IL45716A - Process for the fermentative manufacture of high protein yeast in a nutrient medium - Google Patents

Abstract

1452019 Stirrers HOECHST AG 27 Sept 1974 [28 Sept 1973] 42045/74 Heading B1C [Also in Divisions C3 and C6] The fermentation of n-paraffin hydrocarbons by yeasts of the species Candida may be carried out in a tank provided with at least two stirrers, the ratio of stirrer diameter to tank diameter being from 0.35 to 0.65 and the distance between the two stirrers approximately corresponding to one stirrer diameter. [GB1452019A]

Description

45716/2 PROCESS FOR THE FERMENTATIVE MANUFACTURE OF HIGH PROTEIN YEAST IN A NUTRIENT MEDIUM mnn saaa The present invention relates to a process for the manufacture of high yeast in a nutrient medium protein/by fermentation.

According to known biosynthetic methods, microorganisms, for example certain yeast strains, are multiplied by fermentation in a synthetic nutrient medium containing n-paraffin as the only carbon source. Such methods can be carried out batchuise as well as continuously. The paraffins uaed for this purpose generally contain 8 to 23 carbon atoms.

The yeaet cells thus obtained contain, in the so-called crude protein, usually from 60 to 80 % by weight of amino acids and from 8 to 12 % by weight of nucleinio acids (calculated by multiplication of the nitrogen value found by elementary analyais with the empirical factor 6.25). Such a high portion of nucleinic acids is, however, undesirable, since they produce uric acid, especially in a human organism, which again is the cause of gout. Moreover, they adversely affect the microbial population in the gastrointestinal tube. of high protein content in a nutrient medium We have now found a process for the manufacture of yeast/ having an n-paraffin with 8. to 23 carbon atoms as the carbon source and a nitrogen source under aerobic conditions at a pH-value of from 2.0 to 6.8, which process comprises fermenting yeast strains of the Candida species, the mutants or variants thereof in such a manner that the concentration of n-paraffin in ths phase of logarithmic growth of the yeast cells is between 0.04 and 0.12 % by weight, calculated on the culture suspension.

A concentration of n-paraffins ranging from 0.08 to 0.10 % by weight arid the use of n-paraffins having 10 to 14 carbon atoms in the chain are preferable.

The process of the invention is advantageously carried out in fermentation tanks which hold a nutrient medium containing, in addition to the cited n-paraffin, salts such as potassium nitrate, ammonium sulfate or ammonia, urea or soy bean flour as a nitrogen source. It furthermore contains phosphates, such as potassium hydrogenophosphate or disodium hydrogenophosphate as uiell as magnesium and potassium salts and trace elements as present also, for example, in tap water.

It may be advantageous to add nutrients, such as yeast extract, meat extract, glucose, thiamine and the like, especially to the pre-cultures.

The process is advantageously carried out at temperatures of from 20 to 35° C, preferably from 28 to 32° C.

As yeast strains, Candida species are used, in particular as wall knouin as Candida lypolytica ATCC 20.383 or Candida guilliermondii ATCC 20.382, but also those uihich are less known, such as Candida para-psilosie ATCC 20.384 or rare ones, such as Candida visuianathii ATCC 20.385.

The culture suspension in the fermentation tank is aerated uiith 0.3 to 1.5 liters of air per liter of culture solution and minute (vvm), preferably uiith 0.9 to 1.1 vvm. To ensure a good absorption of oxygen by the cells, intense stirring is advantageous and emulsifiers may also be added. For a good exchange of all the components in the four-phase system cell-salts solution-paraffin-air, it has proved to be advantageous to use one, or even better, two or more stirrers in a fermentation tank, the speeds of uihich being between 100 and 250 r.p.m., preferably betueen 150 and 200 r.p.m. It is furthermore advantageous to arrange tuio stirrers, especially turbine stirrers, in the fermentation tank in such a manner that the distance betueen the stirrers approximately corresponds to a stirrer diameter. The ratio of the stirrer diameter to tank diameter is suitably in the range of from 0.35 to 0.65, preferably from 0.45 to 0.55. The circulation speed of the culture suspension is advantageously exceeding 7 meters per second.

