GB1568267A - Purification of yeast - Google Patents

Description

(54) PURIFICATION OF YEAST (71) We, VEB PETROLCHEMISCHES KOMBINAT SCHWEDT, of 133 Schwedt, German Democratic Republic, a Corporation organised and existing under the laws of the German Democratic Republic, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to a process for the purification of biomass, particularly purification by the separation of culture liquid and/or washing water. It is particularly concerned with a process for the purification of yeast.

The invention can be used in conventional apparatus in which microorganisms are cultivated and purified.

Processes are known by which biomass can be worked up or purified. This also applies to yeast cell suspensions in the industrial production of feeding yeast.

The purification is usually carried out by first mechanically separating the aqueous culture liquid as thoroughly as possible by decantation, the major part of the undesirable substances contained in the cell, e.g. potassium, magnesium, hydrocarbons, carbohydrates, specific to the process thus being likewise separated.

The concentrated biomass suspension is treated with substantially pure water, which is separated by means known per se, preferably by mechanical means in order to concentrate the biomass suspension again. The purification effect is thus mainly governed by the degree to which the culture liquid or the added washing water can be separated mechanically. The higher this degree of separation, the more effective the purification and the fewer washing stages required.

In addition, an increase in the degree of mechanical separation results in a reduction in the energy required for the thermal separation in cases in which the purified biomass has to be delivered as a dry free-flowing pulverulent product and stable in storage, as is generally the case in the production of feeding yeast.

In industry the water separation involved in these purification processes is usually carried out with the use of continuous centrifuges, so-called separators.

Under the industrial conditions in continuous separation, however, the water separation must be limited so that the suspension remains fully capable of flowing so that it can still be pumped to subsequent stages in the apparatus.

Such ready flowability is required to prevent the separators from becoming clogged or the throughput from being seriously reduced.

As these processes are usually carried out at 25-350C, the mechanical separation of water can only be carried out to a degree at which the dry substance still does not exceed 13--15 by weight of the yeast suspension.

The number of washing stages adopted will therefore depend on the degree of purity required. If dry products are to be produced with a water content of 5--100/, by weight a considerable proportion of the remaining water has to be separated by heating, such as vacuum evaporation followed by spray-drying.

These purification processes, however, are likewise subject, as a general principle, to certain limits, insofar that whilst extra-cellular constituents are removable such cannot affect the presence of undesirable substances contained in the cell.

Numerous methods have been described in patent specifications for improving the separation of the various phases and thus improving the degree of water separation. According to Ger. Dem. Rep. Patent Specification 52762, for instance, surface active agents of an anionic, cationic or non-ionic nature are incorporated into the fermenter cycle. To improve the separating effect still further, other measures have been proposed such as employing an increase in the temperature, the addition of intermediate washing processes (Ger. Dem. Rep. Patent Specification 74751), reduction of the osmotic pressure by salting out, evaporation and drying.

Ger. Dem. Rep. Patent Specification 54655 discloses a process for the production of free amino acids by autolysis, hydrolysis or plasmolysis. By increasing the osmotic pressure of the cells by the methods described the cells are caused to break at 45--55"C. Ger. Dem. Rep. Patent Specification 75994 describes a process for improving the oil separation and which involves the heating of the fermentation product.

Finally, German Patent Specification 1,442,090 describes a process for improving the separability of bacterial composition by the coagulation of the bacterial cells from the aqueous medium with intensified thermal treatment.

All the processes described suffer from serious technical and economic disadvantages. Autolysis at 500 C, for example, does not result in the reduction of the water content of the cells because water also has to be added prior to the individual separating stages, and would cause these cells to rupture. The method described in Ger. Dem. Rep. Patent Specification 75994, i.e. that of bringing about the separation of the oil by thermal treatment, merely serves to economize in wetting (surface-active) agents and does not assist in the separation of water. That described in German Patent Specification 1,442,090, involving the addition of salts and acids in conjunction with a temperature treatment, while suitable for bacterial cultures, for the purpose of coagulation, cannot be applied to yeast cultures, as the agglomeration caused by the increase in the size of the yeast cells would render the subsequent processing of the product more difficult. The purpose of the present invention is therefore to avoid or minimise one or more of the disadvantages arising under the conditions mentioned and to develop a simple and more economical purification technology.

