# Patent

## Title
Antibody 67D2

## Abstract

This is a Continuation of: International Appln. No. PCT/EP96/01886 filed May 7, 1996 which designated the U.S.

## Background
The invention relates to low-molecular active ingredients from yeasts and to a method for producing same.

Yeast extracts from yeast fungi of the order Saccharomycetes, as well as their anabolic and respiration-stimulating and generally metabolism-activating effects, are known. Methods for obtaining such extracts have been described, for example, in EP-B 0 065 246. In this method, after a proteolytic enzyme had been added at the time of optimum fermentation, a suspension of the yeast fungus is subjected to an ultrasonic treatment until the temperature had been noticeably increased. Although the use of ultrasonics for the dialysis of yeast cells is an effective means for obtaining the extracts, it has been found that the ultrasonic treatment has quite negative side effects, since practically all compounds in an aqueous solution are subjected to a more or less pronounced oxidation. Thus, no genuine active agents are extracted from the cells in the course of the treatment with ultrasonics, instead, as a rule they are chemically altered compounds.

There is still a demand for yeast extracts and methods for producing them, which result in as genuine as possible a product and have the lowest possible content of substances altered by the production method.

To attain this object, yeast extracts in accordance with claim 1 are proposed, as well as methods for their production in accordance with claim 2.

It has been surprisingly found that it is possible to produce yeast extracts with metabolism-activating effects in a qualitatively and quantitatively improved form, if the cultures are subjected in a defined manner to a temperature change. In accordance with the invention the yeasts, which are maintained at approximately 15.degree. C. prior to preparing the culture, are suspended in water, wherein the ratio of yeast: water is approximately 1:2. Subsequently an amount of sugar, in particular saccharose, which can be fermented with the respective yeast, is admixed in amounts of 5 weight-%. The mass is then heated to the optimum cultivation temperature of 37.degree. to 38.degree. C. and maintained at this temperature for at least 3 hours, and brief stirring and aerating is performed at set intervals. This generation time of the yeasts is approximately 60 minutes.

After three hours of cultivation, the mass then is heated relatively quickly to maximally 45.degree. C. and maintained at that temperature for 60 minutes. Subsequently the suspension is allowed to slowly cool to 25.degree. C. during a period of 60 minutes and is maintained at this temperature for approximately 60 minutes. Thus the total time of the production process lasts approximately 6 hours.

The temperature resistance of the yeasts is improved by increasing the cultivation temperature to maximally 45.degree. C. during a generation time, and the total metabolism is clearly increased. It is assumed that the increased yield of low-molecular, metabolism-active ingredients is the result of the increased metabolization during the increased growth temperature. This assumption is supported in that heat shock proteins can be found in the active ingredient concentrate. Heat proteins, hsp for short, are proteins which are briefly synthesized at greatly increased rates by living cells during thermal or chemical stress. Their exact function is not yet known, but they are essential for surviving stress situations, and it is suspected that they initiate or support the ATP-dependent refolding of denatured proteins. Proteins of the hsp family 60 and 70 are contained in the yeast hydrolyzes. Isolation and proof of hsp are described, for example, in Nature (London) 337, 655 to 659 and 44 to 47 (1989).

Saccharomyces cerevisiae, also in various pure culture forms, or Saccharomyces uvarum or Saccharomyces rosei, are preferably used as the yeasts.

Following the approximately total culture time of 6 hours, the mass is comminuted with the aid of suitable mechanical mills, such as colloid mills. If necessary, comminution can also be performed following the enzyme addition. Then the mass is reacted at approximately 37.degree. to 38.degree. C. with a proteolytic enzyme or a mixture of such enzymes, wherein the ratio of enzyme: biomass should be approximately 0.03:1. The reaction time is a function of the type of proteolytic enzyme used and as a rule is approximately 180 minutes. Papain, ficine or bacterial or fungal proteases are preferably employed.

Following solubilization, the entire suspension is heated inside of 30 minutes to 85.degree. C. for activating the enzyme, and is maintained at this temperature for 30 minutes. After cooling, the mass is centrifuged, wherein the sediment can be washed one more time if required. The residue containing the active ingredients sought is filtered and concentrated at temperatures not above 40.degree. C. in vacuo. Subsequently this solution can be subjected to usual and known spray-drying granulation.

The mixtures of active ingredients in accordance with the invention present clearly elevated metabolic activities in comparison with the mixtures of active ingredients known so far and produced in accordance with other methods, which, in the fibroblast test, as a rule are clearly higher, in part by 50 to 100%, than those of the products known up to now.

The mixtures of active ingredients in accordance with the invention can be employed in human medicine and veterinary medicine in all those cases in which an activation of the metabolism is necessary, for example for encouraging the healing of slow-healing wounds, for improved utilization of food, in particular in stock-breeding and pisciculture. A further area of use is in the field of stimulating the activities of microorganisms in enzymatic processes when treating foodstuffs.

The invention will be described in detail below by means of an example:

10 kg of Saccharomyces cerevisiae of the bottom yeast culture type are reacted with 20 kg of purified and filtered water of drinking water quality in a reactor. This suspension is provided with 5 weight-% of saccharose and well mixed by stirring. Then the suspension is heated to 37.degree. to 38.degree. C. and maintained at this temperature for at least 3 hours, wherein brief stirring and aeration is performed at intervals of approximately half an hour. Following this the mass is heated to 45.degree. C. and maintained for sixty minutes at this temperature, and afterwards again cooled to 25.degree. C. over a period of time of 60 minutes and maintained there for 60 minutes.

Subsequently the balanced suspension is reacted with an appropriate amount of papain, wherein the ratio of enzyme: biomass should be 0.003:1. Following the enzyme addition with simultaneous comminution of the yeasts, the mixture is stirred for 180 minutes at 37.degree. to 38.degree. C. Then the mixture is heated to 85.degree. C. within 30 minutes and maintained at this temperature for 30 minutes, and then cooled and centrifuged. The centrifuged material is drawn off and the sediment is washed again, and again centrifuged. The centrifuged materials are combined and then, following filtration, concentrated at temperatures below 40.degree. C., preferably with the aid of a vacuum evaporator. The concentrated solution which is free of cells is then subjected in a manner known per se to spray-drying granulation.

## Claims

1. An extract from live Saccharomycetes yeast which contains a mixture of low molecular weight active ingredients comprising heat shock proteins of one or both of the families hsp 60 and 70, wherein the active ingredients are metabolically active, said extract being obtained by cultivating Saccharomycetes yeast at a temperature between 37-38.degree. C., subsequently heating the yeast up to 45.degree. C., cooling the yeast to 25.degree. C., and comminuting and enzymatically solubilizing the yeast to obtain the extract.

2. The extract according to claim 1, wherein the yeast is cultivated at a temperature between 37-38.degree. C. for 2-6 hours.

3. The extract according to claim 1, wherein the yeast is heated up to 45.degree. C. for 40-80 minutes.

4. The extract according to claim 1, wherein the yeast is cooled to 25.degree. C. for 40-80 minutes.

5. The extract according to claim 1, wherein the yeast is enzymatically solubilized in the presence of proteolytic enzymes.

6. The extract according to claim 5, wherein the proteolytic enzyme is papain, ficine, bacterial protease or fungal protease.

7. The extract according to claim 1, wherein additional comminuting occurs after the enzymatic solubilizing.