JPH01157395A - Method for culturing microorganism - Google Patents

Abstract

PURPOSE:To produce desired fermentation product, by subjecting the residual mother liquid obtained by separating latex from milk of natural rubber to concentration treatment, etc., to give fermentation medium and inoculating and propagating a fungus or bacterium into the culture medium. CONSTITUTION:The mother liquor (natural rubber serum) of residue obtained by separating a latex from natural rubber milk is concentrated or powdered by spray-drying method and the resultant concentrate or powder is directly used as a fermentation culture medium or added to a fermentation culture medium to prepare a fermentation culture medium. Then a bacterium (containing actinomyces) or fungus (yeast or filamentous fungus) is inoculated into the fermentation culture medium and propagated to produce the microbial cell and/or accumulate a specific fermentation product. The natural rubber serum discharged from plants, etc., in south east asia can be used as a raw material for the culture medium and effective utilization of the natural rubber serum can be contrived according to the above-mentioned method.

Description

[Detailed Description of the Invention] The remaining mother liquor after separating latex from natural rubber sap (
Natural Rubber 5erun (hereinafter referred to as NR8 for short) is, so to speak, industrial waste from natural rubber production, and the neglect of this in natural rubber producing countries is becoming impossible to ignore in terms of environmental conservation. Moreover, NR3
Because it contains large amounts of protein, organic acids, sugars, and their derivatives, it is thought to have a rich nutritional value for microorganisms, and if left untreated, it could cause major environmental pollution. On the other hand, if it can be recycled, it has the potential to become a new and large biomass.

The inventor has investigated the N emissions from Toho Asia's latex factory.
Use R3 as a raw material and concentrate it, or make powdered NR3.
Experiments using liquid or powdered NR3 revealed that it has a growth-promoting effect on various microorganisms,
It was found that it contains essential factors for growth.

In general, microorganisms often require a variety of nutritional factors for their growth, including a variety of amino acids, vitamins, and minerals, and often require multiple nutritional factors, not just humans. Therefore, fermentation media use natural organic nutrients, such as organic nitrogen sources, to provide multiple nutritional factors. These include defatted soybean hydrolyzate (IIVP), Corn 5teep
Liquor (C3L), cottonseed meal, peanut meal, pharlan+cdia, distille
There are a wide variety of sources, including RS 5olable, livestock blood, slaughter waste fluid, and casein thawed product. Moreover, when using these as fermentation raw materials for industrial production, it is essential that they be inexpensive, that they can be supplied stably rather than seasonally or suddenly, and that their quality is stable. It would be desirable to have a universal product that is widely effective against various microorganisms. Of the many natural organic nutritional sources mentioned above, only iVP, C3L, yeast extract, etc. satisfy these conditions, but MVP and C3L are each manufactured as by-products of the food industry. However, recently, due to changes in production methods, costs have increased and supplies have decreased, and yeast extract is expensive and has many disadvantages as an industrial raw material.

The general properties of powder NR3 are: C22% H5.5% N 9.5% ^sh 18.5% according to the elemental analysis values, and looking at the balance of 11 IN, insoluble nitrogen compounds account for 6%. , the insoluble nitrogen-free compound is 1.5χ
, 16.5% soluble organic nitrogen compounds, 28% soluble nitrogen-free compounds, 28% ammonium sulfate, ash 1
8%, moisture 2%.

Soluble nitrogen-free compound! l-! The content of R acids and reducing sugars that are likely to be fermentation raw materials in IJ 28% is small, and NR3 is unlikely to be a C source for fermentation, but it cannot be said to be a simple organic nitrogen source.

According to the experimental results, NR3, regardless of its liquid form,
Even as a powder, it has a wide range of growth-promoting effects on bacteria and fermentation, and contains yeast extract, 1-IVP, and C3L as organic nitrogen sources for the medium.
It has become clear that the purpose of fermentation production can be sufficiently achieved by replacing these with NR8 in fermentation using NR8 and the like. Moreover, in the case of bacteria, the effect was similar for both aerobic and anaerobic bacteria.

Furthermore, in some microorganisms, similar effects were observed even when NR3 was hydrolyzed by protease treatment before being added to the medium, or when it was added to the medium as it was without hydrolysis.

