WO1998017127A1

WO1998017127A1 - Method of using enzymes and yeast fermentation for production of a fermentation-type savory flavored product

Info

Publication number: WO1998017127A1
Application number: PCT/US1997/018671
Authority: WO
Inventors: Ben Cheng; Klaus Pommer
Original assignee: Novo Nordisk Biochem North America, Inc.
Priority date: 1996-10-23
Filing date: 1997-10-14
Publication date: 1998-04-30
Also published as: AU4904997A

Abstract

A method for producing a fermented-type savory flavored product by (a) contacting a protein mixture with an effective amount of one or more proteases and an effective amount of one or more cellulases, (b) incubating the enzyme-treated protein mixture for a sufficient time and at a sufficient temperature to form a protein hydrolysate, (c) adding an effective amount of yeast to the protein hydrolysate of step (a) under conditions sufficient to form a fermented slurry, (d) filtering the mixture of step (c) to obtain a filtrate fraction. The color, flavor and concentration of the filtrate fraction may be adjusted to produce the final fermented-type savory flavored product. Optionally, step (c') is included after step (c) comprising adding an effective amount of one or more proteases to form a hydrolyzed mixture.

Description

METHOD OF USING ENZYMES AND YEAST FERMENTATION

FOR PRODUCTION OF A FERMENTATION-TYPE

SAVORY FLAVORED PRODUCT

FIELD OF THE INVENTION

This invention relates to the field of enzyme process technology, specifically, enzymatic and yeast fermentation of savory flavors.

BACKGROUND OF THE INVENTION

Soy sauce is a fermented food product made from soybeans and wheat. Basically two types of soy sauce exist, fermented soy sauce and chemically produced soy sauce. For obtaining fermented soy sauce, the proteins and carbohydrates contained in the raw materials are hydrolyzed very slowly at below 30 °C for more than 6 months, whereas for obtaining chemically produced soy sauce, the raw materials are hydrolyzed very quickly by hydrochloric acid at above 80 °C for 8-10 hours.

Chemical hydrolysis is thus a very quick and cheap process. However, during the hydrolysis various secondary reactions occur and undesirable compounds are produced. Chemically produced soy sauce is therefore considered to be of poor quality and it is primarily used as an additive to fermented soy sauce.

In order to improve the quality of the chemical soy sauce, semi-chemical soy sauce has been devised. It is made by hydrolyzing raw soybeans with a lower concentration of hydrochloric acid as the first step, followed by fermenting the resultant hydrolyzate with salt-tolerant yeasts in the presence of wheat koji. The odor and flavor of the resultant semi-chemical soy sauce are improved partly by the fermentation procedure, but it is essentially a chemical soy sauce which has the undesirable compounds arising from the chemical hydrolysis.

Fermented soy sauce, on the other hand, is considered to be a natural product having desirable organoleptic properties. Good odor and flavor develops during the fermentation, and the quality is generally considered to be proportional to the fermentation time. Thus, good quality fermented soy sauce is a more expensive product. Traditionally, five varieties of fermented soy sauce exist, namely koikuchi-shoyu (regular soy sauce), usukuchi-shoyu (light-colored soy sauce), tamari-shoyu, saishikomi- shoyu and shiro-shoyu. These five types of fermented soy sauce are recognized by the

Japanese government and each type is classified into three grades, special, upper and standard. Each grade is determined by organoleptic evaluation, contents of total nitrogen, alcohol, soluble solids other than sodium chloride, and color.

Dependent on the type of soy sauce in question, fermented soy sauce is conventionally manufactured through three main process steps, (1) the koji-making step,

(2) the moromi-fermentation (brine fermentation), and aging step, followed by (3) the pressing and refining step.

In the traditional production of fermented soy sauce, spores or cultures of Aspergillus moulds are added to the treated raw materials, and a controlled fermentation takes place (the koji-making step). The mould produces enzymes that are active in the subsequent moromi-fermentation step.

