KR20030030575A - A Method for Lactic Acid Production from Food Refuses - Google Patents

A Method for Lactic Acid Production from Food Refuses Download PDF

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KR20030030575A
KR20030030575A KR1020010062766A KR20010062766A KR20030030575A KR 20030030575 A KR20030030575 A KR 20030030575A KR 1020010062766 A KR1020010062766 A KR 1020010062766A KR 20010062766 A KR20010062766 A KR 20010062766A KR 20030030575 A KR20030030575 A KR 20030030575A
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lactic acid
food waste
fermentation
food
acid production
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윤현희
김광일
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract

PURPOSE: Provided is a production method of lactic acid from food wastes, particularly by enzyme hydrolysis and fermentation, thereby increasing the productivity of lactic acid. CONSTITUTION: The production method of lactic acid from food wastes is characterized by hydrolyzing food wastes to convert it into glucose and converting the glucose into lactic acid by using microorganisms, particularly lactobacillus sp. microorganisms. 80g of lactic acid is manufactured from 100g food wastes within 48 hours, and its productivity is 2g/L*h, higher than conventional lactic acid production processes.

Description

음식물쓰레기를 이용한 젖산 제조방법{A Method for Lactic Acid Production from Food Refuses}A Method for Lactic Acid Production from Food Refuses

본 발명은 음식물쓰레기를 이용하여 젖산을 생산하는 공정에 있어서, 효소가수분해 및 발효방법을 이용하여 젖산의 생산성을 향상시키는 방법에 관한 것이다.The present invention relates to a method for improving the productivity of lactic acid by using enzymatic hydrolysis and fermentation in the process of producing lactic acid using food waste.

젖산(2-hydroxypropanoic acid)은 식품, 의약품, 및 각종 유기화합물의 주부원료로 광범위하게 사용되고 있으며 특히 최근에는 생분해성 및 생체활성 고분자 등의 원료로 그 수요가 증대되고 있다. 젖산의 제조는 미생물발효 혹은 화학적 합성방법을 사용하고 있으며 현재 전체 생산의 절반 이상이 미생물발효방법으로 제조되고 있다. 미생물발효방법은 매년 재생산되는 바이오매스를 원료로 사용한다는 점과 화학적 합성법과는 달리 공해문제가 적은 청정기술이라는 장점을 갖고 있다. 현재까지 젖산 생산의 주원료는 유당 혹은 전분 유래의 포도당이며 이것들은 식품으로 사용될 수 있기 때문에 가격이 비싸고 결과적으로 젖산의 생산원가를 높이는 원인이 되고 있다. 따라서 최근에 젖산 생산의 대체원료로 음식물쓰레기의 활용에 관한 연구가 수행되고 있다. 예를 들면, 1999년 로(Loh) 등은 부엌에서 발생하는 쓰레기를 이용하여 유기산(젖산, 초산 등의 혼합물)을 생산하였으며 회분식 반응기에서 수율 84%를 얻을 수 있다고 보고하였다(참조, C.W. Loh, A. Fakhrul-Razi, M. A. Hassan, and M.I.A. Karimd, Production of organic acid from kitchen wastes,Artf. Cells, Blood Substit., and Immobil.Biotechnol., 27, 455-459, 1997). 이러한 방법은 음식물쓰레기로부터 직접 유기산으로 전환하는 방법으로서 발효시간이 약 4일 정도 소요되어 발효속도가 매우 느리다는 문제점을 갖고 있다. 아울러, 생성된 유기산이 여러가지 성분을 함유하고 있기 때문에 젖산을 정제하는데 있어서 많은 지장을 초래한다. 한편, 1996년 차우(Zhou) 등이 보고한 방법에서는 음식물쓰레기를 포함한 각종 생활쓰레기를 황산을 이용하여 가수분해하고 얻어진 가수분해산물을 중화시킨 후 젖산발효를 수행하였다. 이 방법은 황산 및 중화제 사용에 따른 공정상의 복잡성과 공해물질이 배출되는 문제점을 갖고있다(참조: S.D. Zhou, T.A, McCaskey, and J. Broder, Evaluation of nirogen supplements for bioconversion of municipal solid waste to lactic acid,Appl. Biochem. and Biotechnol.,57/58, 517-524, 1996).Lactic acid (2-hydroxypropanoic acid) is widely used as a housewife raw material for food, medicine, and various organic compounds, and in recent years, the demand for biodegradable and bioactive polymers is increasing. Lactic acid is produced by microbial fermentation or chemical synthesis, and more than half of the total production is produced by microbial fermentation. Microbial fermentation method has the advantage of using biomass that is reproduced every year as raw material and clean technology with less pollution problem unlike chemical synthesis method. To date, the main raw material for lactic acid production is glucose derived from lactose or starch, and since these can be used as foods, they are expensive and consequently increase the production cost of lactic acid. Therefore, recently, research on the utilization of food waste as an alternative raw material for lactic acid production has been conducted. For example, in 1999, Loh et al. Reported that organic wastes (mixtures of lactic acid, acetic acid, etc.) were produced from kitchen waste and yields of 84% were obtained in batch reactors (cf. CW Loh, A. Fakhrul-Razi, MA Hassan, and MIA Karimd, Production of organic acid from kitchen wastes, Artf.Cells, Blood Substit., And Immobil.Biotechnol . , 27, 455-459, 1997). This method has a problem that the fermentation time is very slow because the fermentation time takes about 4 days as a method of directly converting food waste to organic acid. In addition, since the produced organic acid contains various components, it causes many difficulties in purifying lactic acid. On the other hand, in the method reported by Zhou in 1996, various types of household waste including food waste were hydrolyzed using sulfuric acid to neutralize the hydrolyzate obtained, and then lactic acid fermentation was performed. This method has both process complexity and pollutant emissions from the use of sulfuric acid and neutralizers (see SD Zhou, TA, McCaskey, and J. Broder, Evaluation of nirogen supplements for bioconversion of municipal solid waste to lactic acid). , Appl. Biochem. And Biotechnol., 57/58, 517-524, 1996).

