DD219041A3 - METHOD OF CULTIVATING YEAST - Google Patents

Abstract

The invention relates to a process for the cultivation of yeasts with an increased proportion of essential amino acids, especially phenylalanine, histidine and threonine. The previously known methods required a high energy input by stirring and Belueften and gave a biomass with indefinite composition of amino acids. It has been found that by cultivating yeasts on n-alkane-containing hydrocarbons and aqueous nutrient medium under aerobic conditions and stirring the medium with regulation of the specific energy input by bubbling and mixing in the range of 3.5-6.0 kW / t medium, the Regulation of the ratio of the concentration of n-alkanes to the energy input in the range of 3-10 and a transition rate of the n-alkane from the hydrocarbon mixture to the cells in the range of 2.5-5.0 kg per ton of nutrient medium the aim of obtaining a yeast is achieved with an increased content of phenylalanine, histidine and threonine. The positive effect is increased when the yeast producer Candida salmonicola - WSB 779 (ZMPM U-196) is used.

Description

Title of the invention

Method of culturing yeasts

C 12 P 13/04 C 12 P 13/22 C 12 P, 13/24

Field of application of the invention

The invention relates to a method for culturing yeasts with an increased proportion of essential amino acids, especially phenylalanine, histidine and threonine. '. ' ".

The protein biomass obtained from the hydrocarbons is used in the putter for agricultural deer. Biomass protein contains 19 amino acids, including all essential ones. ^! The content of essential amino acids in the protein is unstable and depends on the microorganism culture to be used and the. Cultivation conditions.

Characteristic of the known technical solutions

Different yeast cultures of one species differ in essential amino acid content by 1.2-1.5 fold (GB Patent 1,430,842, US Patent No. 3929571J.

Under the influence of culturing conditions, the content of single essential amino acids changes by 1.5-2.5 times (Japanese Patent No. 43876, Japan Patent No. 13358). ,

To increase the biosynthesis of individual essential amino acids special mutants are obtained whose physiological peculiarity is the

Overproduction of one or more essential amino acids is (Japan Patent No. 37278, Japan, Patent IT No. _1, 37279). ·

Known methods are the cultivation of microorganisms with a regulation of the process parameters. After I ¹ P ßr. 1297619 Yeast Torulopsis and Candida on petroleum fractions im.Apparat with a stirrer and aeration at a speed of

      ' _1 ·

Stirrer of 2000 min. and an air supply of 400 l / h. cultured. In the process, a protein product is recovered. "

Disadvantage of this method is' that _{: the} energy input by stirring and aerating is very high and a biomass is obtained with indefinite composition of the amino acids.

Also known is a method of cultivating microorganisms under aerobic conditions by gradually changing the stirrer energy input with the aim of improving the culturing conditions. First, the intensity of the stirring is increased until the concentration of CO _{2 has reached} a value of 3-11 % in the exhaust air. Thereafter, the energy input is lowered and the addition of air in the apparatus increases (copyright USSR Ur. 413183, Cl. C 12 B-1/08, '74). '.;

Disadvantage of the known method is that it is not possible, the process stable with a recovery of biomass with a certain predetermined content of essential amino acids, including an increased content of phenylalanine. Histidine. and to lead threonine.

, - - 2a -

 · - 2a -

Object of the invention

Ss is the object of the invention. To develop a yeast culturing method which allows a stable biomass recovery process with a given essential amino acid content including elevated levels of phenylalanine, histidine and threonine.

Explanation of the essence of the invention

From the objective-the technical task is derived to develop a method by which the regulation of various parameters in the process allows the production of biomass with an increased content of essential amino acids.

The object is achieved by cultivating yeasts on paraffinic hydrocarbons in the presence of the required mineral elements under aerobic conditions and mixing the medium with regulation of the specific energy input

mixing and aerating in the range of 3.5-6.0. -kW / t medium, 'Regulation of the ratio of. , ; , ... concentration of η-paraffins for energy input

in the range of 3-10 and a transition velocity of the paraffin from the hydrocarbon mixture into the cells in the range of 2.5-5.0 kg paraffin / t, medium. '.