Considering these experimental facts, the effect of NR3 on microorganisms is not only due to the effects of the proteins it contains and the amino acids produced by its decomposition, but also to unidentified substances contained in NR3 and its interaction with proteins. This is thought to be due to the combined effect of

The present invention will be explained below with reference to Examples.

Example 1 Glucose 10 (1, HQSOa + 7112034
+ag, Na1l 2PO4・12H20771+1
(1. Add 110 mg of C to 117 tera Ice decomposition solution of NR3 (320 g of NR) to H/30 phosphate buffer pH.
7,01N, and while leaving the insoluble substances as is, add 20011 g of pronase to this, and incubate at 30°C for 12 hours.
Something that reacted. (Protein decomposition rate is approximately 94%) was added in an amount equivalent to 2 g in terms of NR3, the pH was adjusted to 7.0, and then diluted to II with pure water.

On the other hand, 10g of glucose, H(]SO4・71120
34mg, Na1l2PO4-12112077+
ng, KCI 10+ng, yeast extract (manufactured by Oriental Yeast Co., Ltd.) 5 (l) and polypeptone (manufactured by Daigo Nutrition Co., Ltd.)
5 (J) was added, the pH was adjusted, and then I!J was prepared with pure water as a control.

In both these media, 'I' G C-liquid medium at 30'C.
- 5treptococcus 1ac cultured at night
tis (ATCC19435) was inoculated in 2011, and 30
The cells were incubated statically at ℃. After 12 hours of culture, the bacterial cell side was determined by the dry bacterial weight method, and the amount of lactic acid produced was determined by IIPLc. In the medium containing NR8, the dry bacterial weight was 1.
2h/, Q, lactic acid production amount was 9.2g#. on the other hand,
In the control medium containing yeast extract and polypeptone, dry bacterial weight J1M 1.25 g/l, lactic acid production M9. h/J
or obtained.

Example 2 Enzyme saccharification solution of Charasaba starch (Charasaba starch 100 (
] in 5001 pure water, α-amylase 600
Add u and heat to 70°C and hold for 30 minutes to liquefy. Thereafter, the saccharified solution was cooled to 40°C, glucoamylase was added, and reacted for 24 hours with slow shaking. Add 0.5g and adjust the pH
After adjusting to 6.2, the pure medium was diluted to 100+al and used as a standard medium.

Meanwhile, the enzymatic saccharification solution of Charasaba starch was added to Tarnis 7-10
Calculate the equivalent amount of 0.3 g of NR3 powder (not treated with protease) and 0.5 q of peptone, adjust the pH to 6.2, dilute to 10 On+l with pure water, and use it as the study medium. did.

In addition, 10g of glucose is added to the enzyme saccharification solution of Charasaba starch.
Calculate the equivalent amount, add peptone O, sg, and adjust the pH to 6.2.
The comparison medium was one diluted to 10011 with pure water after conditioning, and the enzyme saccharification solution of charas mackerel starch calculated to be equivalent to 10 liters of glucose, and a medium to which nothing else was added.

These four types of media were sterilized in IDeisscl, and 7y + aononas n + obilis NRR was cultured at 30°C for 20 days in a YM (Difco) liquid medium.
LB14023 was cultured for 2 days after inoculation, and the dry bacterial weight and produced ethanol were measured.

In the medium containing yeast extract and peptone added to the sugar solution, dry bacterial weight 2.05 u/nl and ethanol 5.22+ul were obtained, and in the medium using NR3, the bacterial type i1.611 was obtained.
1 g/111 and 5.01 nl of ethanol were changed.

On the other hand, in a medium containing only peptone in liquid i, the bacterial type MO
, 84 n+q/ll and 3.85 ml of ethanol, but neither bacterial growth nor ethanol production was observed in the medium containing only sugar solution.

Example 3 The same sugar solution used in Example 2 was mixed with 10 g
(Measure the amount equivalent to 1 liter, add 0.3 g of yeast extract, 0.3 g of malt extract, and 0.5 liter of pegtone, adjust the pH to 6°2, and dilute it to 10 On + liter with pure water, and use it as the standard medium. did.

On the other hand, the sugar solution was calculated to be equivalent to 109 glucose, and this was added to N
0.3g of R8 powder (not treated with protease) and peptone O,! After adding o+ and adjusting to ptl6.2,
A medium diluted to 100 nl with pure water was used as the test medium.