음식물쓰레기로부터 젖산을 생산하기 위해서는 음식물쓰레기의 주성분인 전분 및 섬유질을 가수분해하여 포도당으로 전환시키고 이를 다시 미생물 발효시키는 두 단계의 공정이 요구된다. 따라서 효과적인 가수분해 및 미생물발효공정의 개발과 더불어 공정의 단순화가 요구되고 있는 시정이다. 특히 국내에서는 음식물쓰레기의 발생량이 외국에 비하여 상대적으로 많고 특성이 다르기 때문에 독자적인 음식물쓰레기 처리기술이 요구된다.In order to produce lactic acid from food waste, a two-step process of hydrolyzing starch and fiber, which is a major component of food waste, is converted to glucose and fermented again to microorganisms. Therefore, the development of an effective hydrolysis and microbial fermentation process is required to simplify the process. In Korea, since the amount of food waste generated in Korea is relatively high and the characteristics are different, a unique food waste treatment technology is required.

이에, 본 발명자는 국내 음식물쓰레기로부터 효과적으로 젖산을 생산하는 기술을 개발하고자 노력한 결과, 효소를 사용하여 음식물쓰레기를 가수분해하고 이를 미생물발효시켜 젖산을 생산함으로서 높은 생산성 및 수율을 갖게 된다는 것을 확인하고 본 발명을 완성하게 되었다. 특히, 효소가수분해와 미생물발효를 동시에 한 개의 반응기에서 수행함으로서 공정이 단순화되고 생산성이 향상되었다.Thus, the present inventors have tried to develop a technology for producing lactic acid effectively from domestic food waste, as a result of hydrolyzing food waste using enzymes and microbial fermentation to produce lactic acid by confirming that it has a high productivity and yield The invention was completed. In particular, by enzymatic hydrolysis and microbial fermentation in one reactor at the same time, the process is simplified and productivity is improved.

