JP2006333847A - L-lactic acid producing bacteria and L-lactic acid liquid production method - Google Patents

Abstract

[PROBLEMS] To avoid the decomposition of active ingredients and the production of fermentation inhibitors, which were problems in the past, by using the raw material as a medium without heat sterilization, and at the same time, eliminating the need for advanced centralized management and sterilization equipment Development of L-lactic acid producing bacteria that produce L-lactic acid with higher productivity and higher optical purity and a method for producing L-lactic acid liquid using the same.
[MEANS FOR SOLVING PROBLEMS] A newly isolated and identified thermostable microorganism BacilluslicheniformisTY7 is used as a thermophilic L-lactic acid-producing bacterium, and a biomass raw material containing a high content of carbohydrates and other nutritional components is used at 50 to 60 ° C., pH 5. It is a method for producing an L-lactic acid solution that is directly fermented as a culture medium without being sterilized in 8 to 6.5.
[Selection] Figure 3

Description

The present invention relates to an L-lactic acid-producing bacterium identified as a thermostable microorganism BacilluslicheniformisTY7 that has been newly isolated and can be used for L-lactic acid fermentation production from unused organic waste due to high productivity, and L-lactic acid using the same. The present invention relates to a method for producing a liquid.

Lactic acid is an important substance as various food additives and industrial raw materials. In particular, polymer polylactic acid obtained by chemically polymerizing lactic acid has attracted much attention in recent years as a biomass-derived plastic material. The carbon at the 2-position of lactic acid, which is a three-carbon compound, is an asymmetric carbon bonded to a methyl group, a hydroxyl group, and a carboxyl group, and from its conformation, two kinds of optical isomers (enantiomers) of D-lactic acid and L-lactic acid. Body). Worldwide, 90% of the total production is currently produced by fermentation and the remaining 10% is produced by chemical synthesis.

In the production of lactic acid by fermentation, a production medium containing amino acids, vitamins, minerals, etc., together with carbon sources such as glucose and soluble starch, is prepared. After sterilizing by using a possible apparatus and keeping at 120 ° C. and 2 atm for 20 minutes, lactic acid-producing bacteria were inoculated aseptically, and fermentation was mainly performed at 30 to 37 ° C.

For the microorganisms used as fermenting bacteria, it is summarized in the review that many room temperature bacteria of the genus Lactobacillus that mainly produce L-lactic acid (Lactobacillus rhamnosus, Lactococcus lactis, etc.) and the mold Rhizopus oryzae are used. (Non-patent document 1, Non-patent document 2). Further, regarding the production method of L-lactic acid by using bacteria belonging to the genus Bacillus, Bacillus coagulans JCM2257, Bacillus cereus JCM2152, Bacillus subtilis JCM1465, and Bacillus thuringiensis, Bacillus larvae, Bacillus lentimorbus, Bacillus lollimorbus, Bacillus popilliae, Bacillus shphaeric, etc. that produce insect toxins and the like. (Patent Document 1). Apart from this, a method for producing lactic acid continuously at 50 ° C. while performing membrane separation using Bacillus coagulans has been patented (Patent Document 2). By these methods, lactic acid having an optical purity [(L-lactic acid-D-lactic acid) / (L-lactic acid + D-lactic acid) × 100 (%)] of about 90 to 98% was produced.

Polylactic acid has been attracting attention as a biodegradable plastic obtained from renewable biomass materials since around 1990, and its production volume is increasing year by year. Polylactic acid is obtained by dehydration polymerization of L- or D-lactic acid or a mixture thereof. The raw material is lactic acid having high optical activity and low optical activity, and the former is a hard plastic with high crystallinity, and the latter. Produces a polylactic acid having low crystallinity and easy dissolution. For example, poly L-lactic acid is mainly used in engineering plastics that require firmness. That is, lactic acid having different optical purity can be used as a raw material according to the physical properties required for polylactic acid plastic.

On the other hand, in a chemical synthesis method using acetonitrile as a starting material, a mixture of L lactic acid and D lactic acid having an optical purity of 0%, ie, racemic lactic acid, is obtained. In the above-mentioned review, examples of fermentative production of racemic lactic acid by Lactobacillus helveticus and Lactobacillus aminovorus producing D-, L-lactic acid are also reported. Racemic lactic acid and its esters are used as food additives, but in such applications, the level of optical purity is unrelated to their efficacy.

