The present invention related to the invention of a process for the preparation of yeast extract containing low levels of inorganic salts and carbohydrate for applications in fermentation process.
Yeast extract has been known for several decades; its principal application areas are food, pharmaceutical and fermentation processes. It has been used as a flavouring agent in meat products- soups and seasoning and as a well-defined source of nitrogen and amino acids in microbial processes It is known that, yeast extract is prepared by yeast cell lyses and the cell contents are then extracted several times with a suitable solvent (water), and the solvent is evaporated (usually under vacuum) to get yeast extract paste, which is a viscous material, rich in proteins and enzymes. Alternatively, the solvent is evaporated in a spray drier to get yeast extract in powder form. Irrespective of the method chosen for the removal of water, the chemical composition of yeast extract stands to be fairly identical, except for moisture content which would be present at a low level in the spray-dried product as against the paste.
The method of lysis of yeast cells has a bearing on the chemical composition of the final product. As reported in the Polish Patent, Pol. 106,653 (1980), an inorganic salt, preferably sodium chloride is added to cause cell lysis. The yeast suspension is subjected to cell lysis by adding 0.5 to 5% sodium chloride at pH 4 to 7 for first 20-28 hrs at 45-55°C, followed by heat at 60-70°C for 13-23 hrs. and then finally at 75-85°C for 5-15 hrs. After cell lysis, the aqueous layer is separated and concentrated; thus, water-soluble inorganic salts are concentrated along with the cell constituents and hence the concentration of

the inorganic salt in the final product is generally significant. Swiss Patent CH 643, 296 (1984) describes yet another process for the preparation of yeast extract, but the final yeast extract contained 12.9% sodium chloride.
A major area of application of yeast extract is in fermentation processes as a source of nitrogen. Several fermentation processes are directed towards the production of specific biologicals under controlled conditions, with carefully selected nutrients. Several of the metabolic processes are known to be activated or inhibited by specific inorganic ions or salts. For example, the enzyme, urease, has been shown to be inhibited by sodium chloride even at low concentrations as reported in J.Am.Chem.Soc.,(1951), 73, 1646-1650. Thus, it is preferable to develop a yeast extract that is fairly free of inorganic salts, and the requisite salt(s) may be added to the fermentation medium later on, depending upon the specific needs; otherwise, yeast extract that contains a certain concentration of inorganic salt(s) would be inadvertently introduce those electrolytes in fermentation medium, and alter the rate of production of secondary metabolites or the activity of enzymes. Accordingly, the object of this invention is to develop a processing scheme for the preparation of yeast extract that is substantially free of inorganic salts.
Briefly, the process of the present invention is directed toward an improved scheme of preparation of yeast extract containing low concentrations of inorganic salts and carbohydrates, which find applications in fermentation processes and biological waste treatment schemes.

In certain processes for the preparation of yeast extract, addition of an inorganic salt is kept as an option; instead, an organic solvent, such as chlororoform is used. [Tekhnol.Proizvod.Sukkish Diagn. Pitatel'nykh Sred (1974), 6,102-103 (Russ) and in Tekhnol.Proizvod.Sukkish Diagn. Pitatel'nykh Sred (1974), 6, 104-105 (Russ)] or toluene/ethyl alcohol is added as indicated in the Indian Patent, IN 157,237 (1986) to accelerate lysis after some heating. Some of these solvents are flammable and hence relevant precautions are necessary for the handling of large volumes of these solvents during lysis. Moreover, some of these solvents are hazardous. Addition of the solvent for the purpose of lysis requires that, the same solvent be removed during the subsequent stage of concentration of yeast extract by evaporation; one or more of solvent recovery operations would be mandatory which would eventually add to the operating costs. Loss of solvent is bound to occur during any evaporation process, and hence solvent addition or replenishment would be required. Thus, the preparation of yeast extract with organic solvents involves certain expenses arising from solvent handling and replenishment. Moreover, the use of yeast extract containing traces of toxic solvent is discouraged in food and pharmaceutical applications. Accordingly, yet another aim of this invention is to develop an efficient and low-cost processing scheme that does not employ volatile organic solvents for the production of yeast extract.
Almost all the processing schemes, as described above, require that the suspension of yeast, along with a suitable salt or solvent, be maintained at elevated temperatures (40-90°C) for several hours, as disclosed in the Polish Patent Pol. 106,653 (1980), Swiss Patent CH 643,296 (1984),

