Use of yeast cell debris products

(12) PATENT (19) NO (11) 307682 (13) Pg

NORWAY (51) Intel⁷ A23 L 1/27

Patent Office (21) Application no. (22) Start day (24) Running day (41) Alm. avail.

(45) Date announced

19931397

1993.04.15

1993.04.15

1993.10.18

2000.05.15 (86) Int. Entry date and application number (85) Continuation day (30) Priority

1992.04.16, GB, 9208371

<td>(71) Patent holder</td><td>CPC International Inc, PO Box 8000, International Plaza, Englewood Cliffs, NJ 07632-9976, US</td>

<td>(72) Inventor</td><td>John Charles Hobson, Burton-on-Trent, Staffordshire, England, GB Roderick Norman Greenshields, Sketty, Swansea ,, GB</td>

<td>(74) Agent</td><td>Bryn &amp; Aarflot AS, 0104 Oslo</td>

(54) Designation Water-insoluble dye, as well as process for the preparation of yeast cell residues having in vivo morphology of the yeast cell walls and use of said dye (56) Published publications US 4574086, EP 474347 (57)

Coloring agents are disclosed comprising glucan-containing yeast cell residues which comprise a portion of substantially intact yeast cell walls and a color source. The color source may be naturally occurring and may e.g. provided by turmeric or annatto. A process for preparing the colorants comprises providing an aqueous mixture of color source and yeast cell residues, separating insoluble solids from the mixture, and drying the insoluble solids. A process for preparing yeast cell residues for use in the invention comprises extracting yeast waste with acid, treating with alkali, separating whole cells, bleaching and, optionally, acidifying the bleached material.

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BACKGROUND OF THE INVENTION The present invention relates to a water-insoluble dye, its use and a process for preparing yeast cell residues.

The invention relates to the use of waste products from yeast cells in foodstuffs, cosmetic products and pharmaceutical products.

Yeast extract is commercially produced on a large scale by means of lysis (e.g., hydrolysis, autolysis or plasmolysis) of baking yeast or brewer's yeast in suitable form or of another fermenting yeast (e.g. from the production of alcoholic fuels), resulting in soluble material and material rich in virtually intact cell wall bodies. The latter material is usually removed from the soluble material by centrifugation. Light inevitably results in some breakage of the cell walls, so that there is a significant portion of virtually intact cell wall bodies with at least one discontinuous zone in the cell wall surface area (i.e., holes that have occurred in the relevant cell walls). The material that contains cell wall bodies (known as yeast waste, or in general, yeast ref), which has a dark brown color, an unpleasant odor and which is rapidly decomposed, contains a variety of undesirable materials, such as e.g. trace elements, dyes, hop extracts, tartrates, microorganisms, bacteria, protein mucus and a large amount of insoluble constituents such as e.g. yeast cell wall bodies, as well as a certain amount of unlabeled whole cells. Such a yeast waste is normally discarded. The soluble material from which the waste is separated is normally used for the extraction of useful materials, e.g. yeast extract.

PCT / GB91 / 01819 discloses a method of treating yeast waste which provides a purified form of yeast ghosts or shells with substantially intact cell walls (i.e., they retain the in vivo morphology of the yeast cell walls in yeast waste). but without the yeast cell content. That is, the yeast residues in morphology correspond to the morphology of the lysed material (yeast waste, or ref) and not the morphology of the entire yeast cells. Yeast residues essentially comprise yeast B-glucan.

Yeast B-glucans are of course well known. U.S. Patent 4,810,646 discloses a process for producing yeast B-glucans, which comprises separating growing Saccharomyces cerevisiae oil from its growth medium, subjecting the intact, whole yeast cells to alkaline digestion to solubilize the cell's protein portion, and treating the insoluble glucan. with acetic acid to change the B (1,6) bonds. The resulting whole glucan particles are described in US Patent 4,962,094 to be suitable for use as dietary additives and are said to substantially retain the three-dimensional in vivo glucan molecular structure of the yeast cells from which they are obtained. However, the cell walls are effectively destroyed in the method described. In PCT / GB91 / 01819, the yeast residues or shells are obtained from the yeast waste without destroying the cell wall structure.

