JPS59173086A - Alcoholic fermentation process using immobilized yeast - Google Patents

Abstract

PURPOSE:To achieve the selective extermination of sundry germs, in high efficency, without lowering the alcohol productivity of an immobilized enzyme, by treating the enzyme with sulfurous acid, metabisulfite, etc. within a specific pH range. CONSTITUTION:Alcoholic fermentation is carried out by introducing a substrate solution into a reactor filled with an immobilized enzyme. When the reactor is contaminated with sundry germs, a solution containing one or more compounds selected from sulfurous acid, metabisulfate, hypochlorous acid and their salts and having a pH of 2.5-4.5, preferably 3.5-4.0 is introduced into the reactor. As an alternative method, the substrate solution used in the above alcoholic fermentation is added with one or more compounds selected from sulfurous acid, metabisulfite, hypochlorous acid and their salts in an amount to give a pH of about 2.7-5.5.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for fermentative production of 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 big 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 to develop a simple means to solve the problem of bacterial contamination, and found that when sulfite, metabisulfite, and hypochlorous acid are applied in a specific low pH range, immobilized yeast can be destroyed. We have discovered scales that can efficiently kill only bacteria while keeping them alive, and if we add these compounds to the substrate solute, these compounds will exert a bacteriostatic effect and suppress the alcohol fermentation inhibiting effect caused by bacteria. The present invention was completed based on the discovery that the ability of yeast to produce alcohol is not inhibited.

That is, the present invention provides (11) 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, An alcoholic fermentation method characterized by introducing into a reactor a solution with a pH of 2.5 to 4.5 containing one or more of chlorous acid and these salts, and (2) filling with immobilized yeast. A method for carrying out alcoholic fermentation by introducing a substrate solution into a reactor, characterized in that the substrate solution contains one or more of sulfurous acid, metabisulfite, hypochlorous acid and salts thereof. The invention relates to an alcoholic fermentation method.

Yeast is not particularly limited as long as it has the ability to produce alcohol, and examples include Saccharomyces 7ormocensis (Saccharomyces formocensis).
sensis) IFOO216, same cereviche (S.

Cerevlsiael Association Bacteria No. 7 (Ministry of Finance Brewing Association Bacteria No. 7)
ensis).

Robustus female (S, robustus), Roxyi (S, robustus)
S.

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 kale entrapment method is suitable for the method of the present invention. It is preferable to use so-called immobilized and propagated yeast, which is obtained by immobilizing yeast cells of 20% on a carrier and then culturing the immobilized product in a nutrient medium to propagate yeast.The type of carrier is not particularly limited. For example, polyacrylamide, polyvinyl alcohol, agar, carrageenan, colladan, etc. can be used. A method of immobilizing yeast using a curable resin is suitable for the method of the present invention. Examples of photocurable resins include polyesters of unsaturated polybasic acids such as maleic anhydride and polyhydric alcohols, and polyethylene. Polyesters of glycol and methacrylic acid, unsaturated urethanes, nonionic unsaturated acrylic resins, anionic unsaturated acrylic resins, cationic unsaturated acrylic resins, unsaturated polyvinyl alcohol, unsaturated polyamides,
Examples include unsaturated epoxies.

The immobilization method involves mixing any of these photocurable resins with the yeast cell suspension, and adding 250 to 6
The immobilized product obtained by irradiation with 00 nm actinic light 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 that is the fermentation raw material, and the fermentation conditions may be the same as conventional ones.
A substrate solution containing about 51% of ammonium sulfate and about 0.01 to 1 t/dt of ammonium sulfate was passed through the reactor so that the residence time was about 2 to 10 hours, and the temperature inside the reactor was set to 25 to 40%. The temperature can be kept at about 0.degree. C., preferably about 28 to 35.degree. When using other raw materials such as those made from fruit juice lees such as honey phase molasses and pineapple molasses, bimins, 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 left 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. or PH2 containing two or more types
, 5 to 4,5 are introduced into the reactor.