Should foam be produced, a chemical or mechanical foam depression j is recommended, for example by adding 0.01 to 0.1 % by weight of an anti-foaming agent, such as octanol, esters of oleic acid and lauric acid with glycol, glycerol or sorbitol, alcoholic cholesterol solution or silicones, such as polydimethylsiloxane.

The n-paraffin concentration ranging from 0.04 to 0. 12 % by weight, preferably from 0.08 to 0.10 % by weight, can be controlled continuously by various means, for example by measuring the nitrogen consumption, the cell mass portion or preferably by measuring the carbon dioxide release. By this, quick reaction on the lack of carbon initiated by a decreasing COg release is possible and thus precise dosage of the paraffin addition has to take place.

If the pH-v/alue of the culture suspension falls below its permissible limit, it is readjusted to the required value by an addition of alkali, for example sodium hydroxide solution or potassium hydroxide solution.

To check the process, samples are taken from the culture suspension in order to determine the dry weight and the nitrogen content in the yeast cell mass. The data found allow to determine the growth rate and the multiplication period of the cell mass. If the dry weight of the yeast does no longer increase logarithmically in successive samples, the fermentation process is discontinued.

The yeast mass is separated in the usual manner by decanting or preferably centrifuging it while washing it several times with water. So a pasty yeast cell mass is obtained, which still contains 75 to 90 % by weight of water. It may be dried by various methods, for example by means of rollers, a whirl bed or by spraying. The product thus dried merely contains 2 to 5 % by weight of water and 45 to 60 % by weight of crude protein. In this crude protein, the portion of amino acids ranges from 90 to 95 % by weight. In this connection, it is noteworthy that not only this portion of amino acids in general but also the content of j essential and semi-essential amino acids in particular is higher than in comparable products of the prior art. Moreover, the crude ash content of the dried cell mass as obtained according to the invention is substantially lou/er than that of the known products. The crude protein of the invention moreover merely contains 3 to 6 % by ueight of nucleinic acids, whilst known yeast products contain 8 to 12 % by weight of nucleinic acids.

The dry yeast mass obtained according to the process of the invention is therefore particularly suitable as a protein source in foodstuff for man and animal.

The following Examples illustrate the invention.

EXAMPLE 1: The strain Candida lipolytica FH-H-5027 / ATCC 20.383, which has been cultivated on a slanted' tube of agar containing 2.5 % of meat extract, 5.0 % of yeast extract, 10.0 % of meat peptone, 10 % of glucose, 10 % of NaCl and 2.5 % of agar, is suspended with 4 ml of distilled water of physiological sodium chloride solution, and the suspension is transferred by inoculation under sterile conditions to an Erlenmeyer flask having a capacity of I 2 liters and containing 250 ml of a preculture nutrient solution of the following composition: 0.53 % of ( H4)2S04 0.4 % of ΚΗ2Ρ0ή 0.2 % of Na2HP04.12 H20 0.02 % of MgS04.7 H20 0.02 % of C1 1.5 % of n-paraffin and 0.1 % of trace elements (pH 5.5).

Fermentation is carried out in a tank of a capacity of 14 liters, which is filled uith 10 liters of the following nutrient solution: 1 % of ( H4)2S04 0.4 % of KH2P04 0.2 % of Na2HP04.12 Η,,Ο 0.02 % of FlgS04.7 H20 and 0.02 % of KC1.

The pH-value of the nutrient solution is 0.4. It is sterilized for 45 minutes at 121° C. Moreover, 300 g (3 % by weight) of n-paraffin having 10 to 14 carbon atoms are sterilized separately. Prior to the inoculation of the main culture, 1 % by weight of n-paraffin is added to the nutrient solution.

To inoculate the nutrient solution, two times 250 ml of the pre-culture which has been shaken for 48 to 72 hours at 28° C, are used under sterile conditions. The nutrient solution thus inoculated is stirred for 48 to 64 hours at 28° C by means of a turbine stirrer with aeration of 1 vvm of air.