In the moromi-fermentation step, fermentation is traditionally accomplished by the action of enzymes produced by the koji moulds (Aspergilli) added during the koji-making step. It takes more than six months for the entire fermentation and aging of the moromi mash. In order to accelerate the production time, various measures for improving hydrolysis of the raw materials have been suggested, including selecting for optimal moulds to be used, developing improved strains, e.g.. by protoplast or cell fusion, and water/steam treatment of one or more of the raw materials. Also, the addition of enzyme preparations, rather than enzyme-producing microorganisms, has been suggested, as well as use of bioreactors containing immobilized enzymes and microorganisms. There is a need for method for producing a fermented-type savory flavored product, such as a soy sauce-like product, having desired flavor characteristics of traditional fermented products without long-term incubation periods.

BRIEF SUMMARY OF THE INVENTION

The present invention features a method of making a fermentation-type savory flavored product using both enzymatic degradation of protein and yeast fermentation, e.g. , a soy sauce-like product in which the processing time required for fermentation is sharply reduced. As a result of the reduced production time, the risk of microbial contamination is lowered, and a high quality savory flavored product with low or no salt is produced.

Accordingly, the invention features a method of producing a fermentation- type savory flavored product having the steps of:

(a) contacting a protein mixture with an effective amount of one or more protease and an effective amount of one or more cellulase;

(b) incubating the protease and cellulase enzyme-treated protein mixture for a sufficient time and at a sufficient temperature to form a protein hydrolysate;

(c) adding an effective amount of yeast to the protein hydrolysate of step (b) under conditions sufficient to form a fermented slurry; and (d) filtering the slurry of step (c) to obtain a filtrate fraction.

The color, flavor and concentration of the filtrate fraction may be adjusted to produce a fermentation- type savory flavored product.

Optionally, an additional step is included after step (c), comprising the step of (c') adding an effective amount of one or more protease to the fermented slurry of step (c) for a sufficient time and at a sufficient temperamre to form a hydrolyzed fermented mixture. Proteins useful in the method of the invention include, but are not limited to, soy flakes, soy grit, wheat gluten, soy isolate, soy concentrate, peanut protein, pea protein, and corn gluten.

Proteases useful in the invention include one or more of any enzyme exhibiting protease activity, including endoproteases and exoproteases. Preferred proteases include one or more proteases obtained from Aspergillus oryzae (e.g. , Flavourzyme™), Bacillus licheniformis (e.g., Alcalase^®), Bacillus amyloliquefaciens (e.g. , Neutrase^®), and other Bacillus species (e.g. , Protamex™), and Kojizyme™ derived from selected Bacillus and Aspergillus strains. In a specific embodiment, a protease is added at a concentration of about 20-40 LAPU/g protein (w/w total solids) and/or 0.01- 0.5 AU/g protein.

An example of a cellulase useful in the method of the invention is Kojizyme F™ (Novo Nordisk A/S) a cellulase derived selected strains of Aspergillus . Kojizyme F™ is added in an amount of about 0.1-0.4% (w/w total solids).

The enzyme-treated protein mixture of the invention is incubated under conditions sufficient to form a protein hydrolysate. In a preferred embodiment, incubation of the enzyme-treated protein mixture is at a temperature of about 50-60 °C for a period of between about 24 - 48 hr.

The protein hydrolysate is then contacted with an effective amount of yeast to form a fermented slurry. In a preferred embodiment, the yeast fermentation step (c) is conducted at a temperamre of between about 20-45 °C for a period of about 24-60 hr. In a more specific embodiment, the fermentation step is conducted at 30-35 °C for 48 hr.

When a second protease addition is made (optional step (c')), the fermented slurry is contacted with an effective amount of one or more proteases under conditions sufficient to form a hydrolyzed mixture. In one embodiment, step (c') is conducted at a temperamre of about 30-70°C for 12-48 hr. In a more specific embodiment, step (c') is conducted at a temperamre of about 50°C for about 20 hr. In a preferred embodiment, one or more protease(s) are added at a concentration of about 20-40 LAPU/g protein and/or 0.01-0.5 AU/g protein.

Optionally, the hydrolyzed mixture formed at step (c') is subjected to heat treatment for enzyme inactivation. In one embodiment, the hydrolyzed mixture is subjected to heat treatment at a temperamre of about 80-100°C for 2-4 hr.

