JPS60141283A - Cultivation of yeast - Google Patents

Abstract

PURPOSE:To estimate the amount of cultured yeast cells easily, rapidly and accurately, on line, by estimating the amount of the yeast cells from the amount of added substrate and from the yield of cells estimated from the respiratory quotient of the yeast, and adding the substrate according to the change in the estimation. CONSTITUTION:First, the yield of yeast cell is estimated from the respiratory quatient which is molar ratio of the carbon dioxide gas produced by the yeast to the oxygen consumed by the yeast. Thereafter, the amount of the yeast cells is estimated from the estimated yield and the amount of the substrate added to the system, and the yeast is cultured by adding the culture substrate according to the amount of the yeast cells and the change in the amount of the cells. The addition of the culture substrate is preferably controlled by a computer.

Description

[Detailed description of the invention] The present invention relates to a method for culturing yeast.

Generally, in the aerated culture of yeast using carbohydrates as the main carbon source, a method is used in which a substrate is supplied continuously or intermittently, as in the case of, for example, the culture of baker's yeast. As for the method of supplying the substrate, based on conventional experience, a method is used in which an appropriate supply rate is set before culturing, and the substrate is supplied based on that rate.

However, when culturing yeast using carbohydrates as a substrate,
When the amount of substrate supplied becomes excessive, the supplied sugar is converted to ethanol. This is called aerobic fermentation. Therefore, the production yield of yeast cells relative to the amount of substrate supplied (yield relative to sugar)
decreases. Furthermore, when the amount of substrate supplied is insufficient, yeast enters a state of substrate starvation and productivity decreases.

Therefore, in culturing yeast, it is desirable to control the amount of substrate supplied to just the right amount while suppressing the production of ethanol by aerobic fermentation.

In recent years, the concentration of oxygen and carbon dioxide in exhaust gas has been measured, and the rate of substrate supply has been controlled using the molar ratio of the amount of carbon dioxide produced to the amount of oxygen consumed (respiratory quotient = RQ) as an index. A method was proposed.

RQ is the amount of carbon dioxide produced per unit cell and unit time (
Since it is a value determined by taking the ratio of Q co, ) and oxygen consumption (Qo, ), there is no need to directly calculate the amount of bacterial cells, which is advantageous for online measurement control. However, the absolute values of the carbon dioxide gas generation rate (100*) and the oxygen consumption rate (0.0) vary greatly depending on the amount of bacterial cells and the growth rate of the bacterial cells. In addition, the amount of substrate required varies greatly depending on the amount of bacterial cells and the growth rate of the bacterial cells. For this reason, simple on-off control of substrate addition requires
When the amount of bacterial cells is small, the value of 'RQ fluctuates greatly compared to the amount of substrate supplied, and when the amount of bacterial cells increases, there is a risk of insufficient supply of substrate.

In order to prevent the above-mentioned problems, it is desirable to increase the substrate supply rate according to the growth rate and the amount of yeast cells.

However, there are many technical problems in estimating the amount of bacterial cells in culture online, and it is particularly difficult when the bacterial cell concentration becomes high. Therefore, a method of estimating the amount of bacterial cells from various data can be considered. Generally, the method of estimating the growth rate of microorganisms is to calculate it from the amount of carbon dioxide gas generated and the amount of oxygen consumed, but in the case of yeast, the yield of growth due to oxygen respiration and the rate of growth due to fermentation Yields vary greatly, and even under aeration and agitation conditions, respiration and fermentation occur simultaneously depending on the amount of sugar supplied, so the amount of bacterial growth cannot be estimated by simply measuring the amount of carbon dioxide gas produced or the amount of oxygen consumed. Therefore, as a result of considering a method for estimating the amount of bacterial cells online, quickly, easily, and accurately, we found that by considering the mass balance of sugar metabolism in yeast, the growth yield of yeast cells can be expressed as a function of RQ, and the Using this value, we devised a method to estimate the amount of yeast cells proliferating from the amount of inflow substrate. The rationale for this is described below.

When yeast is cultivated aerobically using gluten as a carbon source,
Fermentation is generally suppressed by oxygen based on the Pasteur effect. However, when the amount of sugar intake increases, fermentation occurs even in the presence of oxygen (this phenomenon is called the Crabtree effect, and ethanol fermentation that occurs in this state is called aerobic fermentation).

The mass balance equation when yeast is growing only by oxygen respiration is expressed as follows.

C6) 112011 (Kunle course) +602 → 600
2+6 food 0-1-! +8ATP... (1) The mass balance of mata and the s-coat grown only by ethanol fermentation is expressed as follows.

'aHn06 (?7 curse) → 2002+20H30H
,,OH+2ATP...(2) Here, 002 generation due to oxygen respiration is Qc O, (ox, ), oxygen consumption is QO2, and 0C12 generation due to fermentation is Qco, (Far
m), the value of RQ in the case of growth with only oxygen respiration is as follows.

RQ = Qco, (ox, )/QO, = 1
The value of RQ in the case of growth by fermentation only is as follows.