If necessary, the pH-value is readjusted by adding a sterile 2N sodium hydroxide solution. During the logarithmic phase of the fermentation, a concentration of n-paraffin ranging from 0.08 to 0.1 % by weight is maintained. If the C02 release of the cells decreases, furthe portions of n-paraffin are added automatically until the C02 release again increases. The proceeding fermentation is controlled by taking samples in order to determine the dry weight and the nitrogen content of the yeast cell mass.

When the logarithmic growth phaaa has terminated, the cells are worked up in the usual manner by centrifuging and washing them with water and then spray-drying them. The so -obtained product has a crude protein content of 56.7 % by weight, an amino acid content of 54 ,9 % by weight and a nucleinic acid content of 3.5 % by weight. The essential and semi-essential amino acids alone account for 49.89 % by weight (including glycine: for 54.34 % by weight).

Detailed data are listed in the following Table 1.

T A B L E EXAMPLE 2: the strain Candida wisuanathii FH-H-5123 / ATCC 20.385 is cultivated as in Example 1. Further, 9 1 of the nutrient solution of the composition as indicated in Example 1 are placed into a fermentation tank hawing a capacity of 14 liters, and 0.5 % by weight of sterilized paraffin is added thereto. The nutrient solution is inoculated as in - - Example 1 and stirred at 30 C by means of a turbine stirrer at a speed of 250 r.p.m. with aeration of 1 vvm of air. Where necessary, the pH- value is maintained at 4,0 by adding a sterile sodium hydroxide solution. During the logarithmic phase of the fermentation, a concentration of n-paraffin ranging from 0.08 to 1.0 by weight is maintained. The check of this concentration, the control of the process as well as the separation and work-up of the cell mass obtained are performed as in Example 1. After spray-drying, a product is obtained, uihich has a crude protein content of 46.6 % by weight, an amino acid content of 42.0 % by weight and a nucleinic acid content of 4.1 % by weight. The crude ash content is 3.05 % by ueight.

EXAMPLE 3: The strain Candida parapsilosis FH-H-5356 / ATCC 20.384 is cultivated as in Example 1 first in a fermentation tank having a capacity of 10 liters. The culture suspension thus obtained is then transferred to a fermentation tank having a capacity of 200 liters, u/hich is filled with 150 liters of the following nutrient solution: 1 % of (NH4)2S04 0.4 % of KH2P04 0.2 % of 2H 04 0.2 % of Na2HP04.12 H20 0.02 % of NaCl 0.02 % of l*lgS04.7 H20 and 0.5 of n-paraffin (10 to 14 carbon atoms).

The pH-value of the nutrient solution is adjusted to 5.0 by means of semi-concentrated phosphoric acid. The solution is then sterilized at 120° C under a pressure of 1.4 atmosphere gage for 20 minutes.

After sterilization, the pH value is 4.5 to 5.0.

The culture is then fermented for 40 to 48 hours at 28° C with an > aeration of 1.1 vvm while stirring by means of 2 stirrers at a speed of 250 r.p.m. During the logarithmic phase of the fermentation, a concentration of n-paraffin ranging from 0.08 to 0.1 % by weight is maintained by adding further portions of. n-paraffins, if the carbon dioxide release weakens, until the release again increases. The pH value is maintained at 3.5 to 4.0 by adding a sterile sodium hydroxide solution. The proceeding fermentation and the end point thereof are determined as in Example .1.

The culture suspension thus obtained is used as inoculation material or a fermentation tank having a capacity of 2000 liters. The nutrient solution has the same composition and the same pH value as indicated for the last-mentioned fermentation operation. The fermentation tank is sterilized for 20 minutes at 120° C under a pressure of 1.4 atmospheres gsge with stirring at 210 r.p.m. After sterilization,the pH-value is 4.0 to 4.5.

Subsequent fermentation is performed at 28° C with aeration of 1.0 vvm under a pressure of 0.3 atmosphere gage and using two stirrers at a speed of 210 r.p.m. The pH-value is maintained during the fermentation between 3.0 and 4.5 by means of a sterile sodium hydroxide solution.