After step (d), the collected filtrate fraction is evaporated to form a liquid soy base, and the soy base is adjusted for color, pH, and flavor.

In a specific embodiment of the method of the invention, a fermentation-type savory flavored product is produced by combining the protein hydrolysis of step (a) with the yeast fermentation of step (c) above into a single step. This combined step allows extension of the hydrolysis from 24-48 hours to 60-84 hours without fear of microbial contamination as a result of the alcohol produced from yeast fermentation which inhibits microbial growth.

(a) contacting a protein mixture with an effective amount of one or more protease and an effective amount of yeast;

(b) incubating the enzyme and yeast treated protein mixture for a sufficient time and at a sufficient temperature to form a fermented protein slurry; and (c) filtering the slurry of step (b) to obtain a filtrate fraction.

The color, flavor and concentration of the filtrate may be adjusted to produce a fermentation- type savory flavored product.

Optionally, a second protease addition may be made after step (b), comprising step (c) adding an effective amount of one or more protease to the fermented slurry of step (b) for a sufficient time and at a sufficient temperamre to form a hydrolyzed fermented mixture. The invention additionally encompasses a fermentation-type savory flavored product produced by the method of the invention.

One objective of the invention is to provide a process for producing high quality fermentation-type savory flavored product in a reduced period of time having a low sodium content.

One advantage is that the reduced fermentation time achieved by the method of the invention allows the addition of salt to be reduced or eliminated, thus producing a low or no-salt savory flavored product useful in a number of ways, for example, as a soy sauce substitute. Another advantage of the invention is the production of a low or no-salt savory flavoring without expensive downstream sodium-reducing processes.

The invention uses a high solid content mixture to extend enzyme activity and reduce the risk of microbial contamination. The high solid content has the advantage of slowing proteolytic autocatalysis, thus extending protease activity. The high solid content solutions used in the method of the invention also inhibit bacterial growth and contamination.

Other aspects, features, and advantages of the invention will become apparent from the following detailed description, and the claims.

DETAILED DESCRIPTION

Before the methods and compositions of the present invention are described and disclosed it is to be understood that this invention is not limited to the particular methods and compositions described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims. It must be noted that as used in this specification and the appended claims, the singular forms "a" , "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a color" or "a flavor" includes a plurality of colors and different types of flavoring agents. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials or methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the particular information for which the publication was cited. The publications discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventor is not entitled to antedate such disclosure by virtue of prior invention.

Definitions

The term "protease" means an enzyme that hydrolyses, e.g. , cleaves, a protein substrate, generating two or more protein fragments, polypeptides, peptides, or amino acids. Proteases include exoproteases, e.g. , enzymes that cleave off a single amino acid from either end of a peptide chain, and endoproteases, e.g. , enzymes which attack peptide bonds in the interior of a protein chain. The term protease can include a mixture of enzymes having protease activity, e.g. , Flavourzyme™.

The term "protein hydrolysate" means a mixture of proteins, polypeptides, peptides, and/or amino acids, resulting from hydrolysis of one or more proteins by a protease enzyme. The term "cellulase" means an enzyme able to hydrolyze cellulose into two or more smaller fragments. A preferred cellulase is Kojizyme F™ (Novo Nordisk A/S).

The term "fermentation" means anaerobic catabolism of an organic compound in which the compound serves as both an electron donor and an electron acceptor, and in which ATP is produced by substrate-level phosphorylation. In yeast, fermentation is a process consisting of a sequence of enzymatic reactions which convert glucose to ethanol and carbon dioxide.

The term "effective amount of" a protease, cellulase, or yeast means a sufficient quantity to achieve the desired action. An effective amount of a protease is an amount needed to hydrolyze a protein substrate to achieve the desired hydrolysate mixture of polypeptides, peptides, and/or amino acids. The effective amount of a protease is determined at a given time, temperature, and pH. Similarly, an effective amount of yeast is the amount required to achieve the desired degree of fermentation at a given time, temperamre, and pH.

The term "umami" means savory or tasty ("mouth-watering") characteristics in, for example, a protein hydrolysate.