The term yeast cell residues, as used herein, covers yeast residues or shells where a portion of the yeast cell wall is substantially intact.

The present invention is based on applications of the yeast cell residues described in PCT / GB91 / 01819.

It has been found that yeast cell residues can be used to prepare dyes which are insoluble in aqueous media. The dyes can be prepared from natural substances so as to avoid the use of artificial carriers and / or dyes. The colorants can be used to impart color to a variety of materials, including foods, cosmetics, and pharmaceuticals.

Thus, the present invention provides a dye comprising glucan-containing yeast cell residues comprising a portion of substantially intact yeast cell walls, said yeast cell residues having in vivo the morphology of the yeast cell walls, and at least one naturally occurring color source. The color source is selected such that the desired color is achieved in the colorant and in any product in which the colorant is used and can be any dye or pigment. For applications e.g. in foods, pharmaceuticals and cosmetics, the coloring agents must be acceptable for use in foods and / or pharmaceuticals. The present invention encompasses the use of the water-insoluble coloring agent in a pharmaceutical composition which also comprises a pharmaceutically active substance. The color sources are naturally occurring or are provided by naturally occurring products such as e.g. turmeric and annatto. The color sources may be partially or completely purified from their natural source or used without purification.

A process for preparing a colorant comprises the following steps:

(i) providing an aqueous mixture of at least one color source and yeast cell residues comprising a portion of substantially intact yeast cell walls; (ii) insoluble solids are separated from the mixture and (iii) the insoluble solids are dried.

The aqueous mixture can be treated to optimize the formation of the coloring agent, e.g. by adjusting the pH of the mixture.

The aqueous mixture is preferably prepared by first adding at least one color source and then adding yeast cell residues to water or another suitable aqueous solution. The mixture may be stirred for at least 30 seconds, preferably for 30 seconds to 5 minutes (e.g., 1 minute). The insoluble solids can be separated from the aqueous mixture in any of a variety of ways well known to those skilled in the art, e.g. by centrifugation. The insoluble solids are preferably washed before drying, preferably with water. The insoluble solids can be dried in a variety of ways, e.g. by freeze-drying.

The coloring agents of the present invention can be used to give color to foodstuffs, cosmetic and pharmaceutical preparations.

Yeast cell residues for use in the present invention may be prepared by the method of treating yeast waste having a solids content not exceeding 20% ​​by weight, as disclosed in PCT / GB91 / 01819 and comprising:

(a) the waste is extracted with a food grade alkaline salt, (b) whole cells are separated from the extracted waste to leave a material rich in broken but otherwise intact cell walls, (c) the latter material is treated with an alkaline extractant , (d) said material is bleached with a bleaching agent or a food grade oxidation / reduction binder (e.g. (e) lowering the pH of the bleached material using a food grade acid (such as citric acid, orthophosphoric acid, dilute hydrochloric acid or dilute sulfuric acid).

Typical food grade alkali salts for use in step (a) include sodium, calcium or potassium bicarbonate or sodium, calcium or potassium carbonate. Sodium bicarbonate is most preferred. The yeast waste typically has a solids content of approx. 2-12, e.g. 4-8 (generally about 5) by weight and a viscosity in the range of about 5 to 15 cP (5% aqueous suspension). The waste is generally extracted in step (a) of the process of the invention with the alkali salt, generally at substantially ambient temperature for approx. 1 hour. The alkali salt (which, as previously mentioned is preferably sodium bicarbonate) is preferably used in an amount of up to 2.5 (preferably about 1) wt%, based on the total volume of yeast waste (liquids and solids) and preferably so that the resulting the extracted mixture has a pH in the range of 8-12, more preferably approx. 8-9.