The object of the invention is to reduce the cell water content prior to the separation, at the same time increasing the proportion of dry substance and exerting control over the undesirable substances contained in the cells.

It has been found that specific thermal treatment preceding the separation results in certain advantages and that both effects, i.e. the reduction of the cell water content and an increase in the proportion of dry substance, are generally obtainable simultaneously.

According to the present invention there is provided a process for the purification of yeast cells which have been cultivated on a culture medium in an aqueous environment, and which includes mechanical separation of surrounding extra-cellular water enriched with nutrient to facilitate separation of the yeast from the said extra-cellular water, the process including prior to the said mechanical separation heating the culture medium containing yeast to a temperature of 50 to 90"C for a period of 2 to 20 minutes, the lower part of the temperature range corresponding to the higher part of the period range and vice versa.

Intact yeast cells contain, in aqueous suspension at 25-350C, about 750 by weight of cell water, which it is in principle impossible to remove by the separation process.

A yeast suspension with 14% by weight of dry substance thus still contains water removable by separation. This water, however, is required at 25-350C for the purpose of maintaining the fluidity of the suspension.

The temperature range may be 60 to 800C and the corresponding period range 5 to 15 minutes, and when the yeast suspension is heated sufficiently (at least 5 min.

at 80"C, at least 10 min. at 709C or at least 15 min. at 600C) the volume of the yeast cells decreases by at least 50% involving contraction, denaturing of the cell wall and also of the cell plasma.

In processes according to the preceding paragraph the cells release at least 50% by weight of the cell water and also substances containea in the latter.

After heating for sufficiently long, therefore, the proportion of water removable by separation increases to about 72% by weight, undesirable constituents contained in the cell likewise proving removable from the cell at the same time.

It was found that the reduction in the size of the cells and the release of substances contained in the cells is a process governed by temperature and time.

On the basis of general practical experience, indicating the necessity of a free water phase of usually at least 50% by weight for the maintenance of fluidity, separation thus makes it possible, other conditions being equal, to increase the concentration of dry substance by 25% by weight.

In addition the temperature has a positive influence, as the fluidity of aqueous suspensions is known to increase with the temperature, and it was thus found that the continuous separation process enabled biomass concentrations of 30% by weight and over to be obtained.

As the concentration of solid substance is thus almost doubled by the application of the process of the invention, the number of washing stages and the volumetric flow of the suspension to be purified can thus be reduced to about half, the energy required for obtaining dry products being likewise approximately halved.

The thermal treatment may lead to further economic advantages since, as already mentioned above, substances contained in the cells, and having potassium ions, magnesium ions, phosphate ions as well as carbohydrates or hydrocarbons as their main constituents, are released with the cell water.

The tests performed have shown (in accordance with the examples) that the yeast cells separate, for example, about 2025% by weight of the potassium and magnesium absorbed in the fermenter, and also about 1020% by weight of the phosphorus. If the water resulting after the separation of the yeast which is enriched with potassium ions, magnesium ions and phosphate ions or even the washing water itself is returned to the fermenter, this provides a valuable saving of the potassium, magnesium and phosphorus, for example, which otherwise have to be added, in the form of water-soluble chemical compounds, to the fermentation process.

The advantages attainable by the present invention will be explained by reference to the following examples: Example 1 Purification of a feeding yeast which has been cultivated on cane sugar.

In a stirrer-type fermenter of 12 m3, Terula yeast was continuously cultivated on cane sugar in a known manner. The feed of the process water and of the nutrient and trace components dissolved therein was adjusted to maintain an average residence time in the fermenter of 3.5 hrs.

Further conditions were as follows: Feed of air into fermenter 250 N m3/h.

Temperature of fermentation suspension consisting of yeast, water, nutrient medium, nutrient and trace elements 33"C pH value 4. l.3 The pH was regulated by soda lye, NH3 water and phosphoric acid, the NH3 water at the same time serving as a source of nitrogen for the yeast.