Tekhnol.Prozvod.Sukkish Diagn.Pitatel nykh Sred (1974), 6, 102-103 and 104-105 (Russ) and in the Indian Patent, IN 157, 237 (1986). For example, according to the Polish patent Pol. 106, 653 (1980), the yeast suspension should be maintained at 50-80°C for about 50 hours. The Indian Patent TN 157,237 (1986) indicates that the yeast suspension be kept at 34-39°C for 8 to 24 hours, followed by 20-30 hours at 45-55°C and finally at 80-95°C for 0.5 to 2 hours; thus, the processing time at elevated temperatures vary from a minimum of28 hours to maximum of 56 hours. In industrial practice, heating and maintenance at a particular temperature is usually accomplished by steam, or hot water, or other suitable heating medium. Maintenance of elevated temperatures for several hours is an energy-consuming operation, and the related expenses arising from energy/fuel cost adds to the processing cost of the final product. Therefore, the third object of this invention is to develop an improved and economical process for the preparation of yeast extract at much reduced processing times, without the requirement of elevated temperatures for lysis.
Active yeast contains several carbohydrates. Literature sources indicate that up to 40% of the dry weight of yeast can occur as storage carbohydrates. Trehalose, glycogen and mannans are usually significant constituents of yeast. During the preparation of yeast extract, when the aqueous layer is concentrated after cell lysis, most of the water-soluble carbohydrates are also taken in the same layer and get concentrated. Hence, yeast extract, as the final product after concentration, normally contains about 7 - 10% carbohydrates. One of the significant uses of yeast extract is its utility as a

growth medium in microbiological processes and fermentation industries. It is well known that, in certain fermentation operations, the concentration of the total carbohydrates and the nature of the individual carbohydrates in a fermentation broth play an important role in dictating the direction of the metabolic processes. For example, the yeast Saccharomyces cerevisiae is able to ferment glucose, galactose and suerose but not xylose and arabinose as reported in Process biochem (1981) 16, (4) 42. As a second example, consider biological wastewater treatment schemes where yeast extract may be used as a source of nitrogen. One of the primary objectives of the waste treatment scheme is the reduction of chemical oxygen demand (COD). Many waste streams encountered in petrochemical and organic chemical industries usually contain alphatics and aromatics (primarily carbon compounds) that contribute significantly to the COD; in other words, for the biological treatment scheme to remain effective, almost all the carbon compounds should be metabolized and degraded by the chosen consortium of microbes.
Hence in such waste treatment schemes, it is preferable that the nutrients feed to the system contain a low level of carbohydrates so that the bacteria may be forced to derive most of the carbon requirements from the target pollutants (carbon compounds), and thus the rate of degradation of organic pollutants and consequently the rate of reduction of COD would be much faster. This has been found especially in biotreatment processes developed by SPIC for industrial wastewaters, e.g. Biotreatment of Glycol waste water: [Appl.Biochem. Biotechrol (1991) 28/29, 827-241] and PTA plant waste water [Trans IchemE (1997) 75(B), 245-256]. For such reasons, yeast extract containing a low level

