KR20000073826A - Method for Manufacturing Lactic Acid Using Soybean Component - Google Patents

Abstract

PURPOSE: Provided is a production method of lactic acid using a culture medium containing components, which are derived from beans. Thereby, high concentration of lactic acid is produced with low production cost. CONSTITUTION: A production method of lactic acid(2-hydroxypropionic acid) is characterized by comprising the following steps: (i)inoculating and culturing lactic acid fermentation microorganism in culture medium, which comprises components such as bean powder, bean protein and hydrolysat of the bean protein, derived from beans, and also vitamin; (ii) collecting lactic acid from the culture. 1 liter of culture medium contains 5-300g of components based on 1kg of carbon source content and also comprises 0.1 microg-10mg of vitamin which selected from the group consisting of vitamin B1, vitamin B2, vitamin B6, biotin, nicotinic acid, and aminobenzoic acid. The microorganism in particular, Bacillus, Lactobacillus, Lactococcus or Streptococcus sp., is cultured at 35-45°C of temperature, pH5.0-6.5 by anaerobic fed batch culture or continuos culture.

Description

Method for Manufacturing Lactic Acid Using Soy-Derived Ingredients {Method for Manufacturing Lactic Acid Using Soybean Component}

The present invention relates to a method for producing lactic acid (lactic acid, lactic acid, 2-hydroxypropionic acid) using soybean-derived components. More specifically, in the fermentation process for producing lactic acid by using microorganisms, a component derived from soybean, such as soy flour, soy protein or hydrolyzate of soy protein, is included in the lactic acid fermentation microorganism culture medium and used as a complex nitrogen source. The present invention relates to a method for producing lactic acid with excellent productivity and high economic efficiency.

Lactic acid, which is widely used industrially, can be produced by chemical synthesis or microbial fermentation process. In recent years, the chemical synthesis process of lactic acid, which has accounted for about 50% of the world's production, has been decreasing.While its use is low, fermentation of lactic acid using environmentally friendly carbon sources (starch, glucose, sugar, lactose, etc.) Production methods have been developed, and since it is known that lactic acid can be used as a main raw material for biodegradable polymers and environmentally friendly industrial solvents, various methods for mass production of lactic acid at lower cost have been attempted. In particular, since the raw material cost of lactic acid fermentation has the most important effect on the economics of the entire process, studies are being actively conducted to develop cheaper culture media to lower production costs.

Microbial culture medium for lactic acid production is generally composed of carbon source and nitrogen source, and inorganic salts such as phosphorus, magnesium, sulfur, etc. along with vitamins, nucleic acids, lipids, etc. It is completed by adding a small amount. In particular, anaerobic strains such as Lactobacillus are known to maintain the growth and activity of cells only by providing many kinds of growth factors from the outside. Due to this high nutrient composition, the complex nitrogen source containing a large amount of nutrients such as amino acids and vitamins has been essentially used in the medium for producing lactic acid. Such complex nitrogen source is represented by yeast extract, corn steep liquor and malt. Extracts, whey, etc. may be used, but so far, yeast extracts are known to be the most effective, and yeast extracts are used as nitrogen sources in almost all lactic acid bacteria culture processes. Since yeast extract as a cultivation medium for lactic acid production is very expensive, there has been a problem that must be excluded or reduced in order to reduce the overall production cost in lactic acid production through microbial culture, and many researchers have improved it. I have been trying to.

As a representative example of this effort, in 1996, Hujanen et al., Maltese, Peptone, Grass extract, Casein of barley to reduce the amount of yeast extract used in lactic acid production by microbial fermentation Observing the effect of hydrolyzate, corn steep liquor or alcohol distillate waste feed, when malt extract and a small amount (4 g / L) of yeast extract were used as nitrogen source in culture medium, fermentation rate was decreased but only yeast extract was used. It has been reported that lactic acid at the same concentration (90-100 g / L) as when (22 g / L) can be obtained (M. Hujanen and Y.-Y, Linko (1996), Effect of temperature and various nitrogen sources on L (+)-lactic acid production by Lactobacillus casei, Applied Microbiology and Biotechnology 45: 307-313).

