100rpm, was performed for 2 days.

Samples of food waste (wet moisture content is about 80-85%) Sampling site Mark Wet state (W) Dry state (D) Apartment area WA DA Chonnam National University Cafeteria WB DB Restaurant WC DC Food Waste Composting Office DD

* Wet state moisture content is about 80-85%

② BC production method using enzyme saccharification

-BC production in flask culture

Dried food waste (DB) of Chonnam National University cafeteria was subjected to enzymatic saccharification to obtain 22 g / L of reducing sugar.

Dispense 100 ml of saccharification solution into a 500 ml Erlenmeyer flask and adjust the pH to 5.25 using 2.0 N HCl / NaOH. The medium is sterilized (121 ° C., 15 min) and inoculated with the strain 1%. BC concentration was quantified after 5 days of culture. In order to compare BC productivity of enzyme saccharification liquid, HS medium or other nutrients were added and examined.

As shown in Table 9 below, when only the saccharified solution was used, most species showed BC productivity as 9.3 g / L in political culture. In addition, static culture shows higher BC productivity than stirred culture.

Comparison of BC Productivity Using Food Waste Enzyme Saccharification Solution (Flask Culture) Badge type Flask culture Political Culture Stirring Culture (150rpm) Saccharification 9.3 3.6 Saccharified solution + HS medium (excluding glucose) 8.4 2.9 Saccharified solution + Peptone (0.1%) 7.0 3.6 Saccharified solution + Corn steep Liquor (0.1%) 8.0 7.0 Saccharified solution + Yeast extract (0.1%) 9.2 4.0

-BC production by food waste origin

The collected food waste was dried to examine BC productivity according to the source. Enzymatic saccharification and BC production were performed according to the method of ① and ② above.

Looking at BC productivity by food waste sources, DB and DC produced 8.0 and 7.9 g / L of BC, and DA produced 4.8 g / L of BC (see Table 10). DD's low BC productivity is the place where food waste is collected in Gwangju, which is transported, stored, and composted. Therefore, it is dried and mixed with food waste that has been left for a few days instead of fresh food. Because Therefore, BC productivity should be reviewed for food waste or elapsed time.

Comparison of BC Productivity by Food Waste Source (Plask Culture) By occurrence Reducing Sugar Concentration (g / L) BC concentration (g / L) BC yield (g / g) Apartment (DA) 18.5 4.8 0.26 Chonnam National University Cafeteria (DB) 36.1 8.0 0.22 Restaurant (DC) 37.1 7.9 0.21 Food Waste Composting Office (DD) 16.5 3.3 0.20

-BC production according to food waste

BC productivity was examined using wet and dry food waste and enzyme saccharification solution of food waste after several days of standing. Enzymatic saccharification and BC production were performed according to the method of ① and ② above.

Table 11 shows the results of examining the BC productivity according to the treatment status of food waste at Chonnam National University cafeteria and general restaurant. BC productivity differences between dry and wet food waste are shown. Looking at BC production of food waste at Chonnam National University cafeteria, DB (8.0g / L) produced BC higher than WB (6.1g / L). However, the BC production of WC (11.8 g / L) and DC (7.9 g / L) was higher in WC than in restaurants.

When the food wastes were left in the natural state for 3 days, the production of reducing sugars was reduced and BC production was also reduced. It is believed that anaerobic metabolites inhibited glycation and BC production due to the decay of food wastes caused by the growth of anaerobic microorganisms during natural neglect.

