JPH0579312B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing mannitol from sugars by fermentation using lactic acid bacteria. Mannitol produced by the present invention is a substance useful in foods and medicines. That is, in the food field, mannitol is used as an anti-adhesive agent that takes advantage of its low hygroscopic properties, and in the pharmaceutical field, it is used as a diuretic, a blood pressure lowering agent, etc. Furthermore, mannitol has 60% the sweetness of glucose and is expected to be used as a dietary sweetener. [Prior Art] The following three methods have been reported as current methods for producing mannitol. (1) Synthesis method Currently, mannitol is produced industrially by electrically or chemically reducing sucrose under high pressure. However, this method produces sorbitol, which is a structural isomer, and the production ratio of sorbitol and mannitol is 65% to 35%, so the equilibrium is tilted toward sorbitol. Therefore, in order to obtain the produced mannitol, it must be separated and purified using an ion exchange resin, which is considered to be economically disadvantageous. (2) Fermentation method Many methods for producing mannitol using microorganisms have been reported since the 19th century. Lactic acid bacteria The lactic acid bacteria that produce mannitol are
Lactobacillus brevis (lactobacillus)
brevis), leuconostocmesente roides, lactobacillus famentum
Heterofermentative lactic acid bacteria such as leuconostoc dextranicum and leuconostoc dextranicum are known. With glucose and fructose as substrates,
It is known that when the above bacteria are inoculated at a ratio of 1:2, fructose can be converted to mannitol at a high yield. However, this occurs only when the initial sugar concentration is low, and the production amount is low. To solve this problem, increase the initial sugar concentration to 10% or more,
There are reports of mannitol production (Zeitschriftfu¨r Allg.Mikrobiologie,
6.4. (1966) 323-328) Despite the high concentration of sugar remaining in the culture solution, the production of mannitol stops midway. In other words, it was impossible to produce mannitol with high yield and high accumulation. Yeast The yeast that produces mannitol is Saccharomyces.
Seke; 4.15g, J.Ferment.Technol 16 ;
597-598 (1937)) Torulopsis species; 0.5-2.2 g/dl
Applied Microbiol. 16 1841−1852 (1968)),
Cryptococcus neoformis; 0.13g/dl;
(same as the genus Torulopsis), sugar-tolerant yeast (65% sugar yield including glycerol; Abstracts of the 1984 Fermentation Engineering Society Conference, p. 168), etc. However, mannitol production by yeast that has been reported so far has a low yield of less than 30%, and has the disadvantage that sugar alcohols other than mannitol are produced as by-products. Molds Mannitol-producing molds have been reported to include ascomycetes such as Aspergillus and Penicillium. Among them, a high-producing strain of Aspergillus candidas (8.4 g, 50%) was found (Biotechnol Bioengineer, 9 365-374
(1967)) It takes a long culture time of 12 days, which is economically disadvantageous. (3) Enzymatic method A method is also known in which mannitol is produced by using fructose as a substrate and allowing mannitol dehydrogenase to act. This enzyme has already been purified from lactic acid bacteria, yeast, mold, acetic acid bacteria, etc. These purified enzymes have an optimum pH for mannitol production, ranging from weak acidity to near neutrality (PH4 to 7), but the optimum pH for mannitol production by fermentation is unknown. Mannitol production by enzymatic methods requires expensive coenzymes.
Moreover, in the enzymatic mannitol production reaction, this coenzyme changes from a reduced form to an oxidized form, so a system is required to regenerate the oxidized form into the reduced form. A method using glucose dehydrogenase as a regeneration system has also been reported, but it is still a few meters long.
The substrate concentration is about M, which is not practical. [Problems to be solved by the present invention] As shown in (2) above, in the case of mannitol fermentation using lactic acid bacteria, sugar as a carbon source is increased to 10% or more at the start of culture in order to improve productivity. Time,
Production of mannitol stops during the culture, and sugar remains in the culture solution. For this reason, high yield and high productivity of mannitol cannot be expected. In addition, the culture period is as long as 4 days. The purpose of this research is to solve these problems and produce mannitol with high yield and production in a short time. <Structure of the invention> [Means for solving the problem] In view of the above-mentioned circumstances, the present inventors have prepared
As a result of investigating various culture conditions with the aim of shortening the fermentation time of mannitol by lactic acid bacteria and reducing or eliminating residual sugar when sugar is present at a concentration of By keeping the pH within the range of 5.0 to 6.0, mannitol productivity is increased and residual sugar almost disappears.Also, by adding betaine to the culture medium at this time, the mannitol fermentation time is shortened. They discovered this and completed the present invention. That is, in the fermentation production of mannitol using lactic acid bacteria, the present invention proposes that when the initial concentration of sugar, which is a substrate, is low, the substrate disappears but the production amount of mannitol is low, and in order to increase the production amount, the initial substrate concentration is increased. In order to solve the problem of stopping the fermentation of mannitol, which may occur in some cases, when the substrate sugar concentration in the culture solution is 10% or more, the pH during the culture should not fall below 5. PH6
If the pH exceeds 6.0, by controlling the pH to 5 to 6, the productivity of mannitol can be increased, the sugar remaining in the culture solution can be reduced or eliminated, and in addition, during the culture The present invention relates to a method for producing mannitol, which is characterized by shortening the mannitol fermentation time by adding betaine to the mannitol. As the microorganism used in the present invention, any species or strain of mannitol-producing bacteria belonging to lactic acid bacteria can be used, but a preferred example is Lactobacillus brevis.
brevis, IFO 3960, 3345), Leuconostoc mesenteroides
IFO 3426) etc. The culture in the present invention may be carried out by standing still, with gentle stirring or shaking, or by standing still and introducing air, and the temperature is 15°C.
to 40°C, preferably 25°C to 37°C, and the culture time is 18 to 144 hours, preferably 36 to 96 hours. The conditions listed on the left can be selected as appropriate depending on the bacteria and medium used. As the carbon source, glucose, fructose, and sucrose may be used alone, or a mixture of fructose and glucose may be used, and the ratio may be 2 parts fructose to 1 part glucose. Examples of fructose- and glucose-containing substances include invert sugar of sucrose, isomerized sugar of glucose, and natural products such as cane molasses, beet molasses, and their decomposed substances. In the present invention, the initial PH in the culture solution is not a concern; PH control is performed to adjust the PH in the culture solution to a range of 5.0 to 6.0, and when the pH of the culture solution falls below 5.0, the pH adjustment PH with alkali such as caustic potash, caustic soda, ammonia water, ammonia, etc.
Adjust to 5.0 or higher. If the culture solution reaches a pH of 6.0 or higher, add mineral acids such as sulfuric acid, hydrochloric acid, phosphoric acid, or organic acids such as citric acid or acetic acid to bring the pH to 6.0.
Adjust the amount of betaine, which has the effect of shortening the production time of mannitol, from 0.05% to 5%, preferably 0.1
0.5% is good. When the method of the present invention is carried out, the fructose initially added to the medium is converted to mannitol with a yield of 90% or more. Yield 65% from culture medium containing dl, approx.
Mannitol equivalent to 9.5 g/dl accumulated in 40 hours. In addition, when using Leuconostocmesenteroides, glucose 10g/dl, fructose 20g/dl
Mannitol equivalent to approximately 19 g/dl accumulates in 72 hours with a yield of 63% from a culture solution containing g/dl, and an additional 0.1%
By adding betaine in advance, the culture time can be shortened to 60 hours. Known methods can be used to separate and collect mannitol from the culture fluid thus obtained. Residual glucose, fructose, product mannitol, lactic acid,
All acetic acid was determined by high performance liquid chromatography. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1