In addition, Bogardts et al., In producing lactic acid from whey waste in a milk processing plant, isolates a strain capable of fermenting lactic acid from whey without the addition of complex nitrogen sources such as yeast extract, and uses a continuous fermentation process using the same. It has been reported that it has succeeded in developing a purification process for lactic acid. They would be able to produce 1 kg of lactic acid at a cost of 66 cents, because whey wastes discarded as livestock waste would not have to use separate yeast extracts as carbon and nitrogen sources in the culture medium. Expected (P. Bogardts, et al., Integrated bioprocess for the simultaneous production of lactic acid and dairy sewage treatment, Bioprocess Engineering 19: 321-329, 1998). In this case, the whey waste liquid contained a large amount of lactose as a carbon source, as well as a large amount of protein and other nutrients, so the above-described effects were possible, but in general, whey alone does not provide sufficient nutrients for fermenting high concentrations of lactic acid. It is known that improvements were required.

In addition, Korean Patent Publication No. 97-14592 and Korean Patent No. 23366 disclose a method of excluding yeast extract by using whey or corn steep liquor as a nitrogen source in producing lactic acid. Since whey or corn steep liquor was included in the culture medium at a high concentration of / L or more, it caused a lot of trouble in purifying lactic acid after fermentation. In addition, various research results for the replacement of yeast extracts have been published so far. However, the composition of economical lactic acid culture medium with high productivity and the production of lactic acid using the same have been completely replaced with yeast extract. It was not developed.

In 1996, soybeans produced around 130 million tonnes in 1996, of which 49% were produced in the United States, with more than 50% of the world's oil seeds and protein meals. It takes up more than 60% of), and produces a great amount of corn and wheat, so it has more protein (about 40% by dry weight) and vegetable oil (about 20% by dry weight) than other grains. It is used not only as a food but also as a raw material for a large number of commodities such as animal feed and industrial fats and oils. In addition, soybean meal made from electric soybeans is used in large quantities as a source of food and feed, and in 1996 the price in the United States was $ 289 US per tonne, the lowest price of grain (SOYSTATS, A Reference Guide). (1998) in American Soybean Association's WWW site, http://www.oilseeds.org/asa. Given the nutritional advantages of these soybeans and their low production prices, they are sufficiently competitive with yeast extract, which is about US $ 3.3 per kilogram (1991 price of Amberex 1003, a yeast extract product from Universal Food, USA) used for lactic acid production by microbial culture. It was supposed to be.

In addition, Yoo et al. Tested the effects of casein, gluten, cottonseed, soy protein, or hydrolysates of animal tissues to replace some of the expensive yeast extracts in lactic acid production of Lactobacillus casei. As a result, 3.3g / L of five vitamins (folic acid, vitamin B2, vitamin B5, vitamin B6, nicotinic acid) and soy protein hydrolysate NZ-soy peptone were added to the culture medium. It has been reported that yeast extract concentrations of 10 g / L can be reduced to 5 g / L (IK Yoo, et al., "Effect of B vitamin supplementation on lactic acid production by Lactobacillus casei", Journal of Fermentation and biotechnology, 84 : 172-175, 1997). However, the above-described method was a method that can not completely replace the yeast extract, and the detailed composition of the added vitamin was not disclosed, the situation that needs further improvement in lowering the efficiency and production cost of lactic acid production. Therefore, the necessity of developing a new nitrogen source of lactic acid production culture medium that can completely replace the yeast extract, and using this to develop a method for enabling lactic acid production by high efficiency and low production cost.

Therefore, the present inventors have developed a new medium that can completely replace the use of yeast extract in the culture medium of lactic acid fermentation microorganisms, and as a result of earnest efforts to establish a technology that can economically produce high concentration of lactic acid using this, soybean-derived When the ingredient is used as a nitrogen source in the culture medium and vitamin is added as an adjuvant, it was confirmed that excellent lactic acid production was possible at a lower cost than using the yeast extract, and thus the present invention was completed.

After all, the main object of the present invention is to provide an economical production method of lactic acid that can greatly improve the economics of the process by producing a high concentration of lactic acid at a low medium cost using a culture medium containing soybean-derived components.

1 is a graph showing the concentration of lactic acid over time in the lactic acid fermentation using a culture medium containing soybean-derived components.

Figure 2 is a graph showing the concentration of glucose over time in the lactic acid fermentation using a culture medium containing soybean-derived components.

3 is a graph showing the concentration of lactic acid fermentation microorganisms with time in lactic acid fermentation using a culture medium containing soybean-derived components.

The production method of lactic acid using the soybean-derived component of the present invention includes a step of inoculating the culture medium containing soybean-derived components and vitamins by inoculating lactic acid fermentation microorganisms, and obtaining lactic acid from the electric culture. At this time, as the soy-derived component can be used soy flour, soy protein or hydrolyzate of soy protein, since the electric soy-derived component is different in concentration and composition depending on the prepared state, depending on the type of microorganism used and the amount of glucose used The amount of use may vary, but generally, based on 1 kg of the carbon source content of the culture medium, 50 to 300 g, preferably 70 to 200 g per liter of culture medium is used. Soy protein can be used as a conventional soy protein purified from soybean, hydrolyzate of soy protein can be prepared from the soy flour through an enzyme reaction, for reference, US Patent No. 4,882,180 is a method for producing soy hydrolyzate for food Detailed methods have been described and can be easily prepared using microorganisms and commercialized proteolytic enzymes used in the preparation of soybean-degraded foods such as soy sauce.

