JPS63237781A - Production of microbial cell - Google Patents

Abstract

PURPOSE:To improve the conversion efficiency of soybean into cells useful as feed, food, fertilizer, etc., and to enable the production of bacterial cells at high temperature, by using soybean milk as a main raw material and carrying out mixed culture of bacteria of Bacillus genus and Brevibacterium genus. CONSTITUTION:Microbial cells are produced by the mixed culture of bacteria of Bacillus genus and Brevibacterium genus in soybean milk used as a main raw material. The soybean milk is produced as a waste liquid, etc., in the production of soybean oil and fat, soya protein, etc., and the milk is used as a raw material and added with an auxiliary nutrient source such as corn steep liquor, rice bran and denucleated yeast. The bacterial strain belonging to Bacillus genus is e.g. Bacillus subtilis (IFO-14132) and Bacillus megatherium (IAM-1166) and the strain belonging to Brevibacterium genus is e.g. Brevibacterium ammoniagenesis (IAM-1041).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing microbial cells, and particularly to a method for producing microbial cells useful for feed, food, fertilizer, industrial raw materials, etc.

(Prior art) Generally known methods for producing microbial cells include a method for obtaining baker's yeast using molasses as a carbon source, and a method for obtaining feed yeast using sulfite pulp waste liquid as a carbon source. Usually, raw materials mainly consisting of two types of monosaccharides, which are easily digested, are used as carbon sources, and moreover, single bacterial species are cultured.

In addition, a method has been devised in which starch waste liquid is treated in two stages with amylase-producing bacteria Endomycobsis fibrigel and Candida utilis, which combines waste liquid treatment and yeast production.

In this way, it is known that organic waste or cheap raw materials are used as carbon sources to produce bacterial cells, but the carbon sources of the raw materials used in these processes are basically monosaccharides or saccharification treatments. It is a simple starch and can be easily assimilated by microorganisms.

On the other hand, 1 L of soybean serum, which has a complex composition, has been effectively used as feed, fertilizer, etc., but has never been used as a raw material carbon source for bacterial cell production. In other words, the organic matter contained in soybean whey is composed of sugars such as arapan and galactan that can be assimilated, and the protein content is low, so its uses are currently limited. .

The present inventors focused on 1L of soybean serum, which had limited use until now despite its low cost, and conducted studies to use it as a nutrient source for bacterial cell production.

(Problems to be Solved by the Invention) As described above, soybean whey has a complex composition and contains a large amount of difficult-to-decompose sugars, so it is not easy to use it as a carbon source.

The present inventors have searched for a bacterial cell that can efficiently utilize this soybean alumina and do not produce metabolic byproducts such as organic acids, which are problematic during bacterial cell production, and have conducted intensive analytical research. As a result of repeated efforts, they discovered a method for efficiently producing bacterial cells by combining two specific types of bacteria, and completed the present invention.

(Means for Solving the Problems) That is, the present invention relates to a method for producing microbial cells, which is characterized by using soybean alumina as the main raw material and culturing a mixture of bacteria of the genus Bacillus and genus Previbacterium.

In general, in the production of microbial cells, bacteria of the genus Bacillus have a high ability to assimilate [il], but have the disadvantage that they produce metabolic byproducts such as organic acids, and the cell concentration cannot be increased.

In addition, bacteria of the genus Previbacterium have the disadvantage of having a lower bacterial cell concentration but a lower ability to assimilate sugars.The present invention can only compensate for the disadvantages of each by combining both of these bacteria. Rather, it has a high conversion efficiency to bacterial cells, making it possible to produce bacterial cells at a high concentration. In addition, generally when two kinds of bacteria are cultured in the same tank, one of them becomes leached out and the cell conversion rate decreases, but in the present invention, there is no problem in this respect, and it can be used under mixed culture. It is characterized by a high cell conversion rate.

(Function) The method for producing microbial cells of the present invention will be described in further detail below.

The 1L soybean serum used in the present invention is produced as a waste liquid during the production of soybean oil and fat, soybean protein, etc., and its general component composition is as follows.