of carbohydrates would be preferable. Accordingly, the fourth object of this invention is to develop a process for the production of yeast extract that shall contain a low level of (about 1 %) carbohydrates. It may be pointed out that the carbohydrates normally present in yeast extract does constitute an important source of carbon and hydrogen for microbial growth. If it is specifically required to reduce the concentration of carbohydrates, then a modified procedure (with pH correction) outlined in the next section may be adapted; if the sugar content of the product would be of insignificant consequence, then such a pH correction step may be omitted.
DESCRIPTION OF THE INVENTION:
The yeast cell lysis may be caused by several methods; in this process the suspension of yeast is subjected to high shear, high pressure conditions by passing it through an ultrafine grinding equipment such as a homogenizer, colloid mill, ball mill etc., and thereby cell breakage occurs. The cell components are immediately released into the aqueous solution, which is then clarified to remove the cell debris. The aqueous solution, rich in proteins and enzymes, is then concentrated by evaporation of water to the desired extent, so that the final product shall be a free flowing powder or a paste.
If it is specifically required to prepare a yeast extract with a low carbohydrate content, then the above description is slightly modified in view of the biochemical activity of various hydrolytic enzymes present in yeast. When the proteinaceous cejl contents are extracted in the aqueous layer, certain amount

of water soluble carbohydrates are also extracted. This gives rise to the
carbohydrate content of the product that is usually of the order of 7-10%; the
protein content of the yeast extract (paste) is around 45-60%. To reduce the
level of carbohydrates, the biological activity of the enzymes present in yeast
may be utilized. Thus, while cell lysis is effected, the hydrogen ion
concentration of the solution is adjusted in such a way that the hydrolytic
enzymes of yeast would remain active. In our work, such an enzymatic activity
was found to break down the complex sugar molecules of yeast so that final
level of carbohydrates was reduced to 3-1 %. The pH of a suspension of yeast
is normally acidic (in the range of 3 - 4) and most of the hydrolytic enzymes of
yeast (like invertase) seem to remain almost inactive in this pH range; if the
hydrogen ion concentration of the yeast suspension were not altered before or
after cell lysis, then the pH of the solution would continue to remain acidic so
that most of hydrolytic enzymes continue to remain inactive and the sugar
molecules would not usually be attacked. In this work, it was observed that,
when the pH of the yeast suspension was adjusted to be near neutral before
carrying out cell grinding, then the hydrolytic enzymes, immediately on release
from the cell, seemed to act on the sugar molecules and were able to degrade
them, so that the end products could be organic acids; thereby a decrease in
pH was observed after cell grinding. Thus, in order to prepare a yeast extract
with a low level of carbohydrates, the pH of the yeast suspension was adjusted
to be neutral before cell lysis. After lysis, the aqueous layer was separated and
concentrated as explained before.

It is contemplated that any requisite equipment will either be commercially available or readily fabricated by those skilled in the art, and hence, detailed description of such equipment is believed to be unnecessary.
The following specific examples illustrate the practice of the process:
EXAMPLE 1
One litre of an aqueous suspension of baker's yeast was pH adjusted to neutral with an alkali like NaOH or KOH or NH4OH and subjected to homogenization in a high pressure homogenizer at about 100 bar at room temperature. The suspension was passed through the pressure parts five times so that the cell lysis should nearly be complete. The suspension was kept at room temperature for overnight and then decanted and centrifuged to remove the cell debris. The clear liquid was concentrated by evaporation under vacuum at 70-80°C to furnish the final product of the following composition:
Proteins 59 - 62%, carbohydrates 5 - 6%, amino nitrogen 3 - 4%, moisture 30 - 31%, sodium: less than 0.1%, total nitrogen 8 - 9%, inorganic salts (as NaCI), 0.1%.
Thus obtained yeast extract was tested in fermentation medium and found that it enhanced the microbial growth.

EXAMPLE 2
500 liters of dilute suspension of distillery fermented yeast sludge (contains brewer's yeast) was pH adjusted to neutral with either NaOH or KOH or NH4OH and homogenized at 60 - 200 bar pressure. The suspension was kept at room temperature for 40 hours and allowed to settle. The clear liquid was dark brown in colour, which was decanted and concentrated by evaporation under vacuum at 70 - 80°C. The final product was approximately 25 kg of yeast extract with about 32% moisture. Its composition was,
Proteins 56-58%, carbohydrates 5-6%, amino nitrogen 4-5%, total nitrogen 9-10%, inorganic salts (as CaCi), 0.1%.
On addition to biological wastewater treatment ponds, the yeast extract was found to enhance bacterial growth and resulted in the degradation of target pollutants.
EXAMPLE 3
Yeast extract of low carbohydrate content was prepared as follows. Compressed, frozen (brewer's) yeast cake weighing 500g was suspended in 500 ml of water and the pH was adjusted to 6.5 with 6 N sodium hydroxide solution. The suspension was then homogenized at 60-150 bar pressure allowing four passes through the homogenizer. The pH, immediately after homogenization, was observed to be 5.8. On keeping the suspension