Production of a lactic acid solution by the fermentation method has been carried out in a medium which has been mixed and dissolved in an appropriate amount of components that can be dissolved homogeneously and then subjected to high-pressure steam sterilization. However, saccharides, amino acids, vitamins, and the like necessary for the growth of lactic acid bacteria and molds are unstable and easily decomposed against high-temperature treatment, and when they coexist, the decomposition reaction is further promoted. Also, undesired substances such as browning substances that cause coloration of the waste liquid and furfural that inhibits fermentation are produced in this sterilization process.
If the raw materials for cultivation such as carbohydrates, proteolysates, amino acids, vitamins, etc. are purified with high purity as in the past, they are individually sterilized by autoclaving and then mixed, or by filtration sterilization without applying heat. By adopting a method of sterilization, it is possible to avoid the above-mentioned undesirable decomposition, but in that case, it is necessary to purify the raw material in advance, and the sterilization operation is complicated and the necessary equipment increases. Furthermore, such individual sterilization is impossible when a raw material in which necessary components such as raw garbage and agricultural biomass are mixed is used as a direct fermentation substrate. In addition, filtration sterilization as described above cannot be applied to biomass in the form of semi-solids or suspensions.

On the other hand, as an attempt to improve economy for production, open-system fermentation of raw garbage that does not require equipment costs and sterilization energy has been studied. By keeping the pH of the crushed semi-solid garbage intermittently neutralized (pH amplitude fluctuation control), lactic acid is highly selective in high yield (> 95%) even when lactic acid-producing microorganisms are not inoculated. -Applicants have found that it accumulates in (total organic acids) (Non-patent Document 3).

In this connection, the contents are complex, such as containing a high amount of carbohydrates and other nutritional components at the same time, etc., using raw garbage and agricultural biomass with many germs and natural lactic acid bacteria living as raw materials, equipment, It is a non-sterilized open system that does not require energy costs and can avoid the decomposition of nutrients by high-pressure steam sterilization or the like, and the production of fermentation inhibitors. D-, L-lactic acid (Lactobacillus plantarum) -producing bacteria or L-lactic acid (Bacillus A method for producing lactic acid with a low yield and optical purity according to the application in a high yield by controlling the selective growth of coagulans-producing bacteria is currently disclosed by the applicants (Patent Document 3). However, the productivity at 50 ° C. when B. coagulans was used in this method was as low as 0.9 g / l · h.

J.H.Litchfield, "Microbiological production of lactic acid" Advances in Applied Microbiology 42: 45-95 1996 K. Hofvendahl, B. H-Hagerdal, `` Factors affecting thefermentative lactic acid production from renewable resources '', Enzyme and Microbial Technology 26: 87-107 2000 26: 87-107 (2000). K.Sakai, M.Yoshihiro, H.Yamazumi, Y.Tau, M.Mori, M.Moriguchi, Y.Shirai `` SelectiveProliferationofLacticAcidBacteriaAndAccumulationofLacticAcidduringOpenFermentationofKitchenRefusewithIntermittentpHAdjustment. H.Ohara, M.Yahata, USPatent58010251996MethodforproducingL-lacticacidwithhighopticalpurityusingbacillusstrains (JP7-280660, JP-280661) VanNispen, Joannes G.M., Jonker, Ronald, US Patent 5002881 1989 Process for the fermentative preparation of organic acids. Kenji Sakai, Yutaka Esaki, Yoshito Shirai, Japanese Patent Application 2003-306871 (2003) Method for producing lactic acid solution

Therefore, the problem to be solved by the present invention is to avoid the decomposition of active ingredients and the production of fermentation inhibitors in the raw material medium, which has been a conventional problem by using the raw material as a medium without subjecting it to heat sterilization, and at the same time, advanced fermentation Lactic acid producing bacteria that produce L-lactic acid with higher productivity and higher optical purity than conventional methods in the production method of lactic acid solution that does not require centralized control and sterilization equipment, and a method for producing L-lactic acid solution using the same. .

The present invention has been made in order to solve the above-mentioned problems, and it is the following (1) and (2).
(1) .The partial sequence of 16S ribosomal RNA gene is

An L-lactic acid-producing bacterium characterized by comprising the thermostable microorganism BacilluslicheniformisTY7, that is, the thermophilic L-lactic acid-producing bacterium BacilluslicheniformisTY7 newly isolated and identified by the present inventors.
(2). Using the thermostable microorganism BacilluslicheniformisTY7, it is characterized by fermenting a biomass material containing a high content of carbohydrates and other nutrients as a direct culture medium without sterilization at 50 to 60 ° C. and pH 5.8 to 6.5. To produce L-lactic acid solution.

The lactic acid-producing bacterium and the lactic acid liquid production method of the present invention are a biomass raw material containing a high amount of nutritional components together with carbohydrates, such as food waste and agricultural products or juices thereof, and saccharified liquid obtained by saccharifying the lactic acid with amylase. Inoculate a certain amount of the newly isolated L-lactic acid-producing bacterium BacilluslicheniformisTY7 in a non-sterilized state without sterilization such as high-pressure steam sterilization, and add it at a temperature of 50 to 60 ° C. while maintaining a predetermined pH. By heating, a fermentation broth having an optical purity of 96% of L-lactic acid can be obtained with higher productivity (2.5 g / l / h) than before.
As a result, the fermentation period, which was 4-5 days in the past, has been shortened to about 2 days, and the environmental burden has been further reduced by downsizing the equipment and reducing the energy used, thus eliminating the cost of processing biomass waste. Can do.