By regular analytical control the feed of nitrient and trace salts was adjusted such that the average concentrations of the fermentation suspension taken from the fermenter were as follows: N 100150 mg/litre P 60--80 mg/litre K 80--100 mg/litre Na 50--400 mg/litre Mg 1 20 mg/litre Zn 24 mg/litre Mn 2 mg/litre Fe 5-10 mg/litre.

Cu 0.1-0.2 mg/litre Cl 1500--2000 mg/litre SO4 1OO150 mg/litre Carbohydrate 100--150 mg/litre Dry substance in 20--22 g/litre yeast The fermentation suspension flowing freely out of the fermenter, averaging 2 m3/h, was degasified in an intermediate vessel which precedes a pump and heated with steam to a temperature of 80"C, in a heat exchanger of the type having a nest of pipes. After the heating the product was passed to an intermediate container where it remained for an average of 10 min.

From the intermediate container the heated product was pumped into a continuous separator of the nozzle plate type. By separation in the gravity field of the separator two phases were obtained, the light phase consisting of the process water with a residual content of dry substance amounting to about 1--2 g/litre, while the heavy phase consisted of a yeast suspension with about 10-30% by weight, usually 18-22%, of dry substance. The yeast suspension was given in addition of about the same volume of distilled water, further purification and reduction of the salt content took place, and the mixture was conveyed into a second nozzle plate separator. In the latter, as in the first separation process, two phases were obtained with the same concentration of dry substance in the yeast.

The purified yeast suspension was introduced in a spray-drying plant and worked up into dry and highly pure feeding yeast free-flowing and pulverulent.

The light phases from the separators (process water and washing water) were for the greater part recycled to the fermentation process.

A particular advantage of this process, both for the purity of the seeding yeast produced and from the economic point of view, is the fact that after an adequate heating the yeast cells shrink in size and release, together with the cell water, organic and inorganic substances contained in the cells, as may be seen from the following table: Content in Process Water Before heating After heating Carbohydrate 100--150 mg/litre 400-600 mg/litre Potassium 80-100 mg/litre 250-300 mg/litre Phosphorus 6 80 mg/litre 120-150 mgflitre Nitrogen 100--150 mg/litre 250-300 mg/litre This process thus also ensures, with the same input and expenditure, a reduction of the salt content to 50% by weight in the feeding yeast obtained.

The amount of potassium and phosphorus required for the fermentation was reduced by 20--300/, by recycling the process water from the separation.

Example 2 Purification of a feeding yeast which had been cultivated on n-paraffin.

In an industrial stirrer-type fermenter yeast of the species Candida guilliermondii was cultivated on purified n-paraffins of the chain length C12-C20 as the sole source of carbon.

Fermentation Conditions Temperature 32+2 C pH 4.1+0.1 Aeration 6000 N m3 air/h.

Power supplied to the suspension fermentation by the stirring mechanism 240 kW Time for which fermentation suspension remained in the apparatus 4 h.

The feed of process water, nutrient salt solution n-paraffins, NH3 water and phosphoric acid was adjusted to ensure a residence time in the apparatus as required and the following concentrations maintained in the outflow from the fermenter: N 100-170 mg/litre P 70110 mg/litre K 70120 mg/litre Na 50200 mg/litre Mg 10-20 mg/litre Zn 2--4 mg/litre Mn 2--4 mg/litre Fe 5-10 mg/litre Cu 0.1--0.2 mg/litre Cl 1500-2000 mg/litre S04 lO(l50 mg/litre n-paraffin l(X200 mg/litre Dry substance in yeast 12 g/litre From the fermenter 20 metric tons/hour of yeast suspension were extracted continuously, this then undergoing an intermediate degasification process in an intermediate vessel of 5 m3 capacity and subsequently being heated with steam to a temperature of 80"C in a heat exchanger. This suspension was then temporarily stored in a container of 10 m3 and, after about 10 min, was subdivided into two phases, on similar lines to Example 1, in a separator of the nozzle plate type with a capacity of 2030 metric tons/hour. By the separator the yeast suspension is separated into two phases, one phase consisting of water enriched with salts and the other consisting of a yeast suspension having 1030% by weight of dry yeast substance, usually 18-22% by weight of dry yeast substance.