Shortened Process for Producing Fermented-Type Savory Flavored Product

The invention features a method of producing fermented-type savory flavored product having the steps of (a) contacting a protein mixture with an effective amount of one or more proteases and cellulases, (b) incubating the enzyme-treated protein mixture for a sufficient time and at a sufficient temperamre to form a protein hydrolysate, (c) adding an effective amount of yeast to the protein hydrolysate of step (a) under conditions sufficient to form a fermented protein slurry, (d) filtering the mixture of step (c) to obtain a filtrate fraction; and (e) adjusting the color, flavor and concentration of the filtrate fraction to produce the fermented-type savory flavored product. Optionally, a second addition of protease enzyme is included after step (c) as step (c') adding an effective amount of one or more proteases to the fermented protein slurry of step (c) for a sufficient time and at a sufficient temperature to produce a highly hydrolyzed and fermented mixture.

The raw materials for the production of the fermented-type savory flavoring are soy beans, wheat, salt and water. Whole soy beans or, more commonly, defatted soy bean flakes or grits are usually moistened and cooked under steam pressure. In the method of the invention, the protein mixture of step (a) is composed of water and one or more protein sources, including but not limited to soy grit, soy flakes, wheat gluten, soy isolate, soy concentrates, peanut protein, pea protein, and corn gluten. In a preferred embodiment, the protein mixture is a mixture of 50% soy grit and 50% wheat gluten. Water is added in a proportion of 1 :2 (1 part protein to 2 parts water). The water content of the water /protein mixture may range from about 50-70% .

The protein mixture is preferably heat treated to reduce microbial count and to denature the raw protein. In a specific embodiment, the protein mixture is steamed for 3 hours prior to contacting the protein mixture with a protease. Proteases useful in the method of the invention include Alcalase^®, Flavourzyme™,

Neutrase^®, Protamex™, and Kojizyme™. Alcalase ^® (Novo Nordisk A/S, Bagsvaerd, Denmark) is a commercially available protease exhibiting endoprotease and serine protease activities derived from a strain of Bacillus licheniformis . Flavourzyme™ (Novo Nordisk A/S) is an enzyme exhibiting both endoprotease and exopeptidase activities derived from a strain of Aspergillus oryzae. Neutrase^® (Novo Nordisk A/S) is a protease exhibiting both endoprotease and metallo (Zn) protease activities derived from a strain of Bacillus amyloliquefaciens. Protamex™ (Novo Nordisk A/S) is a commercially available protease complex containing multiple endoproteases. Kojizyme™ (Novo Nordisk A/S) is a protease enzyme derived from selected Bacillus and Aspergillus strains. In a preferred embodiment, the protease is Flavourzyme™. Cellulases and 0-glucanases hydrolyze soya cellulose composed of 0-1,4 glucan chains. The major hemi-cellulose in soya, xyloglucan, is composed of 0-1,4 glucose with α-xylose attached at C-6, and is hydrolyzed by hemi-cellulase. A preferred cellulase is Kojizyme F™ (Novo Nordisk A/S).

In a preferred embodiment, the enzymes Flavourzyme™ and Kojizyme F™ are added to the denatured protein mixmre in an amount effective to form a protein hydrolysate having a savory "umami" taste. The protein content of the hydrolysate is 22- 35% (w/w). The amount of Flavourzyme™ added is based on the protein content of the protein mixmre, e.g. , at a concentration of about 20-40 LAPU/g protein, more preferably 30 LAPU/g protein. The amount of Kojizyme F™ added is based on the total solid material present, e.g. , about 0.1-0.4% (w/w) of total solid material, more preferably in an amount of about 0.2% . Generally, the pH of the enzyme-treated protein mixmre is about 5.0-7.0, preferably 6.3-6.5.

In step (b) of the method of the invention, the enzyme-treated protein mixmre is incubated at a sufficient temperamre and for a sufficient period of time to form a desired protein hydrolysate. The enzyme-treated protein mixmre is incubated at 50-60 °C for an incubation period of about 24-48 hrs. In a preferred embodiment, the enzyme-treated protein mixmre is incubated at a temperamre of about 55 °C for 30-35 hr.