Ash content 8.4-8.6% crude protein
8.7-9.1% Crude fat 0.1-0.4% Total sugars 18.5-20.1% Crude fiber O,O-0.1% pH 3.8-4.1 In the present invention, this Soybean whey is used as the main ingredient, but in addition to it, supplementary nutritional sources such as cornstarch liquor, etc.
Rice bran, denucleated yeast, etc. are used, and cornstarch liquor is most preferably used in the present invention.These auxiliary raw materials include:
Approximately 10 to 3 in terms of visible volume per 1L of soybeans
0% is suitable, which results in a higher assimilation rate of soybean whey.

In the present invention, bacteria of the genus Bacillus and Plevibacterium are used.
) and the Institute of Applied Microbiology, the University of Tokyo (hereinafter referred to as IA)
(abbreviated as M). In addition, as for its type, the genus Bacillus is Bacillus subtilis (
IFO-14132), Bacillus megatherium (IAM
-1166), Bacillus previs (I^ni-1031)
, Bacillus sphaericus (IFO-3525), Bacillus heptareus (IAM-1029), etc., and the genus Plevibacterium includes Plevibacterium ammoniagenes (rA) 1-1041), Plevibacterium acetylicum ([FO-12146)] , Previbacterium linens (IFO-12141), etc. can be used.

The method for obtaining the microorganism cells of the present invention using these raw materials and bacteria uses the above-mentioned 1L soybean serum and auxiliary material F4, and dilutes the soybean 1L serum with water to a concentration of about 10% as total sugars. After putting it in a culture tank and sterilizing it, adjust it to p)I,
Add microcavities of Bacillus and Plevibacterium.

The method of sterilization is not particularly limited and may include steam sterilization, chemical sterilization using free chlorine, etc. According to the steam sterilization method, gm at about 120° C. for about 15 minutes is sufficient.

p)l when culturing is generally in the range of 6.0 to 8.0, preferably 6.5 to 7.5. That is, outside this range, the tVM speed of the bacteria of the genus Bacillus and Plevibacterium decreases significantly.

The pH adjuster to be used is not particularly limited, and sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid, phosphoric acid, aqueous ammonia, etc. can be used, but it can also be used to supply nitrogen and phosphorus.
Preferably, aqueous ammonia and phosphoric acid are used.

Bacteria of the genus Bacillus and genus Previbacterium are mixed in the same amount with the raw material solution at a ratio of 10 to 50 m+/l and placed in a culture tank with a bacterial count of 108 cells/ml.

In this case, if a single strain is cultured without performing mixed culture, not only the high concentration of microbial cells of the present invention cannot be obtained, but also the rate of assimilation will decrease.

Although the reason for this is not clear, it is thought that the decomposition and assimilation of sugars is performed synergistically by bifurcated bacteria.

In addition, in the present invention, bacteria of the genus Bacillus and Plevibacterium with a bacterial count of 10'cell1g#1 are added to the culture raw material, but in the early stage of bacterial cell production, It is sufficient to propagate the bacterial strain to this number using ordinary pre-culture means, and after carrying out the present invention, it is sufficient to use a part of the produced bacterial cells, and the means for doing so are not particularly limited. isn't it.

For example, regarding the means for pre-cultivating the distributed bacterial strain, the medium used may be prepared using the above-mentioned raw materials used in the present invention, but it is also possible to prepare a bouillon medium or a sugar such as glucose as a carbon source. A synthetic medium such as a synthetic medium can be used.

In addition, in the preculture, it is usually preferable to culture both bacteria separately.Furthermore, the culture medium PH is lower than that of Bacillus (p)1.
6.0-7.5, Previbacterium pH 6.5-7.5
Pre-culturing under conditions of 8.0 is preferable for 11f growth of bacteria.

Cultivation is performed with stirring and ventilation.

The type of culture tank may be any type that allows aeration and agitation, and may be either a batch type or a continuous type.

In addition, the dissolved oxygen concentration during culture should be at least 0.1 pp layer,
The culture temperature is preferably in the range of 25 to 35°C.

Bacteria proliferate with foaming as the culture progresses, but in this case, in order to suppress foaming during culture, oils and fats such as fish oil, etc.
There is no problem in adding a silicone antifoaming agent or the like.