결국, 본 발명의 주된 목적은 음식물쓰레기를 자원으로 활용하여 경제적으로 젖산을 생산하는 방법을 제공하는 것이다.After all, the main object of the present invention is to provide a method for economically producing lactic acid by using food waste as a resource.

도 1은 음식물쓰레기의 동시당화발효 실험 결과, 시간에 따라 생성된 젖산의 농도를 나타낸 그래프이다.1 is a graph showing the concentration of lactic acid produced over time as a result of simultaneous glycosylation fermentation experiment of food waste.

본 발명에서 제시한 음식물쓰레기를 이용한 젖산 생산방법은 효소를 이용한 가수분해와 미생물을 이용한 발효공정을 포함한다. 음식물쓰레기 가수분해효소는 알파아밀라제(α-amylase)와 아밀로글루코시다제(amyloglucosidae)를 혼합하여 사용한다. 음식물쓰레기의 종류에 따라 프로테아제(protease)를 첨가하여 사용할 수 있다. 음식물쓰레기의 종류에 따라 전기 효소의 사용량이 달라질 수 있으나, 일반적으로 음식물쓰레기 건조중량 1kg을 기준으로, 알파아밀라제는 100 내지 1000 U, 바람직하게는 300 내지 700 U의 농도로 사용하고, 아밀로글루코시다제는 100 내지 2000 U, 바람직하게는 500 내지 1500 U의 농도로 사용한다.Lactic acid production method using the food waste presented in the present invention includes a hydrolysis using enzymes and a fermentation process using microorganisms. Food waste hydrolase is used by mixing alpha amylase ( α -amylase) and amyloglucosidase (amyloglucosidae). Depending on the type of food waste can be used by adding a protease (protease). The amount of electrical enzymes may vary depending on the type of food waste, but in general, alpha amylase is used at a concentration of 100 to 1000 U, preferably 300 to 700 U, based on 1 kg of dry food waste. The sidase is used at a concentration of 100 to 2000 U, preferably 500 to 1500 U.

젖산 발효균주는 음식물쓰레기의 효소가수분해조건과 유사한 조건에서 활성을 갖는 미생물을 사용한다. 구체적으로, 효소가수분해된 음식물쓰레기를 젖산 발효하는데 사용할 수 있는 미생물균주는 락토바실러스 펜토서스(Lactobacillus pentosus), 락토바실러스 락티스(Lactobacillus lactis), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 아밀로보러스(Lactobacillus amylovorus), 락토바실러스 델부르키(Lactobacillus delbrueckii), 락토바실러스 람노서스(Lactobacillus rhamnosus) 등이 있다.Lactic acid fermentation strains use microorganisms that have activity under conditions similar to the enzymatic hydrolysis conditions of food waste. Specifically, microbial strains that can be used to ferment the enzyme-hydrolyzed food waste are Lactobacillus pentosus, Lactobacillus lactis, Lactobacillus casei, Lactobacillus amyloborus (Lactobacillus amylovorus), Lactobacillus delbrueckii, Lactobacillus rhamnosus, and the like.

