JPH0332359B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for fermentatively producing alcohol using immobilized yeast, in which a decrease in alcohol productivity due to bacterial contamination is prevented by a simple means. Bacterial contamination is always a problem in fermentation production methods, and alcohol fermentation using immobilized yeast is no exception. In a conventional batch culture method in which yeast is not immobilized, the entire apparatus can be easily sterilized by heat sterilization or the like from the point where the fermented liquid is extracted from the fermenter after completion of fermentation. However, when immobilized yeast is used, since the immobilized yeast is always present in the reactor, it is not easy to kill only the miscellaneous bacteria without killing the immobilized yeast. Therefore, in the past, if the inside of the reactor became contaminated with bacteria, the equipment was dismantled, the entire device was sterilized, and immobilized yeast was newly produced and filled into the reactor before operation was resumed. This work is very difficult and is a major problem not only in terms of labor but also in terms of reduced productivity due to reduced operating time. Particularly in the case of alcoholic fermentation, since it is necessary to mass-produce at low cost, this countermeasure against bacterial contamination was the life-or-death of the method using immobilized yeast. The present inventors conducted various studies in order to develop a simple means to solve this problem of bacterial contamination, and found that when sulfite, metabisulfite, and hypochlorous acid are applied in a specific low pH range, immobilized yeast We discovered that it is possible to efficiently kill only the bacteria while keeping the bacteria alive, and furthermore, when these compounds are added to the substrate solute, these compounds exert a bacteriostatic effect and suppress the alcohol fermentation inhibiting effect caused by the bacteria. We discovered that the alcohol production ability of immobilized yeast was not inhibited.
Based on this, we have completed the present invention. That is, the present invention provides (1) a method for performing alcoholic fermentation by introducing a substrate solution into a reactor filled with immobilized yeast, and when the inside of the reactor is contaminated with bacteria, sulfite, metabisulfite, PH containing one or more of hypochlorous acid and these salts
An alcohol fermentation method characterized by introducing a solution of 2.5 to 4.5 into a reactor, and (2) a method of introducing a substrate solution into a reactor filled with immobilized yeast to perform alcohol fermentation. The present invention relates to an alcohol fermentation method characterized by containing one or more of sulfite, metabisulfite, hypochlorous acid, and salts thereof. Yeast is not particularly limited as long as it has the ability to produce alcohol; examples include Saccharomyces formocensis.
formosensis) IFO0216, S. cerevisiae (S.
cerevisiae) Association No. 7 (Ministry of Finance Brewing Laboratory),
S.carlsbergensis, S.robustus, and S.roxyi.
rouxii). The method of immobilizing yeast is not particularly limited, and may be, for example, a method of adsorbing it to porous glass beads, etc., but the gel entrapment method is suitable for the method of the present invention. It is preferable to use so-called immobilized propagated yeast, which is obtained by immobilizing the yeast on a carrier and then culturing the immobilized product in a nutrient medium to proliferate the yeast. The type of carrier is not particularly limited, and for example, polyacrylamide, polyvinyl alcohol, agar, carrageenan, collagen, etc. can be used. The method of immobilizing yeast using a photocurable resin disclosed in Japanese Patent Publication No. 56-43234, Japanese Patent Publication No. 56-43235, Japanese Patent Publication No. 56-131391, etc. is suitable for the method of the present invention. be. Examples of photocurable resins include polyesters of unsaturated polyhydric acids such as maleic anhydride and polyhydric alcohols, polyesters of polyethylene glycol and methacrylic acid, unsaturated urethanes, and nonionic unsaturated acrylics. Examples include resins, anionic unsaturated acrylic resins, cationic unsaturated acrylic resins, unsaturated polyvinyl alcohols, unsaturated polyamides, and unsaturated epoxies. The immobilization method involves mixing one of these photocurable resins with the suspension of yeast cells, and then adding
The immobilized product obtained by irradiation with actinic rays of 250 to 600 nm may be shaped into any shape such as a sphere, a film, a sheet, or a tube. The reactor filled with immobilized yeast, the substrate solution as the fermentation raw material, and the fermentation conditions may be the same as conventional ones, such as sugar concentration of cane molasses, beet molasses, etc., of about 10 to 25 g/dl, and ammonium sulfate.
A substrate solution containing about 0.01 to 1 g/dl is passed through the reactor so that the residence time is about 2 to 10 hours, and the temperature inside the reactor is set to about 25 to 40°C, preferably 28 to 35°C.