Here is the ¹ positive effect when using the yeast Candida salmonicola WS8 779 (deposited in the collection of the Center for Industrial Microorganisms of the Institute ¹¹ WITII genetika of the Central Administration for Microbiological Industry at the Council of Ministers of the USSR under the number ZMPM U-196). larger than other yeast strains.

The process according to the invention is described in more detail below.

Yeast of the genera Candida, Trichosporum or others, is continuously cultivated in a permentor equipped with mixing and aeration device. Carbon sources are petroleum ^{distillates':} 10 - containing 25% normal paraffin used.

Ammonium nitrogen is used as nitrogen source, as source of phosphorus, potassium, magnesium and trace elements various salts containing these elements can be used. in the perforation medium is adjusted so that the concentration of n-paraffins (S ₀ ^par ) is 19 - 39 g / kg. ...

The concentrations of each nutrient and trace. , elements in the per- mentor feed in mg / kg:

N 2700 t 1500. P '370 t 180. ,

,, '. K, 625 -, 375.

, Mg 40 - 25 '

Pe- 3.95 - 2.05, '.' - 4 * -

Zn 9 , O ⁺ - VJL , 5 Mn VJL , 75 t 3 25 CU (V) , 3 t 1 75

Lie yeast is at a dilution rate of 0.2-0.3

-1

Ii, a pH of 2.5 - 4.5, one? Temperature of 30 - 40 ° C and a ventilation rate of 80 160 nr Ι.Έ, / t..h cultured.

The specific total energy input for. Stirring by means of a stirrer and aerating by means of a blower, a suction stirrer or another device which simultaneously mixes and aerates is 3.5 to 6.0 kW / t medium. The total specific energy input is as follows . "Form-el determined:

IL, + - N ₉ + IL,

(kW / t), where

Ϊ ¹

f_ the specific total energy input in kW / t;

L is the energy input (net) "for the mixing process (kW);

F ₂ the energy input (net) for ventilation (kW);

Lj the energy input .- (net) by possible additional mixing devices (kW) ;,

Ip mass content of the per-mentor (t).

, With the setting of the specific total energy. At the same time, the concentration of the carbon source is regulated so that the ratio S ^{r τ} '/ Έ _{a is} in the range 3.0 - 10,,, where

Par - ^s P ^ez ·. ·

S * the concentration of n-paraffins used in relation to the total inflow of the fermenter in kg / t

The transition velocity of the paraffins from the hydrocarbon mixture in the cells is on a. Value between 2.5 - 5.0 kg / t.h set. The transition speed is calculated according to the following formula:. "

kW r 1 -C. ·· ^; , ^r Par.- ^{= C} o * '< ^A "f - - -.) D 10 ³ (kg / th) where

C concentration of Kohlenwasserstoffgemis.ch in the

.Permentor intake (kg / kg)

pi

_G Par. And .C Initial and current concentration ~ '\ ° of paraffins in the hydrocarbon mixture (kg / kg)

D flow rate (h-1)

It has been found that in regulating the culture process ¹ at the specified limits for the specific energy conductivity, for the ratio of the paraffin concentration to the specific energy input and for the transition speed of the paraffins, the process stabilizes and a biomass with an increased content of essential amino acids , is produced.

The yeast suspension, which is removed continuously from the per-mentor, is further processed by known methods. The finished product contains yeast biomass

59 - 62.5 fo Eohprotein, 45.0 - 52.3 % pure protein with up to 3H, 5.7 % phenylalanine, up to 4.3 tfo histidine and up to 6.0% threonine based on the sum of the amino acids. , ,

embodiments

The process shall be described in more detail by the following examples:. ,

Example 1. '. '',

, The yeast Candida salmonicola (ZMPM U-196) WSB 779

became in a Permentor · with a mass content of, 5j5 t and with intensive. Ventilation of 120 m iU / th cultivated. The permentor was equipped with a stirrer, a pneumatic mixer and an external cooling circuit J-- .