The culture time is usually 30 to 50 hours, and the culture is terminated when the remaining fdl (total saccharide concentration) in the culture tank becomes 10 mg/ml or less.

The microbial cells after culturing may be used as they are, or after being separated using a separation means such as centrifugation, depending on the intended use.

The microorganism of the present invention produced in this way can be produced into cheap sake because its main raw material is soybean serum.
Moreover, because of its high concentration, it is useful for feed, food, fertilizer, industrial raw materials, etc.

(Example) The present invention will be further explained below with reference to Examples, but the present invention is not limited thereto.

Moreover, all percentages indicate weight % unless otherwise specified.

Furthermore, measurement methods such as analysis carried out in the present invention will be described in advance.

<Total sugar concentration> Total sugar concentration was measured by the phenol-sulfuric acid method.

Bacterial count (viable bacterial cell count)> This was carried out by the agar plate method using a bouillon medium. The method was to add a predetermined amount of the collected and diluted culture solution to a bouillon medium, and after culturing at a temperature of 300C for 20 to 30 hours, the number of colonies of grown bacteria was counted to calculate the number of bacteria (number of viable bacteria).

<Bacterial cell concentration> A predetermined amount of the culture solution was taken and centrifuged to separate the supernatant and the precipitate containing the bacterial cells. After washing this precipitate with water, it was dried at 105°C until it reached a constant weight, and the bacterial cell concentration was calculated from the weight of the dried product and the amount of culture solution collected.

Bacillus plevis (I^N-1
031) and Previbacterium a7tylicum (IFO
-12146), preculture was first performed.

The preculture method is soybean whey (ingredients: 8.8% crude protein,
Crude fat 0.3%, total sugars 19.5%, PH3.9)
After diluting 20 g with tap water to a pH of about 8001, the pH was adjusted to 7.2 with aqueous ammonia. Add tap water to this to bring the total volume to 10100O, then add 5
00i in a 1-volume shake flask, capped with a cotton plug, and heated to 121°C.
The mixture was steam sterilized for 15 minutes and used as a culture medium.

Into this medium, Bacillus plevis and Plevibacterium acetylicum were inoculated from the slant, and incubated at 30°C.
(2) Culture was performed.

Approximately 18 hours after culturing, the number of bacteria reached 1.
10 X 108 calls/ml, Previbacterium acetylicum 1.15 X 10” cells/ml
It became ml.

101'l! Soybean milk [3,200 g] having the same composition as above was placed in a J-sized culture tank, and water was added to make the total volume 6 L. This liquid was steam sterilized at 121°C for 15 minutes, and then the pH was adjusted to 6.8 with aqueous ammonia.

To this raw material solution, 90+ wl each of Bacillus plebis and Plevipacterium acetylicum after the pre-culture were added, and while stirring at 500 to 1000 rpm while aerating so that the dissolved oxygen concentration was 0.1 PP1 or more, Culture was carried out while maintaining the liquid temperature at 30°C.

After 46 hours from the start of culture, the remaining amount (ILC total sugar concentration) was 7.
2 B/el, and the assimilation rate of total sugars was 93%.

In addition, the bacterial cell concentration at this time was 6.2%, and as a result of measuring the number of bacteria, Bacillus plevis was 8.15
10"cell, 1.s/ml, Previbacterium acetylicum was 1.01 x 10"cells/ml.

Example 2 The genus Bacillus and the genus Plevibacterium 1 [11@], which received the separation line from the il1 institution, were identified as Bacillus subtilis (
First, preculture was performed using Previbacterium ammoniagenes (IFD-14132) and Previbacterium ammoniagenes (l^M-1041).

The preculture method is to add 500ml of broth medium (meat extract 0.5%, peptone 1%, NaCl 0.5%). tlm
The pH was adjusted to 6.8 with aqueous ammonia.

This bouillon medium solution IQOml is 50 hl each! !
The mixture was placed in a flask, sealed with a cotton plug, and steam sterilized at 121°C for 15 minutes to serve as a preculture medium.