[Table] 5 ml of the medium having the medium composition (1) shown in Table 1 was placed in a test tube and heated at 120°C for 20 minutes. One platinum loop of Lactobacillus brevis (IFO 3960) was inoculated into this medium, and static culture was performed at a temperature of 30° C. for 18 hours to prepare a preculture. 50 ml of the medium having the medium composition (2) shown in Table 1 was placed in a 100 ml three-necked glass container and heated at 120° C. for 20 minutes. Add 0.5ml of the preculture solution shown above.
In addition, the cells were cultured for an additional 5 days at 30° C. under microaerobic conditions with stirring at 60-100 rpm. Regarding the culture pH, the pH was adjusted with 2NKOH to keep it constant at the pH shown in Table 2 from the start of the culture. For comparison, the same culture was performed without adjusting the pH of the culture solution. Mannitol in the obtained culture solution was quantified by liquid chromatography. The results are shown in Table 2. Although the amount of liquid increases during culture due to pH adjustment, the amount of each component is shown as a value converted to the initial amount of liquid. From Table 2, when cultured at a constant pH of 5.5,
It was observed that the yield of mannitol was good and residual sugar disappeared. On the other hand, when culturing was performed without adjusting the pH, the pH of the culture solution decreased to around 4, even though the CaCO ₃ added to the medium in advance remained at the end of the culture, and a large amount of substrate was As a result, the yield of mannitol was low.

[Table] Example 2 Effect of PH control on mannitol production by Lactobacillus brevis (IFO 3960) and optimality by controlling the PH to 5.5 and changing the sugar concentration of the medium composition (2) used in Example 1 The relationship with sugar concentration was investigated. The results are shown in Table 3.

[Table] Example 3 Leuconostocmesenteroides (IFO), a lactic acid bacterium that has the ability to produce mannitol.
3426), and an experiment similar to Example 2 was conducted using a high concentration of sugar. The results are shown in Table 4.

[Table] Example 4 Leuconostocmesenteroides (IFO
3426) to investigate the effects of betaine. The experimental method was the same as in Example 3, and the sugar concentration was glucose 10
g/dl fructose 20g/dl. The results are shown in Table 5.

【table】

Claims (1)

[Scope of Claims] 1. Lactic acid bacteria belonging to the genus Lactobacillus or Leuconostoccus and capable of producing mannitol are inoculated into a liquid medium with a sugar concentration of 10% or more, and the
When the pH is below 5, add alkali to lower the pH to 5.
After adjusting the above, when the PH in the culture solution becomes 6.0 or higher, add acid to adjust the PH to 6.0 or lower while culturing, producing and accumulating mannitol in the culture solution, and collecting this mannitol. Characteristic manufacturing method of mannitol. 2. The method for producing mannitol according to claim 1, wherein the liquid medium is a liquid medium containing betaine.