Just keep it at around ℃. When using other raw materials, such as those made from fruit juice lees such as honey phase molasses and pineapple molasses, vitamins, organic nutrients, inorganic salts, etc. may be supplemented as appropriate depending on the raw material. Further, this substrate solution may be sterilized in advance or may be unsterilized. One of the methods of eliminating bacterial inhibition in the present invention is to remove one of sulfite, metabisulfite, hypochlorous acid, and their salts when the inside of the reactor is contaminated with bacteria in such an alcohol fermentation production method. Alternatively, it is characterized in that a solution containing two or more species with a pH of 2.5 to 4.5 is introduced into the reactor. Whether or not the reactor is contaminated with germs can be confirmed by culturing the fermentation liquid discharged from the reactor on a plate or observing it with a microscope. Also,
It can also be estimated by the decrease in alcohol production. In addition, the following sterilization operation may be performed periodically without detecting the presence or absence of bacterial contamination. The disinfectant used is sulfurous acid, metabisulfite, hypochlorous acid, or salts thereof. Suitable salts include alkali metal salts such as sodium and potassium salts, and alkaline earth salts such as calcium. Examples include sodium sulfite, potassium metabisulfite, sodium hypochlorite, and white flour. The concentration is better as it has less effect on the immobilized yeast and has a greater effect on sterilizing bacteria, but this concentration varies depending on the type of disinfectant, the pH of the solution, the amount of sugar remaining in the reactor, etc. It is best to test and determine this in advance. Generally, for example, in the case of potassium metabisulfite, about 30 to 2000 ppm is appropriate, and in the case of sodium hypochlorite, about 30 to 2000 ppm is appropriate. The pH of the solution should be within the range of 2.5 to 4.5. If the pH is higher than 5, the sterilization efficiency will be poor, and if the pH is lower than 2, the damage caused by yeast will be severe. Within this range, especially PH3.5~
A range of 4.0 is appropriate. To adjust the pH of the solution, use hydrochloric acid,
Mineral acids such as sulfuric acid and/or organic acids such as formic acid and acetic acid can be used. This adjustment may be performed before or after introducing the solution into the reactor of immobilized yeast. Next, the results of measuring the bactericidal effect on immobilized yeast and various bacteria by varying the concentration and pH of potassium metabisulfite are shown. The yeast used in this experiment was Satucharomyces formocensis IFO0216, and the immobilization method was the same as in Example 1. The bacteria used were those obtained from a reactor of a device that continuously ferments and produces alcohol using immobilized yeast. As for the experimental method, for immobilized yeast, molasses was used as the sugar concentration.
20g/dl and a solution containing ammonium sulfate 0.1g/dl at 30°C.
For bacteria, 1.0g/dl of glycol, 1.0g/dl of yeast extract,
30 in a medium with a pH of 6.0 containing 1.0 g/dl of peptone, 0.5 g/dl of NaCl, and 0.3 g/dl of malt extract.
The cells cultured at ℃ for 24 hours were used. For each bacteria, the potassium metabisulfite concentration and PH are shown in the table below.
They were added to a molasses solution with a sugar concentration of 2 g/dl at a concentration of 10 ⁷ to 10 ⁸ cells/ml, allowed to stand at 30°C for 24 hours, and the number of remaining viable bacteria was measured. The results obtained are shown in the table below.

[Table] Sugar remaining in the reactor may be mixed into the solution, but this sugar reduces the bactericidal effect. If the sugar concentration exceeds 5 g/dl, the bactericidal effect will be greatly reduced, so care must be taken to keep it below 5 g/dl. Preferably it is 2 g/dl or less. The solution may be introduced from the top of the reactor or from the bottom. After introduction, the liquid is circulated and stirred if necessary, and left for a period of time necessary to kill germs. Although this time varies depending on the type and concentration of the disinfectant, the concentration of sugar, etc., it is convenient for operation to set it to about 30 minutes to 24 hours. Thereafter, this solution is extracted from the reactor, and if necessary, a substrate solution or other medium solution is introduced into the reactor to enhance yeast culture, and then normal operation can be resumed. Another method of eliminating bacterial inhibition in the present invention is characterized in that the substrate solution itself contains one or more of sulfite, metabisulfite, hypochlorous acid, and salts thereof. In this method, sulfurous acid, etc., which were used as the above-mentioned disinfectant, are used as a bacteriostatic agent, and the growth of various bacteria in the substrate solution is prevented and alcohol fermentation is carried out smoothly. The concentration should be sufficient to exert the bacteriostatic effect without reducing the alcohol production ability of the immobilized yeast, but this concentration varies depending on the type of sulfite, etc., the pH of the solution, the sugar concentration, etc. It is best to determine this by testing in advance. However, for example, in the case of potassium metabisulfite, it is usually about 500 to 1500 ppm, and in the case of sodium hypochlorite, it is usually about 500 to 2000 ppm. The lower the PH of the substrate solution, the better, PH2.7~