The separation of whole cells from the extracted waste to produce a material rich in broken cell walls is generally carried out by mechanical methods, of which centrifugation is preferred. The centrifugation is typically operated at approx. 5000 rpm for differential centrifugation or 2500 rpm. for static centrifugation. The use of a bicarbonate in step (a) has been found to be helpful in the separation step (possibly due to gas evolution, which serves to make yeast cell residues easier).

After separation, the material is treated with an alkaline extraction and extractant such as e.g. potassium hydroxide, sodium hydroxide or calcium hydroxide. This treatment with an alkaline extractant (similar to the process known as mercerization) typically comprises a treatment in an alkaline solution having a pH of 8-14, preferably about 12-12.5. The treatment is an aid in removing colored products, dissolving unwanted materials such as protein, opening the cell wall structure and facilitating the bleaching step. The mixture is then preferably heated to a temperature in the range of approx. 65-85 ° C for at least 1 hour. If the final product is required to have a pale creamy color, it is preferred that the alkali used comprises potassium or sodium hydroxide. However, if it is desired for the final product to have a white color, it is preferred that the alkali used comprises calcium hydroxide.

The bleaching step is preferably carried out using hydrogen peroxide or a food grade oxidation / reducing agent (such as ascorbic acid) when the product is to be used for nutritional purposes. The bleaching is preferably carried out so that the bleached material is given a pale creamy or white color. The bleaching step is preferably carried out in a reactor and the amount of material rich in broken cell walls introduced into the reactor is preferably controlled so that the material does not occupy more than approx. half of the reactor volume. This is because the bleaching step typically includes foaming which causes a significant increase in the volume of the material being treated. The foaming is preferably controlled to a considerable extent by the use of a foam-breaking stirrer and can be significantly reduced by the addition of anti-foaming agents.

The bleached material may optionally be treated with a food grade acid (typically hydrochloric or orthophosphoric acid), centrifuged and treated with lecithin. The material can then be further centrifuged before drying. Treatment of the bleached material with lecithin is advantageous, since lecithin is an aid in masking any residual odor or taste arising from the yeast waste.

The lowering of the pH with the food grade acid can be accomplished by washing the material as it is centrifuged, or can be carried out thereafter. The pH is generally lowered to a value of 5-6, either in a single step, or by first lowering the pH to a value of 6 to 7.5 (such as about 7.0) and in a final step to pH approx. 5-6.

Yeast cell residues can also be prepared by the improved process of the present invention which provides a process for making yeast cell residues that cause greater flavor and color removal than previous processes.

The invention thus provides a method for preparing yeast cell residues which have in vivo the morphology of the yeast cell walls and comprising at least a portion of substantially intact yeast cell walls comprising the following steps:

(i) extraction of yeast waste with a solids content not exceeding

2-20 wt.% And a viscosity in the range of 5-15 cP with acid at a temperature of from 50 to 100 ° C for 30 minutes to 21; (ii) treating the extracted waste with an alkali in an alkaline solution having a pH from 8 to 14 at a temperature of from 65 ° C to 85 ° C for 30 minutes to 21; (iii) separating whole cells from the treated mixture to leave a material rich in broken but otherwise intact cell walls; (iv) bleaching said material with a bleaching agent or oxidizer / reducing agent of the aforementioned separation step, and optionally (v) lowering the pH of said bleached material using a food grade acid.

The yeast waste used in step (i) typically has a solids content of approx.

2-10% by weight, e.g. 4-8, e.g. about. 5% by weight and a viscosity in the range of about 5 to 15 cP (5% aqueous suspension). The waste is generally extracted at 50-100 ° C, e.g. 60-70 ° C for 30 minutes to 2 hours, e.g. 1 hour. Many acids can be used in the process, e.g. hydrochloric, sulfuric, phosphoric and citric acids either alone or as a mixture of two or more acids. The acid is present at a concentration effective to perform step (i). Ascorbic acid is preferably used in an amount of up to 2.5, preferably 0.5-1% by weight e.g. 1% by weight based on the total volume of the yeast waste (liquids and solids) and is preferably added as a solid. Ascorbic acid causes both partial hydrolysis and partial oxidation of the yeast waste.