The yeast suspension was mixed in equal parts with washing water heated to 800C and again separated in the same manner as the first stage.

The yeast suspension was then worked up to dry yeast in a nozzle spray-dryer.

The process water and the separated washing water were for the greater part returned as process water to the fermenter.

The dry yeast had a residual paraffin content of below 0.2% by weight. Here again the process water, after the heating, contained increased quantities of the said undesirable substances from the cells, and these substances provided largely removable by the mechanical water separation and also capable of being economically recycled to the fermentation process.

Content in Process Water Content in process water prior to heating After heating n-paraffin 100--200 mg/litre 300--400 mg/litre Potassium 70--120 mg/litre 250-310 mg/litre Phosphorus 70--110 mg/litre 11--170 mg/litre Magnesium l 20 mg/litre 15- 50 mg/litre Nitrogen 100--170 mg/litre 250--320 mg/litre Example 3 Purification of a feeding yeast cultivated on petroleum distillate.

In the fermentation and separation plant described in Example 2, Candida gullllermondii was cultivated under the same biological and technological conditions.

The source of carbon, however, consisted of a petroleum distillate containing .n-paraffins and having a boiling point in a range 240--3400C (n-paraffin content: 21% by weight) in an average quantity of 15% by weight in relation to the fermentation suspension.

In the degasification vessel two layers formed, of which the upper layer mainly contained the yeast and the oil. The lower layer consisted of almost pure process water which proved immediately capable of being recycled to the fermentation process.

The upper layer (of which the composition was about 30% by weight petroleum distillate, 32 g/litre dry yeast substance and about 70% by weight water) was likewise extracted continuously, the petroleum distillate being separated with alkyl sulphonate solution in the known manner in the first nozzle plate separator.

The heavy phase (of which the composition was about 4.2% by weight dry substance, 1% by weight residual petroleum distillate and about 95% by weight water) was heated to 750C by means of steam in the heat exchanger, temporarily stored for about 15 min. in the intermediate container and then subdivided in the second nozzle plate separator into two phases. (The heating in the heat exchanger, of the type having a nest of pipes, could nevertheless equally well precede the separation of the petroleum distillate).

The heavy phase contained 1030% by weight of dry yeast substance usually 19-25% by weight), 2% by weight residual petroleum distillate and 7075% by weight water. This yeast suspension was dried in the spray-dryer and then subjected to known solvent extraction processes for the production of highly pure feeding yeast.

The separated process water likewise contained increased quantities of the said undesirable substances (see table) after the heating operation. In conjunction with the high degree of water separation, this enabled a dry product to be obtained with a low salt content and having particularly favourable properties, as inorganic salts, as is known, impede the extraction operation performed with organic solvents.

The process water removed in the separation was likewise recycled to the fermentation process.

Content in Process Water Prior to heating Subsequent to heating Hydrocarbon 0.3% 0.5% N 100--170 mg/litre 300--400 mg/litre K 70--120 mg/litre 250--380 mg/litre P 7l 10 mg/litre 150-200 mg/litrc Mg 1020mg/litre 15- 50 mg/litre WHAT WE CLAIM IS: 1. A process for the purification of yeast cells which have been cultivated on a culture medium in an aqueous environment, and which includes mechanical separation of surrounding extra-cellular water enriched with nutrient and trace elements to facilitate separation of the yeast from the said extra-cellular water, the process including prior to the said mechanical separation heating the culture medium containing yeast to a temperature of 50 to 900C for a period of 2 to 20 minutes, the lower part of the temperature range corresponding to the higher part of the period range and vice versa.

2. A process according to Claim 1, wherein the temperature range is 60 to 80"C and the corresponding period range is 5 to 15 minutes.

3. A process according to Claim 1 or Claim 2, wherein the culture medium comprises cane sugar, a mixture of n-paraffins having a chain length of C12-C20 or a petroleum distillate.

4. A process according to Claim 1 substantially as herein described and exemplified with reference to any one of Examples 1 to 3.

5. Yeast which has been purified by the process claimed in any preceding

Claims (1)

1. claim.