Into this medium, Bacillus subtilis and Previbacterium ammoniagenes were inoculated from the slant, and
After about 19 hours of shaking culture at 'C', the number of bacteria was 1.25 x 10'C.
cells/+1, and Previbacterium ammoniagenes was 1.21 x 10'cells/II+.

In a culture tank similar to Example 1, add 1 soybean serum (components: crude protein 8.9%, crude fat 0.1%, total sugars 19.4%, p).
Add 3000 g of 14゜0) and 500 g of cornstarch liquor (components: 18.7% crude protein, 1.6% ffl fat, 13.4% total class II, pH 3.9), and add water to this. The total volume was 6L.

After steam sterilizing this liquid at 121°C for 15 minutes, the pH was adjusted to 6.7 with a 10% potassium hydroxide solution.

To this raw material solution, 70 ml each of Bacillus subtilis and Previbacterium ammoniagenes were added after the pre-culture.
Add one by one until the dissolved oxygen concentration is 0. Culture was carried out while stirring at 500 to 1,001,000 rpm and maintaining the liquid temperature at 30° C. while aerating the solution to 1 ppm or higher.

40 hours after the start of culture, the residual m value (total sugar concentration) was 7°1
mg/@l, and the assimilation rate of total sugars was 93.4%.

In addition, the bacterial cell concentration at this time was 6.4%, and as a result of measuring the number of bacteria, Bacillus subtilis was 9.21
10'cells/ml, Previbacterium ammoniagenes is 1.15 x 1G'cell Is/
It was sl.

Example 3 Bacillus hepta reus ([4M-1
029- and Previbacterium linens (IFO-
12141), preculture was first performed.

The preculture was carried out using a broth medium (0.5% meat extract, 1% peptone, 10.5% NaC) made from gUA and shaking culture in the same manner as in Example 2.

Approximately 20 hours after culturing, the number of bacteria reached 1.
05 x 10'cells/ml, Previbacterium linens is 1.11 x IQ8cal1g/Il
It became l.

In a culture tank similar to Example 1, soybean whey (components: crude protein 8.7%, crude fat 0.2%, total sugars 19.6%, pi (
3゜8) 2600g and enucleated yeast (components: crude protein 47,
1% crude fat, 0.4% crude fat, and 30.1% total sugars) were added thereto, and water was added to make the total volume 5L. Add this liquid to 12
After steam sterilization at 1°C for 15 minutes, the PI was adjusted to 7°O with aqueous ammonia.

To this raw material solution, add 90 liters each of the pre-cultured Bacillus heptareus and Previbacterium linens,
Culture was carried out while stirring at 500 to 11,000 rpm and maintaining the liquid temperature at 30° C. while aerating the solution so that the dissolved oxygen concentration was 0.1 pp+i or more.

45 hours after the start of culture, the residual value (total sugar concentration) was 8.5
ml/ml, and the assimilation rate of total sugars was 91.9%.

In addition, the bacterial cell concentration at this time was 6.6%, and as a result of measuring the number of each bacteria, Bacillus hepta reus was 7.88
10" cells/time 1, Previbacterium linens was 1.27 x 1010 cells/ml.

Example 4 A continuous culture test was conducted while supplying a raw material solution prepared from the same soybean whey and corn staple liquor as in Example 2 to the culture tank after producing the bacterial cells in Example 2.

The method involves supplying the raw material solution to the culture tank at a rate of 250 ml/ha, while maintaining the dissolved oxygen concentration at 0. Aerate the liquid to 1 ppm or higher, stir at 500 to 11,000 rpm, and cool the liquid to 30 ml.
It was carried out at ℃.

As a result of continuous operation for about 180 hours, no change was observed in the condition in the culture tank, and the bacterial cell concentration of the culture fluid collected at this time was 6.1%. In addition, as a result of measuring the number of bacteria, Bacillus subtilis was 8.51X 10'cells.
/ml, Previbacterium ammoniagenes is 1
．． 06x 10I0 calls/ml.

Claims (1)

[Claims] A method for producing microbial cells, which is characterized by using soybean whey as the main raw material and culturing a mixture of bacteria of the genus Bacillus and genus Previbacterium.