효소가수분해반응은 통상적으로 회분식 또는 연속식으로 수행할 수 있으며, 40 내지 60℃의 온도 및 pH 5.0 내지 6.5의 범위에서 수행한다. 젖산발효반응은 30 내지 45℃의 온도 및 pH 4.0 내지 7.0의 범위에서, 혐기조건으로 수행한다. 젖산발효시 적절한 질소원과 금속이온 및 무기염이 포함된 배양배지를 사용할 수 있으나, 음식물쓰레기에 이들 성분이 상당량 이미 포함되어 있기 때문에 일반적인 젖산발효에 비하여 극히 소량만 첨가하면 된다. 전술한 방법에 따라 두 단계반응을 거쳐 음식물쓰레기로부터 젖산을 생산할 수 있으나, 보다 효과적인 방법은 효소가수분해와 미생물발효를 한 개의 반응기에서 수행하는 것이다. 이때의 반응조건은 미생물의 활성이 유지되는 조건을 선정한다. 구체적으로, 이와 같은 동시당화발효는 30 내지 45℃의 온도 및 pH 4.0 내지 6.0의 범위에서, 혐기조건으로 수행한다.The enzymatic hydrolysis reaction can be carried out usually batchwise or continuously, and is carried out at a temperature of 40 to 60 ℃ and a range of pH 5.0 to 6.5. Lactic acid fermentation reaction is carried out under anaerobic conditions at a temperature of 30 to 45 ℃ and a range of pH 4.0 to 7.0. When lactic acid fermentation can be used a culture medium containing a suitable nitrogen source, metal ions and inorganic salts, but only a very small amount compared to the general lactic acid fermentation since the food waste already contains a considerable amount of these components. According to the above-described method, lactic acid can be produced from food waste through a two-step reaction, but a more effective method is to perform enzymatic hydrolysis and microbial fermentation in one reactor. The reaction conditions at this time select the conditions under which the activity of the microorganism is maintained. Specifically, such coglycosylation fermentation is carried out under anaerobic conditions at a temperature of 30 to 45 ℃ and a range of pH 4.0 to 6.0.

본 발명은 음식물쓰레기를 이용하여 젖산을 생산하는 공정에 있어서, 효소가수분해 및 발효방법을 이용하여 젖산의 생산성을 향상시키는 방법을 제공한다. 본 발명에 의하면, 음식물쓰레기 건조중량 100g당 약 80g의 젖산을 생산할 수 있으며,48시간 안에 120g/L의 젖산농도를 얻을 수 있으며, 젖산의 생산성이 2 g/L ·h로 반응속도가 기존의 젖산 생산공정에 비하여 현저히 향상되었다. 본 발명은 음식물쓰레기를 포도당 대체원료로 사용함으로써 음식물쓰레기의 자원화와 더불어 젖산 생산의 경제성을 획기적으로 높이게 할것이다.The present invention provides a method for improving the productivity of lactic acid by using enzymatic hydrolysis and fermentation in the process of producing lactic acid using food waste. According to the present invention, it is possible to produce about 80g of lactic acid per 100g dry weight of food waste, to obtain lactic acid concentration of 120g / L in 48 hours, the productivity of lactic acid is 2 g / L · h the reaction rate is Significant improvement over the lactic acid production process. The present invention will significantly increase the economics of lactic acid production as well as the recycling of food waste by using food waste as a substitute for glucose.