The total energy input (net) was 33 kW, the '. specific total energy input thus 33 / 5.5 = 6.0 kW / t The carbon source used was a petroleum distillate with an n-paraffin content of C ^{r #} = 0.145 kg / kg

  puts. The distillate concentration at the input of the

kW: mentors was C. = 0.2 kg / kg, which was a

Paraffin concentration at the inlet of S _Q * = 29 kg / t

equivalent. The concentration of the trace and trace elements was (in mg / kg).

N 2600

P 340, 'K 480.

mg 24 le. 3.0 Zn 7.1 Cu 1.2

The cultivation was carried out at a pH of 3.5 to 3.7, a temperature of 309 to 311 K, a

-1

Yielding rate of 0.25 li and aeration

3 '

rate of 120 m 1 · Ν- / 1; .li.

During cultivation, the concentration of paraffins in the petroleum distillate was determined and the paraffin

Par calculated transition speed. It was found C * to 0.063 kg / kg, which is; , r

^r par

kg / t.h

revealed.

Contained by known methods and purified biomass contained. 62.4% Uohprotein or 52.3 ⁰ Io pure, protein. Der'Gehalt of phenylalanine was 5.4%, based on histidine 3.9 ί> and threonine 5.9% to the sum of the amino acids.

Example 2 .

The yeast C. salmonicola WSB.779, (ZMPM U-196) became '. analogously to Example 1 "in a per-type fermenter with a mass content of 75 t." The flow rate was

1 3

0.25 h, the ventilation intensity 87 nr i.N./t.h. The total energy input (sum of stirring and aeration) was 375 kW, corresponding to Ν = 5.0 kW / t.

s ρθ s ·

The carbon source was a petroleum distillate

Par with an n-paraffin gradient of C _n = 0.155 kg / kg

kW used at a distillate concentration of C. = 0.18 kg / kg. The paraffin concentration in the medium was

Par '

in order to. S = 27.9 kg / t. All other parameters were the same as in Example 1.

During cultivation, the current n-paraffin concentration was in the distillate.

C ^par = 0.074 kg / kg.

The codes-U _{0 a} ,. r_ and the ratio

spec ar

S ^Pai 7u = 5,6 ο 'spec. ^Ji

^r Par = ° ^'18 C ¹ ). 0,25 .1O ³ ^ ^ar 1-0,074

^r par ^{= 3} ' ⁹

The separated and purified biomass contains:

Eohprotein 59.1 ίο

Pure protein 51, 9 ίο ·

Phenylalanine 5.2 ίο based on the

Histidine 4.1 ίο Sum of amino acids

Threonine 5,8 ίο acids

Example 3

The yeast C. salmonicola WSB 779 (ZMPMU-196) was cultured analogously to Example 1 in the fermentor with a mass content of 5 kg. The Dürchflußrate was

-1

0.22 l. The total energy input was "at 19 W.

The carbon source was a petroleum distillate with

Par

an n-alkane concentration of G * = 0.138 used. The concentration of the distillate in the fermentation feed was C _o ^kW = 0.26 and correspondingly S ₀ ^Par = 35.9.kg / t. The other parameters were identical to those of Example 1. The actual concentration of the n-alkanes in the bottled petroleum distillate was C ^Par = 0.084 and the indices of the process were as follows:

V ^{s 3} ' ^{8 kw / t}

^r par

The separated and purified biomass contained:

Soh protein 60.1 $

Seine protein 51, 3 $

Phenylalanine 5.7 % based on the

Histidine 4.3 # sum of amino

Threonine 5.9 ί ° acids.

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Example 4 .

The yeast Lodderomyces elbngisporus IMET-H 128 (Candida guilliermondii "Druzba") was cultured in a per-mentor with a mass content of 75 t analogously to Example 2

 The total energy input for stirring and aeration was 405 kW. It was a petroleum distillate with a

Par n-alkane concentration of C = 0.19 used, the concentration of the distillate in the feed

kw medium C = 0.158. The concentration of

Va τ paraffins in the feed was thus S _Q = 30 kg / t.

The ^Cultivation was carried out at a pH from 4.0 to 4.2; a temperature of 305 - 307 K and

a ventilation rate of 87 m. i.BT./t.h.