Whether or not the reactor is contaminated with bacteria can be confirmed by culturing the fermentation liquid discharged from the reactor on a plate or observing it with a microscope. It is also possible to estimate it based on the decrease in alcohol production. In addition, the following sterilization operation may be performed 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 metal salts such as calcium. Examples include sodium sulfite, potassium metabisulfite, sodium hypochlorite, and white flour. The concentration is better as it has less influence on the immobilized yeast and has a greater effect on sterilizing bacteria, but this concentration varies depending on the type of disinfectant, the amount of sugar remaining in the PR1 reactor of the solution, etc. It would be better to test and determine this in advance.

In general, for example, in the case of potassium metabisulfite, 3
Approximately 0 to 2000 ppm, and in the case of sodium hypochlorite, 30 to 2000 ppm is appropriate.

The pH of the solution is in the range 2.5 to 4.5. PH5
If the pH is above 2, the sterilization efficiency will be poor, and if it is below 2, the damage to yeast will be severe.
．． A range of 0 is appropriate.

Next, the results of measuring the bactericidal effect on immobilized yeast and various bacteria by varying the concentration and P'H of potassium metabisulfite are shown.

The yeast used in this experiment was Satucharomyces formocensis IFOO216, and the immobilization method was the same as in Example 1. The one obtained from the reactor was used. The experimental method for immobilized yeast is to use molasses as a sugar concentration of 20? /dt and ammonium sulfate at 0.1
Use a solution that has been pre-cultured at 30°C for 24 hours in a solution containing f/dt, and for bacteria, glucose 1.0 r/d)
, yeast extract 1.0 f/dt % Vept 71. O
t/dl, NaC60,! M/dt and malt extract 0.31! , /dt and cultured at 30° C. for 24 hours. and,
Potassium metabisulfite concentration and pH shown in the table below for each image
The bacteria were added to a molasses solution with a sugar concentration of 2 f/dt at a concentration of 107 to 108 cells/-, and the number of viable bacteria remaining was measured at 30°C for 24 hours. The results obtained are shown in the table below.

Sugar remaining in the reactor may be mixed into the solution, but this sugar reduces the bactericidal action. In particular, if the sugar concentration exceeds 5 ff/dt, the bactericidal effect will be greatly reduced, so care must be taken to keep it below 5 ff/dt. It is preferable to set the t/c) to 2 t/c or less.

The solution may be introduced from the top of the reactor or from the bottom. After introduction, the solution is circulated and stirred as needed for a period of time necessary to kill germs. Although the 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, the solution may be extracted from the reactor, and if necessary, a substrate solution or other medium solution may be introduced into the reactor to enhance and culture the yeast, and then normal operation may 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 sulfurous acid, meta-1 (sulfurous acid, hypochlorous acid, and tonera salt).

In this method, sulfite, which was used as the bactericidal agent described above, is used as a bacteriostatic agent to prevent the growth of germs in the substrate solution and to facilitate alcoholic fermentation.

The concentration should be determined so that the immobilized yeast has sufficient antibacterial effect without reducing its alcohol production ability, but the concentration should be determined in advance because it varies depending on the type of sulfite, etc., the PH1 sugar concentration of the solution, etc. It is best to determine this through testing. 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, and the pH is between 2.7 and 5.
．． A value of about 5, particularly about 3.5 to 5, is appropriate.

The substrate solution may otherwise be the same as usual, and the fermentation conditions using this substrate solution may also be the same as usual.

You may use only one of the two methods mentioned above.
It goes without saying that they may also be used together.

In the method of the present invention, alcoholic fermentation is carried out while exerting a bactericidal effect by adding a specific bactericide such as potassium metabisulfite to the substrate, thereby greatly increasing the number of times the reaction system is sterilized. The other method is to use a specific disinfectant such as potassium metabisulfite at a specific pH as a disinfectant solution, which eliminates the need to disassemble the reactor during disinfection. I have to.