[실시예]EXAMPLE

본 발명에서 고안한 효소가수분해 및 미생물발효방법을 이용하여 음식물쓰레기로부터 젖산을 생산하는 실험을 수행하였다. 본 실험에 사용한 음식물쓰레기는 대학교 식당에서 수거하여 분쇄한 후 냉동실에 보관하고 필요한때 해빙시켜 사용하였다. 음식물쓰레기의 수분함량은 85-90% 이었다. 음식물쓰레기 가수분해효소로는 SAN Super 240L(α-amylase, amyloglucosidase, protease 복합효소)를 사용하였으며 이 효소의 활동도(activity)는 240 AGU/㎖이었다. 젖산생산균주는Lactobacillus delbreukii(KCCM No. 40069 , NRRL-B445)를 한국미생물보존센터로부터 분양 받아 사용하였다. 종균배양은 MRS 배지를 사용하여 37℃, pH 6.4 에서 24시간 동안 배양하였다. 동시당화발효 실험에 사용한 배지의 조성은 yeast extract 15g/L, K2HPO40.5g/L, KH2PO40.5g/L, CH3COONa ·3H2O 1g/L, MgSO4·7H2O 0.5g/L, MnSO4·7H2O 0.05g/L, ammonium citrate 1g/L, FeSO4·7H2O 0.03g/L이었다. 기질은 음식물쓰레기를 사용하였다. 발효용액은 121℃에서 20분 가량 멸균하여 사용하였다. 멸균 후 효소를 첨가하고 10%(v/v)의 종균배양액을 접종하였다. 발효는 200 ㎖ 유리병을 사용하여 42℃에서 수행되었으며, pH 조절을 위하여 CaCO3를 첨가하였다. 발효액의 당성분 및 젖산농도는 HPLC로 분석하였다. 사용한 컬럼은 HPX-87H(Bio-Rad, 미국)이고 이동상은 0.005M H2SO4이었다. 도 1에 보듯이, 음식물쓰레기 100g으로부터 80g의 젖산이 48시간 내에 생산되었다.Experiments were carried out to produce lactic acid from food waste using the enzymatic hydrolysis and microbial fermentation method devised in the present invention. The food waste used in this experiment was collected from the university cafeteria, crushed, stored in a freezer and thawed as needed. The water content of food waste was 85-90%. SAN Super 240L ( α -amylase, amyloglucosidase, protease complexase) was used as food waste hydrolase. The activity of this enzyme was 240 AGU / mL. Lactobacillus delbreukii (KCCM No. 40069, NRRL-B445) was used as a lactic acid-producing strain from Korea Microorganism Conservation Center. The seed culture was incubated for 24 hours at 37 ℃, pH 6.4 using MRS medium. The composition of the medium used for the co-glycosylation fermentation experiment was yeast extract 15g / L, K 2 HPO 4 0.5g / L, KH 2 PO 4 0.5g / L, CH 3 COONa3H 2 O 1g / L, MgSO 4 7H 2 O 0.5 g / L, MnSO 4 · 7H 2 O 0.05 g / L, ammonium citrate 1 g / L, FeSO 4 · 7H 2 O 0.03 g / L. The substrate used food waste. The fermentation solution was sterilized for 20 minutes at 121 ℃. After sterilization, enzymes were added and seeded with 10% (v / v) spawn culture. Fermentation was carried out at 42 ° C. using a 200 ml glass bottle, and CaCO 3 was added for pH control. Sugar content and lactate concentration of the fermentation broth were analyzed by HPLC. The column used was HPX-87H (Bio-Rad, USA) and the mobile phase was 0.005MH 2 SO 4 . As shown in FIG. 1, 80 g of lactic acid was produced within 48 hours from 100 g of food waste.

Claims (4)

음식물쓰레기로부터 효소가수분해와 미생물발효를 이용하여 젖산을 생산하는 방법How to produce lactic acid from food waste by enzymatic hydrolysis and microbial fermentation 제 1항에 있어서,The method of claim 1, 음식물쓰레기를 가수분해하기 위하여, 음식물쓰레기 건조중량 1kg을 기준으로, 알파아밀라제를 100 내지 1000 U의 농도로 사용하고, 또는 아밀로글루코시다제를 100 내지 2000 U의 농도로 사용하는 것을 특징으로 하는 음식물쓰레기로부터의 젖산 생산방법In order to hydrolyze food waste, alpha amylase is used at a concentration of 100 to 1000 U, or amyloglucosidase is used at a concentration of 100 to 2000 U, based on 1 kg of dry food waste weight. Process for producing lactic acid from food waste 제 1항에 있어서,The method of claim 1, 가수분해된 음식물쓰레기를 젖산 발효하기 위하여, 락토바실러스(Lactobacillus) 속의 미생물을 사용하는 것을 특징으로 하는 음식물쓰레기로부터의 젖산 생산방법Process for producing lactic acid from food waste, characterized by using microorganisms of the genus Lactobacillus to ferment the hydrolyzed food waste. 제 1항에 있어서,The method of claim 1, 온도 30 내지 45℃ 및 pH 4.0 내지 6.5의 범위에서, 혐기조건으로 회분식 또는 연속식으로 효소가수분해와 젖산발효를 수행하는 것을 특징으로 하는 음식물쓰레기로부터의 젖산 생산방법Process for producing lactic acid from food waste, characterized in that the enzyme hydrolysis and lactic acid fermentation are carried out batchwise or continuously under anaerobic conditions in the temperature range of 30 to 45 ℃ and pH 4.0 to 6.5.
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