In addition, it is essential to add a small amount of vitamin as a supplement when using soy-derived ingredients in the culture medium, such as vitamin B1, vitamin B2, vitamin B5, vitamin B6, biotin, nicotinic acid, aminobenzoic acid, and their equivalents. Four or more from each group should be included in the range of 0.1 μg to 10 mg per liter of culture medium. In this case, the vitamins may be used in combination with purified vitamins, or may be added in accordance with the range of addition of the corresponding vitamin from the conventional industrial medium components containing a large amount of each vitamin component. The culture medium may be a culture medium containing a carbon source and trace metal ions and inorganic salts that can be represented by conventional sugars, glucose, lactose, which can cultivate lactic acid fermentation microorganisms, and the lactic acid fermentation microorganism is Bacillus Conventional lactic acid fermentation microorganisms of the genus Lactobacillus, Lactococcus or Streptococcus can be used. Specifically, lactic acid fermentation microorganisms to which a culture medium containing soybean-derived components can be applied include Lactobacillus rhamnosus, Bacillus coagulans, Bacillus lebolacticus, and Lactobacillus. Lactobacillus amylophilus, Lactobacillus amylovorous, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus helveticus Lactobacillus lactis, Streptococcus faecalis, Streptococcus thermophilus, Streptococcus inulinis, Streptococcus cremoris, and the like.

In addition, the culture of the lactic acid fermentation microorganisms may be used in a conventional batch or continuous culture, the culture in anaerobic conditions at a temperature of 35 to 45 ℃ and pH 5.0 to 6.5 by the culture conditions suitable for each lactic acid fermentation microorganisms. Perform. After culturing the fermented microorganisms of lactic acid by the above-described method, the lactic acid solution can be easily obtained from the culture through a series of known recovery processes such as centrifugation, membrane separation, acid treatment, electrodialysis, and evaporation. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Comparative Example 1: Lactic Acid Production Using Yeast Extract

In order to confirm the production characteristics of lactic acid when a culture medium containing yeast extract is used, a batch culture using a culture medium containing heat sterilized yeast extract and a culture medium containing yeast extract filtered and sterilized to avoid heat sterilization The lactic acid fermentation microorganism used in the culture was Lactobacillus rhamnosus ATCC 10863, and the above strains were dispensed in 1 ml each of MRS (Difco, USA) medium containing glycerol, and then -75 ° C. It was stored in the freezer and used as a culture seed for all fermentations. Culture medium for batch culture is glucose 150g / L, yeast extract (yeast extract, Difco, USA) 15g / L, KH ₂ PO ₄ 2.5g / L, Na ₃ -citrate 2H ₂ O 0.5g / L, MgSO _{4 · 7H 2 O 0.2g / L,} MnSO 4 · H 2 O 0.03g / L, FeSO 4 · 7H were prepared such that the ₂ O 0.03g / L, and _{H 2 sO 4 0.01㎖ / L,} 2 -fold dilution of 100㎖ Inoculated with the culture spawn in MRS medium and shaken for 12 hours, and then centrifuged and washed once with distilled water in order to reduce the effect of the culture medium, inoculated into a fermenter of 2 liters of the initial fermentation volume. In order to neutralize the lactic acid produced by the culture of lactic acid fermentation microorganisms, the pH of the culture medium was maintained at pH 6.0 using an aqueous ammonia solution, and the culture was performed for about 60 hours while maintaining the fermentation temperature at 42 ° C. The cell concentration in the culture solution was properly diluted and then converted from the absorbance measured at a wavelength of 620 nm using a spectrophotometer (Ultrospec-3000, Pharmacia, UK). The concentration of glucose and lactic acid in the culture solution was centrifuged. The microorganisms were separated to remove microorganisms, and then measured by HPLC with an RI detector attached: Aminex HPX-87H (Biorad, USA) was used for the column, and 10 µl of 10-fold diluted culture was injected. And analyzed. Cell concentration and glucose and lactic acid concentration over time during the culture process for about 60 hours were measured by the above-described method (see FIGS. 1 to 3 (●)). As shown in Figures 1 to 3, by batch culture using a culture medium containing a yeast extract sterilized for 15 minutes at 121 ℃ lactic acid of a maximum concentration of 112g / L was produced within 60 hours.