The best mode for carrying out the present invention is to dispose of other nutritional components together with saccharides such as crushed suspensions of raw garbage and agricultural waste, squeezed liquids thereof, or saccharified liquids obtained by saccharifying them with amylase. The biomass raw material contained is used as a culture medium without sterilization such as high-pressure steam sterilization, and an L-lactic acid-producing bacterium BacilluslicheniformisTY7 newly isolated in this culture medium is inoculated so as to be 10 ⁵ to 10 ⁷ , The ratio of lactic acid per organic acid produced is high by maintaining the pH at 5.8 to 6.5, preferably pH 6.0 while maintaining the pH at 50 to 65 ° C., preferably 50 ° C. This is a method for producing an L-lactic acid solution having a high optical purity of 9% or more.

That is, the present invention provides an effect that an L-lactic acid solution having a high optical purity of 90% or more and high optical purity can be efficiently produced from the non-sterilized biomass raw material by the above means.

These means and effects are brought about by the identification of a novel L-lactic acid-producing bacterium TY-7 shown in FIGS.
TY-7 is a gram-positive gonococcus having a length of 2.1-3.8 micrometers and a width of 0.6 to 0.8 micrometers, and formed endospores. Catalase activity and VP test were positive. The above was in good agreement with the properties of bacteria belonging to the genus Bacillus.
Table 1 shows the types of carbohydrates that can be used by this bacterium to produce acid. From the comparison of API50CHB positive rate table provided by Japanese Biomeryu, it was found that this isolate was Bacilluslicheniformis with 99.9% identification probability. It was also found that glycerol, galactose, glucose, fructose, cellobiose, maltose, sucrose, trehalose, raffinose, starch, glycogen, etc. were used as important carbohydrates in biomass.

表２には本分離菌の16SリボゾーマルRNA遺伝子の部分配列（１２０３ｂｐ）の分析結果を示した。これにより本分離菌の16SリボゾーマルRNA遺伝子の部分配列はBacilluslicheniformisstr.B-6-4Jと100%相同であることが分かったのである。
以上のことから本分離菌TY-7はBacilluslicheniformisと命名されるべきであることが示された。

Table 2 shows the analysis results of the partial sequence (1203 bp) of the 16S ribosomal RNA gene of this isolate. This revealed that the partial sequence of the 16S ribosomal RNA gene of this isolate was 100% homologous to Bacilluslicheniformisstr. B-6-4J.
From the above, it was shown that this isolate TY-7 should be named Bacilluslicheniformis.

図１のグラフには、各pHに調整したMRS培地（１Ｌ中にグルコース２０ｇ、ペプトン１０ｇ、肉エキス８ｇ、酢酸ナトリウム５ｇ、酵母エキス４ｇ、リン酸２カリウム２ｇ、クエン酸三アンモニウム２ｇ、硫酸マグネシウム０.２ｇ、硫酸マンガン０.０５ｇ、ソルビタンモノオレエート１ｍｌを含む）を用いて新規Ｌ乳酸生産菌（BacilluslicheniformisTY-7）を静置培養した際の、培養時の初発ｐＨと生育度（６１０ｎｍにおける濁度）の関係を示す。
これにより、本菌の最適ｐＨが６．0であり、生育可能ｐＨ範囲が５．０〜6．０付近であることが判明したのである。
図２のグラフには、pH６.２に調整したMRS培地中で，温度勾配培養装置を用いて各種温度で新規Ｌ−乳酸生産菌（BacilluslicheniformisTY-7）を静置培養した際の、その培養時の温度と生育度（６１０ｎｍにおける濁度）の関係を示す。
これにより、本菌の生育上限温度が、６０℃前後であることが判明したのである。
図３のグラフには、非殺菌実生ゴミ糖化液を５０℃でpHを一定に保ってＬ−乳酸生産菌（BacilluslicheniformisTY-7）を植菌した場合の発酵時間と生成した有機酸、全乳酸、Ｄ及びＬ乳酸量との関係を示すものである。これにより約４３g/lの可溶性糖質（内グルコース３０g/lを含む）と共に不溶性の基質を含む原料から、およそ４２g/lのＬ乳酸が９７%以上の光学純度で生成し、この際、最大生産性は２．５g/l/hであり，Ｄ−乳酸及び酢酸の生成がほとんど見られないことが判明した。

