JPS5817589B2 - Koubonoseizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new species of Candida mtsuiensis belonging to the genus Candida.
s) into a medium containing one or more selected from ethanol, acetic acid, sugars, and hydrocarbons, and cultured aerobically to produce yeast, and is suitable for food and feed use. The purpose is to supply high-quality microbial protein.

Up to now, known methods for producing bacterial protein include using carbohydrates, normal paraffin, methane, methanol, and ethanol as carbon sources.

Each method using each carbon source has its own characteristics.

Which raw materials are used depends on location and other factors.

However, no matter which raw material is used, from an industrial viewpoint of bacterial protein production, a bacterial strain that can grow rapidly at high temperatures and low pH is required.

In addition, the produced bacterial cells must have a high protein content, high nutritional value, and be safe.

The present inventors searched the natural world for bacterial strains that satisfy these conditions, and as a result, found a strain that grows well on sugars such as ethanol, acetic acid, glucose, starch, and starch decomposition products.

The above-mentioned substances have been used as food or drinks by humans for many years, and it is thought that there are no toxicity problems attributable to the raw materials.

Furthermore, this strain can grow using hydrocarbons such as normal paraffin as a carbon source.

Recently, safety tests have been carefully conducted on bacterial protein derived from normal paraffin over a long period of time, so it is thought that its safety will be confirmed in due course.

Since this strain can utilize various carbon sources, it provides an advantageous method of switching carbon sources depending on factory location conditions and economic conditions.

The mycological properties of this strain are as follows.

(a) Growth status in each medium ■ Growth in MY liquid medium Cultured in MY liquid medium at 28°C for 3 days: Shape is mainly circular or oval (1,8-4) X (1,,8-5) μ
The main cells are small (2×2) μ cells.

Produces many elongated cells. Film formation is vigorous (wr
inked or heavy).

Propagate by budding method.

(2) Growth on MY medium Cultured on MY agar medium at 28°C for 5 days; the shape is round or oval, (2-4) x (2-5)μ, and many elongated cells are produced.

Growth is good, colonies are white, powdery in appearance, and the surface is rough and scattered.

The colony is plate-shaped, and the periphery is filamentous.

■Slide culture on potato agar medium Pseudohyphae and hyphae are actively produced on potato/glucose agar medium.

(b) Ascospore formation Stone bone medium, Gorodkova medium, sodium acetate medium, V-8
When tested in culture medium, no ascospore formation was observed.

(c) Without the formation of apo-spores d) Each physiological property ■ Optimal growth conditions Optimum pH; pH 3 to 6 Optimum growth temperature; 30 to 43°C ■ Growth range pH; 2 to 9.5, temperature 15 to 47 °C■ Assimilates nitrate.

■ Decomposition of fat Decomposes arbutin.

■ Urea decomposition ability (urease; in Christen medium) is negative.

■ Gelatin has almost no liquefiability.

■ Osmotic pressure resistance: Does not grow on MY agar medium containing 20% sodium chloride.

Slight colony formation was observed on the MY agar medium containing 20% salt after 3 weeks of observation.

■ No carotenoids are produced.

■ Significant organic acid production Acid production is observed.

[Phase] There is no formation of starch-like substances.

■ Vitamin requirement: Can grow without vitamins.

However, growth is weaker than in a medium containing all vitamins.

0 Reaction in lidmus milk medium Forms a film.

Do not peptonize. The color changes to blue-purple.

(e) Fermentability and assimilation of various carbon sources (1) Fermentable glucose +, galactose +, sucrose +, maltose +, lactose −
, raffinose +, (2) Assimilation of carbon compounds glucose +, galactose + (S), sucrose, maltose +, lactose + (S) -nrubose +, cellobiose, trehalose +, melibiose +
, raffinose +, melezitose +, inulin -, soluble starch, xylose
+, Lr arabinose +, D-arabinose +, D-ribose, L-rhamnose
+(S), ethanol +, glycerol
+, erythritol +, adonitol +
, dulcitol, D-mannitol +, D-
Sorbitol +, α-methylg) Recocide +, salicin dried, lactic acid +, succinic acid
+, citric acid +, inositol, (S): This strain belongs to the genus Candida because it does not form ascospores, does not produce carotenoid pigments, grows by the budding method, and forms pseudohyphae. It's yeast.

This strain is unique in that it can use nitrate as its only nitrogen source.

J. Lodder called yeasts of the genus Candida that can use nitrate as a nitrogen source as “TheyeastsJ (19
They are summarized in Table 21 on page 909 of the 1971 edition).