Comparison of BC Productivity by Food Waste Status (Plask Culture) Chonnam National University Cafeteria Restaurant WB 3 days left DB WC 3 days left DC Reducing sugar concentration (g / L) 26.0 17.4 36.1 33.4 25.6 37.1 BC concentration (g / L) 6.1 4.9 8.0 11.8 5.0 7.9 BC yield (g / g) 0.24 0.28 0.22 0.35 0.20 0.21

④ BC production using bioreactor

9 is a view showing the amount of reducing sugar, glucose and bacterial cellulose (BC) with the incubation time in the production of bacterial cellulose (BC) using the enzyme saccharification solution. The preculture was incubated for 3 days by inoculating cells into a 500 ml flask containing 100 ml of HS medium. This culture was inoculated with 5L of food waste saccharification solution in 10L jar fermentor (BioG, Hanil R & D Co., Korea) and 1% of the supernatant obtained from the preculture and incubated at 30 ° C for 80 hours. Optimum culture conditions were adjusted to oxygen partial pressure (air volume) 0.28atm (0.3vvm), stirring speed 460rpm, pH 5.0. As a result of the culture, BC production of 7.1 g / L was obtained at 80 hours (see FIG. 9).

Example 4

Development of functional material by solidifying food waste saccharification residue

Food waste, which has undergone enzymatic saccharification, has a large specific surface area since it is decomposed into a fibrous structure of the polymer and is changed into a structure having a large number of pores. It is structurally stable for a long time because only a stable substance remains difficult for biodegradation to proceed any further. Due to these physical properties, it is excellent in hygroscopicity and adsorption, so it is a moisturizer and microbial carrier. Conversion to biotope materials is possible. It is also possible to convert fertilizers or compost by adding other nutrients.

As described above, the present invention can produce a large amount of high value-added bacterial cellulose (BC) at low cost by using food waste enzyme saccharification liquid without adding other nutrients, and industrialization is expected to be greatly expected, and the solidification process of saccharified residues is expected. It can be converted into a functional material through, and by using the culture balance generated in the production process of the bacterial cellulose (BC) as a nutrient source of poor load, wastewater treatment plant has the effect of generating little waste.

Specifically,

First, in the United States and Japan, raw material costs are high due to the production of BC using commercial materials (glucose, etc.).

Second, since the saccharified residue is converted to functional material through solidification process, and the culture residue generated in BC production process is used as a nutrient source for poor load and wastewater treatment plant, there is almost no waste generation in the process.

Third, BC production using saccharified liquid is expected to be industrialized as the level produced by the existing optimized complex medium is achieved even if BC is produced without the addition of other nursing homes.

Fourth, food waste treatment methods include incineration, feed, and composting, which have been converted to low-value products (food, compost, methane production, etc.) through large energy costs. However, this conversion process can produce high value products (1 million won / kg-BC) such as BC,

Fifth, because the enzyme used for enzyme saccharification is also produced using waste fiber, the cost of enzyme production is lower than that of other commercial substrates. Enzymatic saccharification is more corrosive, higher energy cost, and neutralizing agent than acid hydrolysis. The cost of such chemicals has a very low effect.

Claims (3)

Crushed rice straw, waste paper, waste paper, and then crushed liquid culture or ethanol, rice straw, bean waste, which are finely crushed fiber waste, and then crushed fiber waste. Using state culture in the bioreactor (ⓐ) Fpase (FPase), CMCase (CMCase), xylanase (beta) -glucosidase (β-glucosidase), avicelase (Avicelase), amylase ( fibrinase production process (ⓐ) in a bioreactor producing fibrinase of amylase); Fpiaase (FPase), CMCase (CMCase), xylanase, beta-glucosidase, β-glucosidase, avicelase, amylase produced by the fibrinase production process In the saccharification reactor converting the food waste, which has been selected and pulverized into the complex of fibrinolytic enzymes of 10 to 20% by weight based on the dry weight of the fibrinolytic enzyme, saccharifies food waste in the saccharification reactor (ⓑ) and converts it into monosaccharides and disaccharides. Saccharification solution production process (ⓑ); The saccharified liquid converted in the saccharified liquid production process is centrifuged to remove saccharified residue, and the saccharified liquid from which the saccharified residue is removed is cultured to produce bacterial cellulose in a bioreactor ⓒ (BC). ) Production process (ⓒ); The saccharified liquid generated in the saccharified liquid production process is centrifuged before being introduced into the bacterial cellulose production process, and comprises a solidification process (ⓓ) for converting the remaining saccharified residue after the centrifugation into a functional material. Method for producing bacterial cellulose using food waste enzyme saccharification liquid. delete delete