After the incubation period of step (b), yeast is added to the protein hydrolysate and the fermentation process begun. A source of yeast for use in the invention includes, e.g. , Saccharomyces cerevisiae. Yeast is generally added in the form of a yeast solution, e.g. , dried or powdered yeast dissolved in water to form a solution. A suitable yeast food, such as glucose, may also be added to the yeast solution. In one embodiment, 1 % yeast (w/w total solids) is dissolved in 2.5 parts water (e.g. , 1 g yeast in 2.5 g water), and about 2% (w/w total solids) glucose added to the yeast solution with blending to form a homogenous solution. The yeast solution is added to the protein hydrolysate and the yeast/hydrolysate mixed. Generally, a pH range between 5.0-6.5 is desirable to achieve the desired fermentation.

Fermentation is allowed to proceed at a temperamre and for a sufficient period of time to form a fermented protein hydrolysate slurry. Generally, the mixmre is conducted at a temperamre of about 20-45 °C for a period of about 24-60 hr. In a preferred embodiment, the fermentation is conducted at 30-35 °C for a period of 48 hr, an optimal temperamre for the yeast to produce ethanol during fermentation. A time period of between 1-3 days at that temperature will allow yeast to convert most of the available glucose to ethanol without the risk of acetic acid production. At the end of the fermentation phase, increasing batch temperamre is used to stop fermentation. A strong ethanol note, e.g. , an alcohol-like aroma, characterizes a successful fermentation.

Following the fermentation step, an optional hydrolysis step is conducted at a higher temperamre to form a fermented hydrolysate mixture, e.g. , 30-70°C for 12-48 hr. One or more proteases may be added, and the water content, salt and pH adjusted. For example, a protease and salt may be added at this time. In a preferred embodiment, Flavourzyme™ is added to the fermented slurry at a concentration of about 15 LAPU/g of protein, and salt is added in an amount of about 3 % (w/w total solids).

At the optional hydrolysis step, the temperamre is preferably increased to about 50 °C and the slurry fermented with agitation for 20 hr. This is optionally followed by a short heat treatment at 85-95 °C for 2-8 hr to generate the desired flavor and to inactivate the enzyme activity. Preferably, the hydrolysis is conducted at 90 °C for 2-4 hr. Prior to the heat treatment, the pH is adjusted to between about 5.5-6.5. Preferably, the pH is adjusted to about 5.5.

The hydrolyzed mixmre is filtered, and a filtrate fraction obtained. The insoluble fraction may be mixed with water one or more times and re-filtered. The combined filtrates are evaporated to form a liquid savory flavored base having a protein content of between 7-10% (w/v). The base is adjusted as desired, including by adjusting pH, salt content, flavor, and color. The soy base produced by the method of the invention may be used directly or mixed with other products, e.g. traditionally-fermented soy sauce, to produce the final product. The above-described method may also be modified by combining the protein hydrolysis step of step (a) with the yeast fermentation step of step (c), as described in Example 2 below. This combined step allows the hydrolysis period to be extended from 24-48 hours to 60-84 hours while inhibiting microbial contamination by means of the alcohol produced from the yeast fermentation. In a preferred embodiment, a protein mixture is contacted with one or more protease(s), and yeast, and the resulting mixmre incubated for 60-84 hours at a temperamre of between about 40°C-60°C. In a more specific preferred embodiment, the protein mixmre containing protease(s), and yeast is incubated for 72 hours at 50 °C. The resulting fermented slurry is treated as described above (Example 1), producing a high quality fermented-type savory flavoring which can be used directly or mixed with other products to form the final flavoring product.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use various constructs and perform the various methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric pressure. Efforts have been made to ensure accuracy with respect to numbers used, (e.g. , molecular weights, amounts, particular components, etc.) but some deviations should be accounted for.