RQ= QQOt (Ferm)/Q(1,=CD
(Since Qo, =O), the RQ value when respiration and fermentation occur simultaneously is as follows.

Yield by respiration is YOX, yield by fermentation is yy, r
, then +1), (from equation 21, YF8r, koYox- 9 ).

Here, let Y be the growth yield of bacterial cells when respiration and fermentation occur simultaneously. Generally, Yox,': 0.5, so substituting this into equation (3) yields the following.

Estimating the bacterial cell concentration based on this value is as follows.

X=Xo ten houses (','””” (FSi-(v+av)
S)/. 385RQ-2 (v-)av) ) at・・・・・・・・・・・・(
5) Here, X is the bacterial cell concentration at time t, x
O is the initial bacterial concentration [l/l], and A is the flow rate of substrate addition (1
/ hr), sR is the standard concentration of the additive solution C9/l”), S
is the residual substrate concentration in the tank [sh/l], and v is the initial culture solution volume [j? ), aV represents the increase in liquid volume 1') at time t.

(lV=F(it becomes.

In an actual culture system, the carbon dioxide concentration and oxygen concentration in the exhaust gas are measured, the RQ value is calculated from the measured values, and the RQ value is approximately 1 (for example, 1.0 to 1.5, preferably 1.
The amount of substrate added is controlled to be in the range of 0.05 to 1.10. Furthermore, the bacterial cell concentration can be calculated based on equation (5) from the value of RQ and the flow rate of the additive liquid, and the flow rate of the additive liquid can be changed based on the calculated value. The above has the advantage that it can be easily carried out by controlling culture measurement using a computer, and calculations are also simple.

FIG. 1 is a frame diagram showing a method for controlling yeast culture according to the method of the present invention. In the figure, ■ indicates the process of inputting the ring and CO2 concentration values, ■ indicates the calculation of the value of RQ, and ■ indicates the yield Y.
■ indicates the process of calculating the value of the bacterial mass X, ■ indicates the process of stopping the pump, ■ indicates the process of determining the pump operating time (t = kx), Show the process.

An actual culture example will be explained below.

Example 1 Baker's yeast (Saccharomyces c.
erevl,s:Iae.

■To 2044) was used, and glucose (To 2044) was used as the medium.
Initial charge R) 10 c'/l + ammonium sulfate 2
9/l! , potassium phosphate 19/l, magnesium sulfate 0.59/l! , sodium chloride 0.1f7/J, calcium chloride dihydrate 0.1l/l, and various vitamins.

A solution containing a metal salt was used.

The culture conditions were as follows: using a 14j' jar 7 armer;
Temperature 30°C, pm5. It was controlled to o. RQ was calculated from the analysis value of the exhaust gas, and when this value was 1.05 or less, the substrate addition pump was activated, and when it was 1.10 or less, it was stopped. In addition, the amount of bacterial cells was calculated from the RQ value and the substrate addition rate, and the substrate addition rate (pump operation time) was increased in proportion to it.

The result was a bacterial cell concentration of 479/l after 110 hours of culture.

The bacterial cell yield was (L48 (g bacterial cells/g glucose).0 When the culture solution was collected during the culture and the bacterial weight was measured by the turbidity method, it was found to be similar to the value estimated based on the RQ value. Agreed.

In the above culture, measurements, controls, etc. were performed using a personal computer.

[Brief explanation of the drawing]

FIG. 1 is a diagram explaining the steps of the method of the present invention. Patent applicant: Toyo Soda Kogyo Co., Ltd. Engraving of drawings (no change in content) No. 11 JP-A-Sho GO-141283 (4) Procedural amendment (method) % formula % 1 Indication of case Patent Application No. 245529 of 1982 2 Name of the invention Yeast cultivation method Relationship with the case concerning the person making the amendments Patent applicant address 560 Oaza Tomita II, Shinnanyo City, Yamaguchi Prefecture 746 Toyo Soda Kogyo Co., Ltd. Patent Information Department Telephone number (585) 3311 6 Amendments Target (1) Full text of the specification (2) Full text of the drawings 7 Contents of amendments As shown in the attached sheet (no changes to the content, type engravings are to be submitted for the specification, and drawings in black drafting ink are to be submitted for the drawings) 68 Attachment List of documents (1) 1 copy of engraving specification (2) 1 copy of engraving drawings

Claims (2)

[Claims] (1) In the method of culturing yeast while feeding a culture substrate, the bacterial yield is estimated from the value of the respiratory quotient, the amount of yeast cells is estimated from the estimated yield and the feeding amount of the substrate, and the bacterial cell yield is estimated from the value of the respiratory quotient. A method for culturing yeast, characterized by feeding a culture substrate according to the body mass and changes thereof. (2) Control the amount of fed-batch substrate so that it becomes smaller while the estimated value of bacterial mass is small, and so that the amount of fed-batch substrate increases as the estimated value of bacterial mass increases. , the culture method according to claim 1.