According to it, C.curiosa, C.aqua-t.
ica, Comelini i, C, utilis, C
, vartiovaarai etc. (Hansenula
(excluding those that fall into this category) fall into this group, but as shown in Table 1, they differ from this strain in significant respects in fermentability and assimilation.

Furthermore, since it is clearly different from the above-mentioned strains in terms of maximum growth temperature, this strain is judged to belong to a different species from these strains.

In addition, Cantida ethanothermophilum, Cantida yucasothermophilum, and Cantida acidothermophilum shown in JP-A No. 49-71188 primarily utilize nitrate and decompose arbutin. Gender is also different.

In addition, as shown in Table 1, there are significant differences in the fermentability and assimilability of carbon sources.

Therefore, this strain was recognized as a new species, and it was classified as Candida m1tsuiensis MT 1027 (Candida m1tsui).
encis n, Sp-MT1027).

Furthermore, this strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 3122.

In addition to the above-mentioned strains, all mutant strains belonging to Candida mitziensis can be used as the bacteria used in the present invention.

To produce yeast industrially, continuous, semi-continuous, or batch-type aerated agitation culture methods are generally used. Either method may be used as long as it is cultured.

A high culture temperature is preferable from an industrial perspective.

This strain is a yeast that can grow up to 47°C, and especially
It is preferable to culture at 0 to 43°C.

It can be cultured at 45°C without significantly reducing the speed.

The pH of the culture solution is preferably low in order to prevent the proliferation of bacteria.

Since this strain grows well at pH 3 to 6, it is preferable to culture it at pH 3 to 4.

The culture medium used in carrying out the present invention contains one or more selected from ethanol, acetic acid, glucose, molasses, starch, and normal paraffin, particularly normal paraffin having 9 to 18 carbon atoms, as the main carbon source. It can be used at a concentration of 0.5 to 10%.
The position is appropriate.

As a nitrogen source for the culture solution, for example, ammonium sulfate,
One or more selected from inorganic nitrogen sources such as ammonium chloride, sodium sulfate, ammonium sulfate, ammonium phosphate, aqueous ammonia, and organic nitrogen sources such as urea, peptone, amino acids, etc. can be used in combination. .

Examples of inorganic salts to be added to the culture solution include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, and heptahydrate.

In addition to the above, yeast extract, corn stable liquor, meat extract, malt extract, etc., and trace metal salts such as iron, zinc, manganese, and calcium can be added when preparing the culture solution, if necessary.

An example of the composition of the culture solution used in the present invention is as follows.

carbon source (eg glucose) 10 to 409/l, nitrogen source (eg ammonium sulphate) 5 to 10 EI/l
, dipotassium hydrogen phosphate 0.2 to 29/l, potassium dihydrogen phosphate 0.2 to 1 &/l, yeast extract or cornstarch liquor 0.2 to 2 El/l
l, ferrous sulfate heptahydrate 2 to 50 m9/l, sodium chloride 50 to 200 to /11 calcium chloride 1o
It is added to distilled water, tap water or well water at a rate of 1 to 100 m/l.

Adjust the pH of the initial culture solution to about 6.

After sterilizing this, it is cooled and inoculated with a seed culture (for example, Candida mitziensis MT 1027).

The culture is carried out under aerobic conditions such as shaking culture or aerated agitation culture at a suitable temperature between 30 and 47°C.

Since the pH changes as yeast grows, it is controlled to an appropriate pH value of 3 to 6 using an alkaline solution such as aqueous ammonia or an acidic solution such as hydrochloric acid.

In batch culture, when most of the substrate is consumed, yeast cells are collected from the culture by means such as centrifugation, washed, and dried.

In continuous culture, the medium is supplied at an appropriate rate, and aeration and agitation are performed while maintaining an appropriate residence time.

The bacterial cells are collected from the culture as described above and dried.

Examples are shown below, but they are not intended to limit this patent.

Example 1 3 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, 1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, and 1 ml of corn steep liquor were dissolved in 11 of tap water.
Dispense 0ml into a 500ml shake flask and add 12
Sterilize at 0°C for 5 minutes.

After cooling, ethanol equivalent to 1% was added aseptically, and the pre-cultured Candyk mitsuiensis MT1027 was inoculated and cultured with shaking using a reciprocating shaker at 38°C.

The pH was adjusted to pH 4 once a day with aqueous ammonia.

・After culturing for 2 days, the bacterial cells were collected by centrifugation, washed, and then transferred.

It was dry.

As a result, 7.29/l of dry bacterial cells were obtained. The yield based on ethanol was 72%.

Analysis of this bacterial cell revealed that the crude protein content was 45%.