Example 1. Formation of Fermented-Type Savory Flavored Product

Formation of protein hydrolysate. Starting with a 50% soy grit and 50% wheat gluten mixture, the protein materials are mixed with water at a ratio of 1 :2 (one part protein material to 2 parts water), and steamed for a sufficient amount of time to reduce microbial contamination and denamre the proteins. Generally, the steaming is conducted at 90-100°C for approximately 3 hours. The steamed protein materials are transferred to a container, and enzymes added. In one embodiment, Flavourzyme™ is added at 30 LAPU/g of protein, and Kojizyme F™ is added at 0.2% of total solids. The pH of the mixture is about 6.3-6.5. The container is maintained at 55°C for 32 hours. Fermentation Step. A yeast solution was prepared by dissolving 1 g of yeast in

2.5 g of water and adding 5% yeast to total solids (w/w). Glucose was added at 0.2% (w/w) of total solids. The mixmre was blended to distribute the yeast solution in the protein hydrolysate homogeneously. The resulting pH of the protein-yeast mixmre is about 5.3-5.8. The temperamre was lowered to about 30-35 °C and the mixmre fermented for 48 hours.

Formation of Hydrolyzed Mixture. At the end of 48 hours, Flavourzyme™ was added to the fermented protein mixmre at a concentration of 15 LAPU/g of protein, salt (sodium chloride) was added at 3% (w/w of total solids), and water was added at the original concentration (e.g., 1 protein : 2 water). The pH of the solution was adjusted to 5.5, and the temperamre increased to 50°C. The mixture was incubated with agitation for 20 hours, at which time the water content is adjusted to the original protein concentration with 3 kg of water added to the fermented hydrolysate, the pH adjusted to 5.5, and the mixmre heated at 90 °C for 2-4 hours.

Filtration and Flavor Adjustment Steps. The hydrolysate was filtered and the filtrate recovered as liquid savory flavored base. Water was added to the insoluble filtered material, mixed, and the resulting solution re-filtered. This step was repeated three more times. The filtrates were combined, and the liquid base evaporated to a protein concentration of 7% (w/v). The pH of the liquid base was adjusted to pH 4.5 with lactic acid. The final savory flavored product was produced by adding flavor and color agents, including, salt, dextrose, alcohol, and color. Depending on the desired characteristics of the final product, typically salt is added in the amount of between 10-

15% (w/v), alcohol is added to a final concentration of 2% (w/v), dextrose is used to adjust the solid content of the flavoring to the specified Brix or percent solid, and Carmel color is added to adjust the product color.

The soy hydrolysate liquid made by the above described process has a typical amino nitrogen: total nitrogen ratio of 50-60% (AN/TN=55%), salt content of between 99

- 252 mg/15 ml. In comparison, commercial soy sauce has an AN/TN ratio ranges between 44-62%, with a salt content of 1,380-2,560 mg/ml. The amino nitrogen to total nitrogen ratio is an indication of the degree of hydrolysis. A higher AN/TN ratio indicates a higher degree of hydrolysis, and generally provides a better product taste. After adding salt, alcohol, and dextrose to the soy hydrolysate liquid, the final product was judged to have a very similar taste to the commercial soy sauce products having fruity, apple cider, and fermented flavors. Fruity and apple cider flavors are derived from the ester products. The fermented note is similar to the alcohol-like aroma produced at the fermentation step. All of these flavors are desirable as characteristic of traditional well fermented soy sauce. The fermented-type savory flavored produced by the method of the invention may be used alone or combined with other types of flavorings such as soy sauce, as desired.

Example 2. Formation of Fermented-Type Savory Flavored Product With Combined Protein Hydrolysis and Yeast Fermentation Process

Formation of fermented protein hydrolysate. A protein mixmre of 50% soy concentrate and 50% wheat gluten was mixed with water at a ration of 1 part protein to 3 parts water. The mixmre was steamed for 3 hours to reduce microbial count and denamre the protein. Alcalase^® was added to the protein mixmre at 1 % protein (w/w), Flavourzyme™ was added at 15 LAPU/g of protein, and active dry yeast (Saccharomyces cerevisiae) was added to the protein mixmre at 1 % of total material weight. Additionally, glucose and salt were added at 1 % of total weight. The pH of the protein mixmre was 6.3-6.5. The protein mixmre was incubated at 50°C for 72 hours.