The alkali treatment in step (ii) can be carried out with an inorganic alkali such as e.g. potassium hydroxide, sodium hydroxide or calcium hydroxide. The treatment is typically carried out in an alkaline solution having a pH of 8-14, preferably approx. 12-

12.5. The yeast waste mixture is preferably diluted to from 1-5% (eg from 2-3%) weight / volume prior to treatment. The mixture is heated to 65-85 ° C for 30 minutes to 2 hours, e.g. about. 1 hour. Thus, step (ii) is completed in less time than the corresponding step of the known process. If the final product is required to have a pale creamy color, it is preferred that potassium or sodium hydroxide be used as the alkali. If the final product is to have a white color, it is preferred that calcium hydroxide be used as an alkali.

The separation of whole cells from the extracted waste to produce a material rich in broken cell walls (step (iii)) is generally carried out by mechanical methods, of which centrifugation is preferred. The centrifuge is typically operated at approx. 5000 rpm for differential centrifugation or approx. 2500 rpm for static centrifugation. The alkalinity is preferably reduced before centrifugation, but preferably not below pH 9.5.

The bleaching step (step (iv)) is preferably carried out using hydrogen peroxide or a food grade oxidation / reducing agent such as e.g. ascorbic acid when the product is to be used for nutritional purposes. The bleaching is preferably carried out such that the bleached material is given a pale creamy or white color which is advantageous when the resulting product is to be used as a food grade material such as e.g. functional fibers. The bleaching step is preferably carried out in a reactor and the amount of material rich in broken cell walls and introduced into the reactor is preferably controlled so that the material does not occupy more than approx. half of the reactor volume. This is because the bleaching step typically comprises foaming which causes a significant increase in the volume of the material being treated. Preferably, the foaming is substantially controlled by the use of a foam-breaking stirrer and can be significantly reduced by the addition of anti-foaming agents.

The bleached product can further be treated by neutralization with ascorbic acid (preferably as a concentrated aqueous solution), centrifugation and washing.

The process according to the invention provides a yeast cell residue product with less odor and a lighter color than previous methods.

Example 1

Brewer's yeast was subjected to autolysis to break up the cell membranes. The lysed product was then separated by centrifugation to prepare yeast waste, a fraction containing cell bodies having a viscosity of 10 cP (5% aqueous solution).

The yeast waste was then treated with sodium bicarbonate to prepare an extracted mixture with a pH of 8.5. The mixture was then stirred for approx. 1 hour at room temperature and centrifuged for further separation.

The centrifuged material resulted in two streams, namely a cell wall-rich material and unbroken cells. The cell wall-rich material was resuspended in 2.5% sodium hydroxide and then 40% sodium hydroxide was added to adjust the pH of the material to 12.5. The cell wall-rich material was then indirectly heated in a water bath to ca. 65 ° C for approx. 1 hour and then bleached by treatment with hydrogen peroxide for approx. 1 hour of mixing. Concentrated hydrochloric acid was then added to the bleached material to obtain a pH of 7.0. The material was further centrifuged and the pH further lowered to 5.0. The resulting product was substantially free of whole yeast cells and consisted mainly of yeast residues or shells with substantially uninvolved walls. The yeast residues contained a smaller amount of yeast cells relative to the entire cells in the waste.

Water (200 ml) was acidified to pH 2 with dilute hydrochloric acid and turmeric (0.3 g) was added to the resulting solution. The yeast cell residues (8.0 g) were added to the solution and the mixture stirred for 1 minute, centrifuged, washed twice with water and lyophilized to give a stable, canary yellow lacquer color.

Example 2

Water (200 ml) was acidified to pH 6 with dilute hydrochloric acid and annatto (0.3 g) was added to the resulting solution. Yeast cell residues (8.0 g) as prepared in Example 1 were added to the solution and the mixture stirred for 1 minute, centrifuged, ice washed twice with water and lyophilized to give a stable orange lacquer color.