In the graph of FIG. 1, MRS medium adjusted to each pH (20 g of glucose, 10 g of peptone, 8 g of meat extract, 5 g of sodium acetate, 4 g of yeast extract, 2 g of dipotassium phosphate, 2 g of triammonium citrate, magnesium sulfate) The initial pH and degree of growth (at 610 nm) when a new L-lactic acid-producing bacterium (BacilluslicheniformisTY-7) was statically cultured using 0.2 g, 0.05 g of manganese sulfate, and 1 ml of sorbitan monooleate (Turbidity) relationship.
As a result, it was found that the optimum pH of this bacterium was 6.0 and the viable pH range was around 5.0 to 6.0.
The graph of FIG. 2 shows the culture time when a new L-lactic acid-producing bacterium (BacilluslicheniformisTY-7) was statically cultured at various temperatures in a MRS medium adjusted to pH 6.2 using a temperature gradient culture apparatus. Shows the relationship between the temperature and the growth degree (turbidity at 610 nm).
As a result, it was found that the upper limit temperature for growth of this bacterium was around 60 ° C.
The graph of FIG. 3 shows the fermentation time and the organic acid, total lactic acid, and the fermentation time when the non-sterilized seedling saccharified saccharified solution is inoculated with an L-lactic acid-producing bacterium (BacilluslicheniformisTY-7) at a constant pH of 50 ° C. The relationship with the amount of D and L lactic acid is shown. As a result, about 42 g / l of L-lactic acid is produced with an optical purity of 97% or more from a raw material containing an insoluble substrate together with about 43 g / l of soluble carbohydrate (including 30 g / l of internal glucose). The productivity was 2.5 g / l / h, and it was found that the production of D-lactic acid and acetic acid was hardly observed.

Open lactic acid fermentation of non-sterilized food waste is a biomass raw material that contains a high amount of carbohydrates and other nutrients. 20% (V / W) water and glucoamylase (final concentration 0.03%) are collected from raw garbage collected from commercial facilities. ) And rotating and stirring at 50 ° C. for 8 hours, and then using the filtrate obtained from a 100 mm ^2- mesh sieve (: real saccharified saccharified liquid), the whole volume 2L small multipurpose culture device, pH meter and 6-dot type It was performed with a recorder (manufactured by ABLE).
In 5 ml of the aforementioned MRS liquid medium, a new L-lactic acid producing bacterium (BacilluslicheniformisTY-7) is statically cultured at 24 ° C. for 24 hours, washed and collected, and 100 μl of the collected bacterial solution is added to 30 ml of standard garbage, and at 50 ° C. And 24 hours static culture. 30 ml of this was placed in 1.5 L of the above-mentioned seed saccharified liquefied saccharified solution added to a 2 L small-sized multipurpose culture apparatus without sterilization, and rotated and stirred at 60 rpm under constant control of pH 6.0 with 10% NH _3. Maintained at 50 ° C.
As a result, a lactic acid solution having a high optical purity of 97% and a high lactic acid ratio per organic acid of 100% could be obtained.

Table 3 compares the fermentation results of standard saccharified saccharified liquefied liquids of Bacillus coagulans and a new isolate, Bacillus lichemiformisTY-7, which have been conventionally used. As a result, the yield to glucose, yield to sugar, optical purity, L-lactic acid selectivity, etc. were almost the same as Bacilluscoagulans, but the maximum productivity reached 2.8 times.

This makes it impossible to avoid decomposition by high-pressure steam sterilization due to the presence of carbohydrates for lactic acid fermentation and nutrients necessary for the growth of microorganisms, despite the presence of room-temperature spoilage microorganisms such as garbage and agricultural products. From biomass, it is possible to obtain lactic acid exhibiting the desired optical purity with low equipment cost and low energy with high yield and high productivity.

The graph which shows the relationship between the initial pH at the time of culture | cultivation, and growth degree (turbidity in 610 nm) of BacilluslicheniformisTY-7 in this invention. The graph which shows the relationship between the temperature at the time of culture | cultivation, and the growth degree (turbidity in 610 nm) of BacilluslicheniformisTY-7 in this invention. The graph which shows the relationship between the fermentation time at the time of inoculating BacilluslicheniformisTY-7 and keeping the pH constant at 50 degreeC in the non-sterilization seedling waste saccharified liquid in this invention, and the amount of organic acids produced, total lactic acid, D and L lactic acid .

Explanation of symbols

Claims (2)

The partial sequence of 16S ribosomal RNA gene is

An L-lactic acid-producing bacterium characterized by comprising the thermostable microorganism BacilluslicheniformisTY7. L-lactic acid characterized by using a thermostable microorganism, BacilluslicheniformisTY7, and fermenting a biomass material containing a high amount of carbohydrates and other nutrients as a direct culture medium without sterilization at 50 to 60 ° C and pH 5.8 to 6.5 Liquid manufacturing method.

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