overnight at room temperature without agitation, the pH had further decreased to 5.4. After clarification, the clear aqueous layer was concentrated by evaporation under reduced pressure. The above process was conducted at room temperature. The final product had the composition as follows:
Proteins 42.5%, carbohydrates 0.9%, total nitrogen 6.90%, amino nitrogen 3.1%, moisture 38.2%, inorganic salts (as NaCI), 0.1%
On addition to fermentation medium, the yeast extract was found to enhance bacterial growth.
Although several examples of the invention have been given, it will be appreciated that changes and modifications thereof may be made without departing from the scope of the invention. Therefore, the invention shall not be limited to the specific examples and embodiments of the process shown and described herein, but shall be defined in scope by the following claims:
WE CLAIM
1. A process for the preparation of yeast extract wherein an aqueous suspension of yeast (baker's, brewer's or any other suitable yeast species) is pH corrected to neutral and is ground physically by subjecting it to high shear and/or high pressures to cause cell lysis, the ^pqueous suspension is then maintained at temperature between 27-30°C for 10 hours. The clear layer is separated by decantation and

overnight at room temperature without agitation, the pH had further decreased to 5.4. After clarification, the clear aqueous layer was concentrated by evaporation under reduced pressure. The above process was conducted at room temperature The final product had the composition as follows:
Proteins 42.5%, carbohydrates 0.9%, total nitrogen 6.90%, amino nitrogen 3.1%, moisture 38.2%, inorganic salts (as NaCI), 0.1%
On addition to fermentation medium, the yeast extract was found to enhance bacterial growth.
Although several examples of the invention have been given, it will be appreciated that changes and modifications thereof may be made without departing from the scope of the invention. Therefore, the invention shall not be limited to the specific examples and embodiments of the process shown and described herein, but shall be defined in scope by the following claims:
WE CLAIM
1. A process for the preparation of yeast extract wherein an aqueous suspension of yeast (baker's, brewer's or any other suitable yeast species) is pH corrected to neutral and is ground physically by subjecting it to high shear and/or high pressures to cause cell suspension is then maintained at temperature between 27-
-
°C for 10 hours. The clear layer is separated by decantation and

concentrated by evaporation to give a paste or powder of the yeast extract product containing less than 50% moisture.
The process as described by claim 1 wherein a colloid mill, a ball mill, a high pressure pump, a homogenizer or any other mechanical equipment is used to develop sufficient pressure in the order of 20-1000 bar or grinding action so as to rupture the yeast cells.
The process as described by claim 1 wherein the aqueous suspension after cell grinding is maintained at ambient to 90°C, for a period of time ranging from 0-48 hours, with or without agitation.
The process as described by claim 1 wherein the pH of the yeast suspension is adjusted, to 6 - 8 before cell lysis, by using 6 N sodium hydroxide, so as to reduce the carbohydrate content of the final product. The pH of the solution is adjusted by slow addition of dilute sodium hydroxide (6 N) between 6-8, before, during and after cell grinding so as to maintain the biological activity of the hydrolytic enzymes near optimum.
The process as described by claim 1 wherein the cell debris are optimally separated from the aqueous layer by any combination of known separation devices, such as a clarifier, a thickener, a centrifuge or a device using a semi-permeable membrane, and the aqueous layer

is concentrated by any combination of known methods such as evaporation, pervaporation, drying or by semi-permeable membranes.
The process for the preparation of yeast extract substantially as herein described.