About 5.5, especially about 3.5 to 5 is appropriate. Other than this, the substrate solution may be the same as usual, and the fermentation conditions using this substrate solution may be the same as usual. It goes without saying that the two methods described above may be used alone or in combination. In the method of the present invention, alcoholic fermentation is carried out while exerting a bacteriostatic effect by adding a specific bactericide such as potassium metabisulfite to the substrate, thereby controlling the reaction system. The number of sterilizations is greatly reduced.The other method is to use a specific sterilizing agent such as potassium metabisulfite at a specific pH as a sterilizing solution, which reduces the number of sterilizations in the reactor during sterilization. Eliminates disassembly work. The method of the present invention greatly reduces the labor involved in sterilization work during alcohol fermentation, and also greatly contributes to cost reduction. Examples are shown below. Example 1 A suspension of Satucharomyces formocensis IFO0216 was added to a urethanized prepolymer of polyethylene glycol, isophorone diisocyanate, and 2-hydroxyethyl methacrylate with an average molecular weight of about 5000, and benzoin ethyl ether was added as a photosensitizer. were added and uniformly dispersed using a homogenizer. This dispersion was irradiated with a low-pressure mercury lamp with a dominant wavelength of 360 nm for about 3 minutes to produce a film-like immobilized yeast with a thickness of about 1 mm. This immobilized yeast is cut into rectangular shapes and arranged in a rectangular cylindrical tank with a length and width of 90 mm and a height of 700 m via spacers, and these tanks are connected in four stages to form one reactor. A reactor consisting of three reactors connected in series was used. An unsterilized substrate solution containing molasses with a sugar concentration of 20 g/dl and ammonium sulfate 0.1 g/dl was passed through the reactor so that the residence time in the reactor was 5 hours.
Alcohol was produced continuously at 30°C with ventilation. Then, once a day, the alcohol-containing liquid flowing out from the reactor was removed to a synthetic medium for bacteria containing the above-mentioned moldicidin, and cultured, and the presence or absence of bacteria was monitored for 350 hours. It was discovered that bacteria had developed. Therefore, the reaction solution was extracted from the reactor, and a molasses solution with a pH of 3.5 and a sugar concentration of 2 g/dl was charged into the reactor, and then sodium hypochlorite was added to the reactor so that the chlorine concentration was 500 ppm. After circulating at 30℃ for about 3 hours, this liquid was extracted and molasses with a sugar concentration of 20g/dl and
Filled with a substrate solute containing 0.1 g/dl ammonium sulfate and heated at 30°C.
Enhancement culture was repeated four times with aeration while maintaining the temperature, and then normal operation was resumed. The alcohol concentration of the reactor effluent immediately before this sterilization treatment was 7.2 g/dl, and the number of bacteria was 4×.
However, the alcohol concentration of the reactor effluent after this treatment was ^7.7 g/dl, and the number of miscellaneous bacteria was 0. Example 2 Saccharomyces cerevisiae association No. 7 bacteria was used instead of Saccharomyces formocensis IFO0216 to immobilize it in the same manner as in Example 1, and this immobilized yeast was installed in the same reactor as in Example 1 to produce alcohol by fermentation in the same manner. During this process, it was confirmed that bacteria had grown inside the reactor 400 hours after the start of production. Then, the reaction liquid was extracted from the reactor and put into the reactor.
Filled with molasses solution with a sugar concentration of 5 g/dl at pH 3.5,
Potassium metabisulfite was added to give a concentration of 1000 ppm as SO ₂ . After the liquid was circulated at 30°C for 3 hours, the liquid was extracted and normal operation was resumed in the same manner as in Example 1. The alcohol concentration of the reactor effluent immediately before this sterilization treatment was 7.4 g/dl, and the number of bacteria was 5x.
However, the alcohol concentration of the reactor effluent after this treatment was ^7.9 g/dl, and the number of miscellaneous bacteria was 0. Example 3 Using the same reactor equipped with the same immobilized yeast as in Example 1, potassium metabisulfite was added to the reactor.
Alcohol was continuously produced for 600 hours in the same manner as in Example 1 except that SO ₂ was added at 1000 ppm, and the same substrate solution (PH5.0) as in Example 1 was supplied. During this period, the alcohol concentration of the alcohol-containing liquid flowing out from the reactor was approximately constant at about 80 g/dl, and the number of bacteria was also approximately constant at 10 ² cells/ml.

Claims (1)

[Claims] 1. In a method for performing alcoholic fermentation by introducing a substrate solution into a reactor filled with immobilized yeast, when the inside of the reactor is contaminated with bacteria, sulfite, metabisulfite, An alcoholic fermentation method using immobilized yeast, which comprises introducing into a reactor a solution with a pH of 2.5 to 4.5 containing one or more of chlorous acid and salts thereof. 2. A substrate containing one or more of sulfurous acid, metabisulfite, hypochlorous acid, and salts thereof, in a method of carrying out alcoholic fermentation by introducing a substrate solution into a reactor filled with immobilized yeast. An alcohol fermentation method using immobilized yeast, characterized by carrying out alcohol fermentation using a solution.