Example 2 Dissolve 5 g of sodium acetate, 6 g of ammonium sulfate, 1 g of dipotassium hydrogen phosphate, 1 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, 5 m9 of ferrous sulfate, and 1 ml of cornstarch liquor in 11 tap water and make a small jar fer. The mixture was placed in a mentor tube and sterilized at 120°C for 5 minutes.

Candida mitziensis MT cultured separately after cooling
1027 suspension was inoculated and cultured with aeration under conditions of a temperature of 35° C., an aeration volume of 0.4 VVA, and a stirring speed of 500 rpm.

When growth began, the pH became alkaline, so acetic acid was added to maintain the pH at 4.

48 hours after the start of culture, acetic acid 1.5 ml/11. Sodium acetate 79/l, ammonium sulfate 6 g/l, dipotassium hydrogen phosphate 1 g/l, potassium dihydrogen phosphate 1 g, #, magnesium sulfate 0.5 p/l 3. Iron sulfate 5m9/l, :J-7
A culture medium having a composition of 1 ml/l of stabilizer was continuously supplied and cultured continuously.

First 40 ml, then 60 ml/h at the rate of Anr.
The medium was supplied at a rate of r, and continuous operation was performed for one week.

Culture 11 was centrifuged, bacterial cells were collected and dried to obtain 5.48 g of bacterial cells.

Since the amount of acetic acid contained in the continuous culture medium was 18.8 p/l, the bacterial cell yield was 29.1%.

Example 3 20 g glucose 1 3 g ammonium sulfate 1 potassium dihydrogen phosphate
g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate 0.5. ! li', salt 0.1. ? 1 ml of corn staple liquor was dissolved in 11 tap water, placed in a small jar fermenter, and sterilized at 120° CI for 5 minutes.

Separately, 50 ml of a suspension of Campita mitziensis MT 1027 precultured in a medium with the same composition was aseptically inoculated at a temperature of 43° C. and an aeration volume of 0.5 VVM.

Aerated agitation culture was performed while maintaining the agitation speed at 600 rpm.

During cultivation, the pH was automatically adjusted to 4 with aqueous ammonia.

From 20 hours after the start of culture, the medium with the above composition was sterilized and then
It was fed continuously at a rate of mVhr.

The residence time is 10 hours.

Sampling was performed 50 hours after the start of continuous culture, and the bacterial cells were collected using a centrifuge, washed with water, and then dried.

10.8 from the culture solution of 11. ! Dry cells of i' were obtained.

At this time, the residual sugar was 0.033%. The yield of bacterial cells based on the raw material glucose was 54%.

The crude protein of this bacterial cell was 40. Example 4 20g soluble starch, 7g ammonium sulfate.

Potassium dihydrogen phosphate 1.5.9. Magnesium sulfate
0.5 g of supernatant salt and 1 g of yeast extract were dissolved in 11 tap water, and 50 ml of the solution was dispensed into a 500 ml shaking flask and sterilized at 120°C for 10 minutes.

Candida mitziensis MT 1027, which had been precultured, was inoculated into a medium with the same composition, and cultured with shaking at 45°C.

After shaking culture was continued for 2 days while adjusting the pH to 5 twice a day with aqueous ammonia, the bacterial cells were collected by centrifugation, washed, and dried.

As a result, 8.2 p/l3 of dry bacterial cells were obtained.

The yield based on soluble starch was 41 parts.

Example 5 7 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate/superhydrate salt, and 1 g of yeast extract were dissolved in 14 tap water,
Dispense 50 ml of this into a 500 ml shaking flask, and add mixed normal paraffin (C130, 3%,
2 ml of C1450.5%, C1548.8%, Cl60.4) was added and sterilized at 120°C for 10 minutes.

The precultured Candida mitziensis MT1027 was inoculated into a medium with the same composition, and cultured with shaking at 35°C.

After culturing with shaking for 4 days while adjusting the pH to 4 twice a day with ammonia water, the bacterial cells were collected by centrifugation, washed, and dried.

As a result, 22.59/l of dry bacterial cells were obtained.

Example 6 Example 4 was repeated except that 20 g of molasses was used instead of 20 g of soluble starch.

As a result, 10.5 g/l of dry bacterial cells were obtained.

The yield on molasses was 53%.

Claims (1)

[Claims] 1. A strain selected from Candida mitziensis or its mutants is inoculated into a medium containing one or more selected from ethanol, acetic acid, glucose, molasses, starch, and normal paraffin as a main carbon source, and A method for producing yeast characterized by atmospheric cultivation.