Next, since the yeast extract of 26g contained in the culture medium in the previous experiment was heat sterilized at 121 ° C. for 15 minutes, the vitamin contained in the yeast extract may be denatured by heat, and thus, the yeast extract filtered by sterilization using a filter. Using the same method was carried out in batch culture to determine the effect (see: (○) of Figures 1 to 3). As shown in Figures 1 to 3, when using a culture medium containing yeast extract filtered by sterilization to avoid heat sterilization, lactic acid at a concentration of up to 119 g / L is produced within 60 hours, so that the yeast extract is not filtered by heat sterilization By doing so, nutrient components such as vitamins contained in the yeast extract did not occur, and it was confirmed that not only the lactic acid production rate of lactic acid fermentation microorganisms were particularly helpful for cell growth, and to observe the effects of the present invention in detail. In Example 2, the culture results using the sterile yeast extract were used as a control.

Example 1 Lactic Acid Production Using Soybean-Derived Ingredients

In order to observe the lactic acid productivity improvement effect of soybean-derived components in comparison with yeast extract, lactic acid fermentation microorganism culture was performed in a 250 ml flask: the volume of the culture was 100 ml and calcium carbonate was used to neutralize the resulting lactic acid. A culture medium was prepared in the same composition as that of Comparative Example 1, except that yeast extract was added and 5 g / L ammonium chloride was added. flour I, SIGMA, USA), soybean flour II (SIGMA, USA), Amisoy ™ (QEUST, USA) or Soytone (Soytone ™, DIFCO, USA) in each flask ) Is added to the same concentration, and as a control, heat sterilized yeast extract is added at a concentration of 15 g / L, and then inoculated with 0.2 ml of the culture medium and incubated at 42 ° C. for 72 hours. The resulting lactic acid and the concentration of residual glucose was measured in the same manner as in Comparative Example 1. At this time, soybean powder 1 is degreased soybean powder, soybean powder 2 is baked soybean powder 1, amisoy is hydrolyzed soy protein with acid, and soyton is hydrolyzed soy protein using enzyme.

On the other hand, in order to confirm the effects of vitamins on lactic acid production using soybean-derived components, seven vitamins (vitamins B1, B2, B5, B6, biotin, nicotinic acid and amino, which are present in yeast extracts in comparison with soybean components) Benzoic acid, SIGMA, USA) was prepared at the same concentration as the yeast extract used as a control, then sterilized with a filter to prevent thermal denaturation, added to the medium, and the resulting lactic acid and residual glucose were cultured in the same manner. The concentration of was measured, and the results were compared with the culture results without the addition of vitamins (see Table 1).

Production of Lactic Acid Using Soy-Derived Ingredients ingredient* Concentration (g / L) Cell concentration (g-dry cell / L) Lactate Concentration (g / L) Residual Glucose Concentration (g / L) Yeast Extract + V 15 8.26 95 40 Yeast Extract (Control) 15 8.26 86 42 Soy flour 1 + V 19.7 - 50 97 Soy flour 1 19.7 - 48 103 Soy flour 2 + V 19.7 - 64 77 Soy flour 2 19.7 - 51 101 Amisoi + V 12.3 3.01 0 152 Ami Soi 12.3 0.96 0 156 Soyton + V 17.1 9.60 96 37 Soyton 17.1 0 0 156

*: V denotes the experiment with vitamins, and in the experiment with soy flour

The turbidity of the medium is too high to determine the microbial concentration.

As shown in Table 1, when using a culture medium containing the enzymatic hydrolyzate (soyton) of the soybean-derived components, it was confirmed that the addition of vitamins is essential, the culture medium added vitamins to yeast extract The synergistic effect of lactic acid production was shown to be due to the significant loss of vitamins in yeast extracts, since the medium was sterilized at 121 ° C. for 15 minutes as compared to the use of culture mediums containing only yeast extracts. In addition, when the soy flour was included in the culture medium, the production of lactic acid proceeded without adding a separate vitamin. Since the soy flour was less processed than the hydrolyzate (amisoy and soyton), there was some nutritional component. It was analyzed that the production of lactic acid was made without adding vitamin. However, the reason why the vitamin addition effect is insignificant is that the amino acids in soy flour are present in the form of protein that is not hydrolyzed, and thus it is assumed that the microorganisms are difficult to use. In addition, the use of a culture medium containing amisoyi showed no lactic acid production activity at all, which was presumed to be due to the loss of important components necessary for microbial growth and lactic acid production in the acid treatment step for hydrolysis. When using a culture medium containing vitamins added to Soyton, the same result as in the culture medium containing yeast extract was obtained. Therefore, the culture medium and the electroculture medium containing the hydrolyzate by the enzyme of soy protein are obtained from this result. By adding the appropriate vitamins to the yeast extract, it was predicted to have an equivalent lactic acid production effect that can replace the yeast extract, and after performing some basic experiments to find the optimal concentration of the added vitamins, the following Example 2 Proceeded.