At the end of 72 hours, Flavourzyme™ was added at 15 LAPU/g of protein, and the mixmre incubated at 50 °C for 24 hours, to form a fermented protein slurry.

Following incubation, water was added to restore the 3: 1 water: protein ratio, and the mixmre agitated for 2 hours.

The hydrolysate was filtered and the liquid sauce based recovered as described above (Example 1). The resulting final savory flavored product was formulated by addition of salt, dextrose, alcohol, and/or coloring.

Claims

What is claimed is:

1. A method for producing a fermented-type savory flavored product, comprising the steps of:

(a) contacting a protein mixmre with an effective amount of one or more 5 proteases and an effective amount of one or more cellulases;

(b) incubating the protease and cellulase enzyme-treated protein mixture for a sufficient time and at a sufficient temperamre to form a protein hydrolysate;

(c) adding an effective amount of yeast to the protein hydrolysate of step (b) under conditions sufficient to form a fermented slurry; and o (d) filtering the mixmre of step (c) to obtain a filtrate fraction.

2. The method of claim 1, further comprising a step after step (c) of

(c') adding an effective amount of one or more protease to the fermented slurry of step (c) for a sufficient time and at a sufficient temperature to form a hydrolyzed 5 mixmre.

3. The method of claim 1 , wherein the protein mixmre is comprised of one or more proteins selected from the group consisting of soy flakes, soy grit, wheat gluten, soy isolate, soy concentrate, peanut protein, pea protein, and corn gluten. 0

4. The method of claim 1 , wherein one or more proteases are selected from the group consisting of endoproteases and exoproteases.

5. The method of claim 3, wherein the protease is obtained from Aspergillus 5 oryzae, Bacillus licheniformis, Bacillus amyloliquefaciens, and other Bacillus species.

6. The method of claim 5, wherein the protease is added at a concentration of about 20-40 LAPU/g protein (w/w total solids).

7. The method of claim 1, wherein the cellulase is derived from strains of Aspergillus and Bacillus.

8. The method of claim 7, wherein the cellulase is added in an amount of about 0.1-0.4% (w/w total solids).

9. The method of claim 1, wherein incubation of the enzyme-treated protein mixmre is at a temperamre of about 50-60°C for a period of between about 24 - 48 hr.

10. The method of claim 1 , wherein the yeast fermentation step (c) is conducted at a temperamre of between about 20-45 °C for a period of about 24-60 hr.

11. The method of claim 10, wherein the fermentation step is conducted at 30- 35°C for 48 hr.

12. The method of claim 2, wherein step (c') is conducted at a temperamre of about 30-70°C for 12-48 hr.

13. The method of claim 12, wherein step (c') is conducted at a temperature of about 50°C for about 20 hr.

14. The method of claim 2, wherein the protease is added at a concentration of about 20-40 LAPU/g protein.

15. The method of claim 2, wherein the protease is added at a concentration of about 0.01-0.5 AU/g protein.

16. The method of claim 2, further comprising an additional step after step (c') of heat treating the hydrolyzed mixture at a temperamre of about 80-100°C for 2-4 hr.

17. The method of claim 1, wherein the filtrate is evaporated to form a liquid base, and the base is adjusted for color, pH, and flavor.

18. A fermented-type savory flavored product produced by the method of claim

1.

19. A method of producing fermented-type savory flavored product comprising the steps of: (a) contacting a protein mixmre with an effective amount of one or more protease and an effective amount of yeast;

(b) incubating the enzyme and yeast treated protein mixmre for a sufficient time and at a sufficient temperamre to form a fermented protein slurry;

(c) adding an effective amount of one or more protease to the fermented slurry of step (b) for a sufficient time and at a sufficient temperamre to form a hydrolyzed fermented mixmre; and

(d) filtering the slurry of step (c) to obtain a filtrate fraction.

20. The method of claim 19, wherein incubation of the enzyme and yeast treated protein mixmre is at a temperamre of 30-70°C for a period of between about 60-84 hours.

21. The method of claim 20, wherein incubation is at a temperamre of 50 °C for 72 hours.