The proceeding process is controlled by takiig samples for checking sterility, measuring the pH-value and determining the dry weight and nitrogen content of the yeast cell mass.

The concentration of n-paraffin is maintained between 0.08 and 0.1 % by weight during the logarithmic phase of the growth, and checked as disclosed above in accordance with the carbon dioxide release.

When the logarithmic phase of growth has terminated, the culture suspension is heated to 55° C for a short time and then cooled to 18° C. The pH-value ranges from 3.5 to 4.5. , 1B50 liters of the culture suspension thus obtained having a content of 23 g per liter of dry mess are centrlfuged in a solid-jacket screw centrifuge at 4450 g and a throughput of 1060 liters per hour. The moist yeast cells thus separated are suspended in demlnerallzed water to yield a suspension having a content of 10 to 15 % by weight of dry mass, and the suspension was stirred vigorously at 15 to 18° C in an adequate vessel, whereby accompanying substances and constituents of the nutrient solution are washed off within about 1 hour. After another centrifuging operation, a 20 to 25 $-by-weight yeast suspension is prepared with demlnerallzed water, stirred under the same conditions and again centrlfuged. The yeast cells thus washed are made into a 40 to 50 jS-by-weight suspension which was then spray-dried.

Thus, 36 kg of a light cell mass having a faint yeast-like smell are obtained, which contains 47.9 % by weight of crude protein, 43.6 % by weight of amino acids - among which 57.Θ % by weight are essential amino acids -, 3.9 % by wBight of nucleinic acids and 3.7 % by weight of crude ash.

EXAMPLE 4: The strain Candida guilliermondii FH-H-5151 / ATCC 20.382 is fermented as in Example 3 in a fermentation tank having a capacity of 2000 liters, and containing 1900 liters of culture suspension. The process is carried out as in Example 3. 38 kg of a cell product having a faint yeast-like smell are obtained, which contains 48.8 % by weight of crude protein, 45.1 % by weight of amino acids and 3.6 % by weight of nucleinic acids. The crude ash content is 3.5 % by weight.

Claims (9)

HOE 73/r 300 Ide claim: of ^ p rotei- n COntent , i n a nutri ent

1. A process for the manufacture of yeast/hawing an n-paraffin with medi um 8 to 23 carbon atoms as a carbon source and a nitrogen source under aerobic conditions at a pH-value of 2.0 to 6.8, which com- prises fermenting a yeast strain of the Candida species, the mutants or variants thereof in such a manner that the concentration of n-paraffin in the phase of the logarithmic grouth of the yeast cells is between 0*04 and 0.12 % by weight, calculated on the culture suspension.

2. A process as claimed in claim 1, wherein the concentration of n-paraffin is between 0.08 and 0.1 % by weight.

3. A process as claimed in claim 1, wherein n-paraffin having 10 to 14 carbon atoms is used as a carbon source.

4. A process as claimed in claim 1, wherein the culture suspension is aerated with 0.3 to 1.5 volumes of air per volume of suspension and minute.

5. A process as claimed in claim 1, wherein the culture suspension is stirred at a speed of 100 to 250 r.p.m.

6. A process as claimed in claim , wherein fermentation is carried out in a tank provided with at least two turbine stirrers, the ratio of stirrer diameter to tank diameter being between 0.35 and 0.65 and the distance between the two stirrers corresponding to one stirrer diameter.

7. A process as claimed in claim 1, wherein Candida lipolytica FH-H-5027 / ATCC 20.383, the mutants or variants thereof are used.

8. · A process as claimed in claim 1, wherein Candida viswanathii FH-H-5123 / ATCC 20.385, the mutants or variants thereof are used.

9. A process as claimed in claim 1, wherein Candida parapsilosie FH-H-5356 / ATCC 20.384, the mutants or variants thereof are ueed. HOE 73/r 300 A process a 8 claimed in claim 1, wherein Candida guilliermondii FH-H-5151 / ATCC 20.382, the mutants or variants thereof are used Attorneys for Applicant