JPH0368670B2 - - Google Patents

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 yeast is cultured using carbohydrates as a substrate, if the amount of substrate supplied becomes excessive, the supplied sugars are 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. Also,
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, a method has been proposed in which the oxygen and carbon dioxide concentrations in exhaust gas are measured and the substrate supply rate is controlled using the molar ratio of the amount of carbon dioxide produced to the amount of oxygen consumed (respiratory quotient = RQ) as an index.

RQ is a value obtained by taking the ratio of the amount of carbon dioxide produced (Q _CO2 ) and the amount of oxygen consumed (Q _O2 ) per unit cell and unit time, so there is no need to directly calculate the amount of cells. However, it is advantageous for online measurement control. However, the absolute values of the carbon dioxide generation rate (I _CO2 ) and the oxygen consumption rate (I _O2 ) vary greatly depending on the amount of bacterial cells and the growth rate of the bacterial cells. Furthermore, the amount of substrate required also varies greatly depending on the amount of bacterial cells and the growth rate of the bacterial cells. Therefore, with simple on-off control of substrate addition, when the amount of bacterial cells is small, the RQ value fluctuates greatly compared to the amount of substrate supplied, and when the amount of bacterial cells increases, there is a risk of insufficient substrate supply. There is.

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 as the bacterial cell concentration increases. 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 quickly, easily, and accurately online, 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 cultured aerobically using glucose 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.

C ₆ H ₁₂ O ₆ (glucose) + 6O ₂ → 6 _CO2 + 6H ₂ O + 38ATP (1) In addition, the mass balance when propagating only by ethanol fermentation is expressed as follows.

C ₆ H ₁₂ O ₆ (glucose) → 2CO ₂ +2CH ₃ CH ₂ OH + 2ATP …(2) Here, the CO ₂ generation due to oxygen respiration is Q _CO2 (ox.) Oxygen consumption is Q _O2 , and the CO ₂ generation due to fermentation is Q _CO2 (Ferm.)
Then, the value of RQ in the case of growth using only oxygen respiration is as follows.

RQ = Q _CO2 (ox.) / Q _O2 = 1 The value of RQ in the case of growth by fermentation only is as follows.

RQ = Q _CO2 (Ferm.) / Q _O2 = ∞ (because Q _O2 = 0) When respiration and fermentation occur at the same time, the RQ value is as follows.

1 < RQ (= Q _CO2 (ox.) + Q _CO2 (Ferm.) / Q _O2 = 1 + Q _{CO2 (Fern.)} / Q _O2 ) < ∞ Yield by respiration is Yox., yield by fermentation is
If YFerm. is used, then YFerm.Yox./19 is obtained from equations (1) and (2).

Here, if respiration and fermentation are occurring at the same time, the growth yield of bacterial cells is Y, then Y=1×Yox.+Q _CO2(Fern.) ×3×Y _Fern. /Q _O2 /1+Q _C
O2(Fern.) ×3/Q _O2 =Yox.+(RQ-1)×3×YFerm./1+(RQ-1)×
3 = (6/19+3/19RQ)/3RQ-2・Yox.…(3). Since it is generally assumed to be Yox.0.5, substituting this into equation (3) yields Y=1/38・16+3RQ/3RQ−2 ……(4).

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

X=X ₀ +∫ ^t _p [1/38・16+3RQ/3RQ−2 {FS _R −(V+dV)S}/(V+dV)]dt …(5) Here, [g/],
Xo is the initial bacterial concentration [g/], F is the flow rate of substrate addition [/hr], S _R is the reference concentration of the added solution [g/],
S represents the remaining substrate concentration in the tank [g/], V represents the initial culture solution volume [], and dV represents the amount of increase in the fluid volume at time t [].

dV=Fdt 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 these values, and the RQ value is approximately 1 (for example, 1.0 to 1.5,
The amount of substrate added is preferably controlled to be in the range of 1.05 to 1.10. Furthermore, the bacterial cell concentration can be calculated based on equation (5) from the RQ value and the flow rate of the additive solution, and the flow rate of the additive solution 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 flowchart showing a method for controlling yeast culture according to the method of the present invention. In the diagram
indicates the process of inputting O ₂ and CO ₂ concentration values, indicates the process of calculating the RQ value, indicates the process of calculating the yield Y value, indicates the process of calculating the bacterial cell amount X value, and indicates the process of stopping the pump, is the pump operating time t determined (t=
kX) indicates the process of pump operation.

An actual culture example will be explained below.

Example 1 Baker's yeast (Saccharomyces
cerevisiae, IFO 2044), glucose (initial charge) 10 g/, ammonium sulfate 2 g/, potassium phosphate 1 g/, magnesium sulfate 0.5 g/, sodium chloride 0.1 g/
, various vitamins and metal salts were dissolved in 0.1 g of calcium chloride dihydrate.

The culture conditions were controlled at a temperature of 30°C and a pH of 5.0 using a 14-volume jar fermenter. RQ was calculated from the analysis value of exhaust gas, and the substrate addition pump was activated when this value was 1.05 or less, and stopped when it was 1.10 or more. 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.

As a result, after 110 hours of culture, the bacterial cell concentration was 47 g/g, and the bacterial cell yield was 0.48 [g bacterial cells/g glucose].

When the culture solution was sampled during the cultivation and the bacterial weight was measured by turbidity method, the result was in good agreement with the value estimated based on the RQ value.

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

[Brief explanation of drawings]

FIG. 1 is a diagram explaining the steps of the method of the present invention.

Claims (1)

[Claims] 1. In a method of culturing yeast while feeding a culture substrate, the growth yield is estimated from the value of the respiratory quotient, and the amount of yeast cells is estimated from the estimated yield and the amount of the substrate fed. A method for culturing yeast, characterized in that a culture substrate is fed in accordance with the amount of bacterial cells and changes thereof. 2. A patent that controls the amount of fed-batch substrate so that it decreases while the estimated value of bacterial mass is small, and increases as the estimated value of bacterial mass increases. The culture method according to claim 1.