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 IFOO216 was added to a urethanized prepolymer of polyethylene glycol, inphorone diisocyanate, and methacrylic@-2-hydroxyethyl with an average molecular weight of about 5000, and benzoin was added as a photosensitizer. Add ethyl ether and disperse the straw uniformly with a homogenizer.
7. This dispersion was irradiated with a low-pressure water iron nail having a main wavelength of 360 nm for about 3 minutes to produce a film-like immobilized yeast with a thickness of about 1 layer.

This immobilized yeast was cut into a rectangular shape with a length and width of 90 m.
A reactor is formed by arranging rectangular cylindrical tanks with a height of 700 m via spacers, and connecting these tanks into 4 stages to form a reactor.
A series of reactors connected in series was used as the reactor.

An unsterilized substrate solution containing molasses with a sugar concentration of 20 f/dt and ammonium sulfate 0.1 t/dt was passed through this reactor so that the residence time in the reactor was 5 hours, and ventilation was performed. Alcohol was continuously produced from moths at 30°C. Then, once a day, the alcohol-containing liquid flowing out from the end reactor was removed to the synthetic medium for feeding seedlings to which the above-mentioned moldicidin was added and cultured, and the presence or absence of germs was monitored, and 350 hours passed. It was discovered that bacteria had developed. Then, the reaction liquid was extracted from the reactor and the pH of the reactor was 3.
In Step 5, a molasses solution with a sugar concentration of 217 dt was charged, and then sodium hypochlorite was added so that the chlorine concentration was 500 ppm. After circulating the liquid at 30°C for about 3 hours, the liquid was extracted and molasses with a sugar concentration of 20 f/dt and 0.
．． Pour a substrate solute containing ammonium sulfate at 1 f/dt and heat at 30°C.
Enhancement culture was repeated 4 times with aeration and maintained at a constant temperature, and then normal operation was resumed.

The reactor effluent immediately before this sterilization treatment had an alcohol concentration of 7.2 f/di and a brood count of 4 x 10/
-, but the reactor effluent after treatment of jin was alcohol-1
The concentration was 7.7 t/dt, and the number of common bacteria was 0/d.

Example 2 Instead of Satucharomyces formocensis IFOO216, Satucharomyces cerevisiae association 7 aromatic bacteria was immobilized in the same manner as in Example 1, and this immobilized yeast was installed in the same reactor as in Example 1 and alcohol fermentation was carried out in the same manner. During production, it was confirmed that bacteria had grown in the reactor 400 hours after the start of production.

Then, the reaction liquid was extracted from the reactor, and PH3 was added to the reactor.
In step 5, add molasses solution with a sugar concentration of 5 f/dt,
Potassium metabisulfite was added to 1000 ppm. 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 t/dt and the number of miscellaneous bacteria was 5 x 107 cells/ml, but the alcohol concentration of the reactor effluent after this treatment was was 7.9 fi'/dt, 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 as 802 and 1
Alcohol was produced continuously for 600 hours in the same manner as in Example 1 by supplying the same substrate solution (pH 5,0) as in Example 1, which was added at a rate of 0.000 pprn.

During this time, the alcohol concentration of the alcohol-containing liquid flowing out from the reactor was approximately constant at about 80 f/dt, and the number of miscellaneous bacteria was also approximately constant at 10/-.

Patent applicant New fuel oil development technology research association representative
Patent Attorney 1) Masahiro Naka 15- 491-

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, P containing one or more of chlorous acid and towara salt
! (25-4; Alcohol fermentation method 2 using immobilized yeast, characterized by introducing the solution of 5 into a reactor. Alcohol fermentation is carried out by introducing a substrate solution into a reactor filled with immobilized yeast. a method characterized in that the substrate solution contains one or more of sulfurous acid, metabisulfite, hypochlorous acid, and salts thereof;
Alcohol fermentation method using immobilized yeast