In addition, calculate the amount of sugar solution equivalent to 10 g of glucose,
After adding 0.5g of water and adjusting the pH to 6.2, dilute with pure water for 10 minutes.
Glucose diluted to 0+nl and sugar solution
09 equivalent extension was calculated, and a medium to which nothing was added was used as a comparison medium.

Put these four types of culture medium into n+eissel, sterilize it, and use 10g of crucose/! Yeast extract 3g/l, malt extract 3g/l, peptone 5 (1/l (pH 6, 2)
Saccha cultured at 30°C for 2 days in a preculture medium consisting of
romyces uraurn IFO0565 5n
After persimmon inoculation, the cells were cultured for 2 days, and the dry bacterial weight and the produced ethanol were measured.

In the medium containing yeast extract, Full 1-extract, and peptone added to the sugar solution, a dry bacterial weight of 4.901'mg/ml and ethanol of 4.891111 were obtained, and in the medium using NR3, the bacterial type i4. 041B! II/1ml and ethanol 4.581111 were changed. On the other hand, in a medium containing only peptone in a sugar solution, the bacterial weight was 0.64+n.
g#a I and ethanol 1,99n+l were obtained. In addition, even in a medium containing only sugar solution, the bacterial weight is 0.33 + aq
/11 and 0.84 nl of ethanol was changed.

Example 4 Glucose 1.0%, hexametaphosphate sorter 0.1
7%, C10,1%, H(ISO4-
Based on a medium (ptl 7.0) consisting of 7H200.04%, ■ Nothing else added ■ Defatted soybean hydrochloric acid hydrolyzate (MVP) 0.5 nl/
Added to maintain the old concentration ■ Yeast extract 0.25
% of glucose was added ■ Four types of media were prepared with 0.25% of NR3 powder added, and after sterilization, 201 of each medium was dispensed into 500 nl shaking flasks, and 1.25% of glucose was added.
0%, yeast extract 1°0%, volibeprine 1.0%,
Preculture medium consisting of NaCl 015x (pH 7,0)
Pseud.

monas aeruginosa KYU-1 (FE
RN P 0701) was inoculated, 100 μm of Pross cultured at 30°C with overnight shaking was added, cultured with shaking at 30°C for 3 days, and the weight of the produced dry bacteria was measured. In medium ■, no bacterial growth was observed. In the medium (■), the dry bacterial body weight reached 2.3 ig/if, in the medium (■), the dry bacterial body weight reached 2,919/11, and in the medium (■), the dry bacterial body weight reached 3.1 ng/n+I.

Example 5 Glucose 10%, K112 PO40.1%, H
GSOa ・711200.24%, FeSO
4-711200,01'X,Mn5O4,41+2
Q O, 001%, vitamin Bt 100r/l,
Add 1.0 liquid NR3 to a medium consisting of biotin 3r/I.
11/Add at the old concentration, adjust the pH to 7.0 and sterilize, total 1! 300 for J's mini jar fermenter
I staked out n l.

On the other hand, in a medium having the same composition except for NR3, 0% of the defatted soybean hydrochloride hydrolyzate used in Example 4 was added instead of NR8.
．． The mixture was added to a concentration of 75n+l/old, sterilized after adjusting the pl+ to 7.0 as before, and 300nI was added to a mini-jar fermenter with a total volume of 11 to serve as a control. Both jars contained 1.0% glucose, 1.0% yeast extract, 1.0% polypeptone, and NaCl O,5
Brcvi in preculture medium (all 7.0) consisting of %
bacteriun+ flavuIIATCC140
67 and cultured at 30°C overnight with shaking.
Add n+I, temperature 34℃, ventilated stirrer Lt1/2
VVM, t200rpn+, and pH control were carried out by controlling the pH to 7.5 while feeding ammonia water. After 30 hours of culture, glutamic acid of 46.5% relative to sugar accumulated in the culture medium using defatted soybean hydrochloride hydrolyzate, and glutamic acid of 348.2% relative to sugar accumulated in the culture medium using liquid NR3. Was.

Claims (1)

[Claims] 1) Concentrate the remaining mother liquor after separating the latex from the natural rubber sap or pulverize it by spray drying and use it as a fermentation medium, or add a portion to the fermentation medium and incubate the medium with bacteria (actiradiation). A method of cultivating microorganisms that involves inoculating and multiplying fungi (including bacteria), fungi (yeast or filamentous fungi), producing bacterial cells, and/or accumulating specific fermentation products.