The actual concentration of n-alkanes in the distillate was C ^Par = 0.073.

The indicators of the process were as follows:

S ^Pai 7n = 5,6 ο ' spec .. ^'

^r par

The separated and purified biomass contained: _

Crude protein 60.1 Io .

Re in protein 47.2 Io, _s

Phenylalanine 4.8 % of the

Histidine 3.5 % sum of the amino acids

threonine

- 11 -

The yeast Lodderomyces elongisporus IMET-H 128

(C. guilliermondii "Druzba") was analogously to Example 4 in a per- mentor with a mass content of

Cultivated 20 kg. , ,

The flow rate was 0.2-h and the aeration rate 150 1 i.K./kg. H.

'The total energy input for stirring' and 'venting' was 86 W. The carbon source

Par oil distillate used with C = 0.12.

kW The concentration of the distillate was C- '= 0.158, and the current paraffin concentration was C ^Par = 0.046. , .

The indices of the process were 'as follows' /

^r Par ^{= 2} > 5-.kg/t~. H.

The separated and purified biomass contained:.

Eohprotein - 61.3% Seine protein 45.4.%: Phenylalanine 4.5% based on the histidine 3.0% sum of the amino acids .. Threonine · 5.1% ^;

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Example 6

The yeast Lodderomyces elongisporus IMET-H 128

(C guilliermondii "Druifba ¹¹ ) was cultured analogously to Example 5. The total energy input for stirring and aeration was 120 W and the flow rate

-1

0.275 h. All other parameters were as in Example 5. The current concentration of n-alkanes in the distillate was C ^Par = 0.045. The indicators of the process were with it like. follows:

= 3.2. .,; -: - r _{par #} = 3.4 kg / t. H : . ,

The separated and cleaned. Biomass contained:

; Crude protein 60.1 % .

Pure protein 46.2 ⁰ Io . , Phenylalanine 5.0 % based on the histidine 3.2 ⁰ Io sum of the amino acids

Threonine 5.0 %

Example 7

The yeast Lodderomyces elongisporus IMET-H 128 (C. guilliermondii "Druzba") was cultured analogously to Example 5. The total energy input for stirring and aeration was 60 W, all other parameters correspond to those in Example 5..

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The actual concentration of η-alkanes in the distillate was C ^ar = 0.049. The indicators of the process were as follows:

= 3 W / kg · ... S ₀ ^Par / N__ = 6.3

r _par = 2.4 g / kg. H

The separated and purified biomass contained:

Crude protein 59, 8 'Io

Pure protein 45, 8 Io

, , "Phenylalanine 4.1 # based on the

Histidine 2.8 % sum of

Threonine 4.3 ί> amino acids

There were thus by one division of the Eermentationsprozesses to the invention without additional expenditure parameter levels of the essential amino acids phenylalanine, histidine and threonine to 5.7 # 4.3 ° fo and 5.9% when using the yeast C. salmonicola WSB 779 and ίο to 5.0, ^, 3.5 and 5.1% during cultivation of the yeast Lodd. elong. IMET Ή 128 (C. guilliermondii "Druzba"). In the cultivation and a known method, only 4.1 % , 2.8 % and 4.3 ⁰ Io were achieved.

- 14 '-

Claims (2)

Brfindungsanspruch _; 1. A method for the cultivation of yeast having an increased G content of phenylalanine, histidine and threonine on n-alkane-containing hydrocarbons in .. an aqueous nutrient medium with mixing and aeration, characterized in that in the biomass of the specific energy input for mixing and aerating in the range 3.5 - 6, Ό kW / t "per- tion medium, the ratio of the concentration of n-alkanes in the feed to the specific energy input in the range 3.0-1.0.0 and the transition speed / do not insert the n-alkanes from the hydrocarbon into the yeast cells in the range 2.5 - 5.0 kg / t · h. 'is. ,:,,, 2. The method according to item!, Characterized in that the yeast Candida salmonicola (WSB-779 ZMPM U 196) is used. '. ν