Example 2 Production of Lactic Acid According to Vitamin Amount

A culture medium having the same composition as in Comparative Example 1 was prepared, except that 19.3 g / L of Soyton was used instead of the yeast extract and 10 ml, 20 ml, or 50 ml of vitamin solution was added. The culture was carried out by the same method as 1, the concentration of lactic acid, glucose and lactic acid fermentation microorganisms in the culture medium was measured. The composition of the vitamin solution used is shown in Table 2, the results are shown in Figures 1 to 3 compared with the results of Comparative Example 1. (▼) in Figures 1-3, 10 ml of vitamin solution; (▽) 20 ml of vitamin solution; And (■) shows the concentrations of lactic acid, glucose and lactic acid fermentation microorganisms in the culture medium when 50 ml of the vitamin solution was added.

Composition of vitamin solution vitamin Common name Concentration (mg / L) Vitamin B1 thiamine hydrochloride 13.4 Vitamin B2 riboflavin 23.4 Vitamin B5 d-pantothenic acid hemicalcium salt 554.9 Vitamin B6 pyridoxine hydrochloride 5.8 Biotin (vitamin H) d-biotin 0.1 Nicotinic acid nicotinic acid (niacin) 171.5 Aminobenzoic acid p-aminobenzoic acid (PABA) 11.4

As shown in Figures 1 to 3, when producing lactic acid using a culture medium containing soyton, a hydrolyzate of soy protein, by adding an appropriate amount of the seven vitamins shown in Table 2, corresponding to the effect of yeast extract It can be seen that lactic acid production capacity can be induced. In addition, the growth of lactic acid fermentation microorganisms was shown to be most effective when using a culture medium containing yeast extract, but the production of high concentration of lactic acid was most effective when using a culture medium containing vitamins and soy protein hydrolysates and vitamins. It turned out. In addition, when 50 ml of vitamin solution is added, it shows the fastest lactic acid production, but even when 20 ml of vitamin solution is added, 150 g / L of glucose can be sufficiently converted to lactic acid to produce 120 g / L of lactic acid within 60 hours. It was found that there was a result corresponding to the effect of yeast extract.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described and demonstrated in detail above, the present invention provides a method for economically producing lactic acid at a low medium cost by using the soy-derived component as a complex nitrogen source of lactic acid fermentation microorganism culture medium. According to the present invention, in the production of lactic acid through the cultivation of lactic acid fermentation microorganisms, lactic acid of 120 g / L can be produced in 60 hours at a low medium cost compared to when using the yeast extract as a nitrogen source. This result is important in that the yeast extract, which is a medium component that takes up the overall economic efficiency of the whole process, can be completely replaced by using soybean, which is a large component of the overall process. The cost can be drastically reduced.

Claims (6)

A method of producing lactic acid using a soybean-derived component comprising the step of inoculating a culture medium containing soybean-derived components and vitamins by inoculating lactic acid fermentation microorganisms to obtain lactic acid from the electric culture. The method of claim 1, Soy-derived component is characterized in that the soy flour, soy protein or a hydrolyzate of soy protein Lactic acid production method using soybean-derived ingredients. The method of claim 1, Soy-derived component is characterized in that it comprises 50 to 300g per liter culture medium based on 1kg of carbon source content of the culture medium Lactic acid production method using soybean-derived ingredients. The method of claim 1, The vitamin is characterized in that it comprises at least 0.1 to 10mg per liter culture medium, each of four or more from the group consisting of vitamin B1, vitamin B2, vitamin B5, vitamin B6, biotin, nicotinic acid and aminobenzoic acid Lactic acid production method using soybean-derived ingredients. The method of claim 1, Lactic acid fermentation microorganism is a genus of Bacillus, Lactobacillus, Lactococcus or Streptococcus, characterized in that Lactic acid production method using soybean-derived ingredients. The method of claim 1, At a temperature of 35 to 45 ℃ and pH 5.0 to 6.5, characterized in that the culture in batch or continuous under anaerobic conditions Lactic acid production method using soybean-derived ingredients.