The present invention relates to the use o highly purified natural improving agents in the productio of bread and other yeast raised baked goods. It als relates to the recovery of extracts containing highl purified improving agents for use in bread and other yeas raised baked goods, which are derived from the residues o "waste" products of fermentation and food productio industries and to the use of these highly concentrated foo quality improving agents in the production of bread an other yeast raised baked goods. BACKGROUND ART

It is well known that the making of bread uses mechanical operations such as kneading the dough, dividing the dough into pieces, and moulding into a predetermined form before proofing and baking. The physical properties of the dough, such as elasticity, extensibility, non-stickiness and ability to be. moulded are dependent on a combination of factors including the quality of the flour, the quality of the gluten, and/or the presence of other food additives. In addition the quality of the final baked product as judged by parameters such as crumb softness, volume, texture, taste, mouthfeel etc., is equally dependent upon the above physical properties and is affected by both the chemical and is biological parameters.

Using traditional methods of breadmaking, the period of bulk fermentation is important. It is a resting period, during which time the yeast ferments, interacts with the gluten in the dough, and the dough itself changes from a rough, dense mass with poor gas retention and lacking extensibility, into a smooth extensible dough with good gas retaining properties. Such properties are essential to the production of a loaf with good volume, soft yet resilient with fine crumb cell structure; Any method of bread making which omits bulk dough fermentation can only succeed in

SUBSTITUTE SHEET

Traditionally produced breads using longer bulk fermentations in the absence of, or in the presence of very small quantities of, chemical or other added improvers exhibit desirable quality characteristics including flavour, - crumb structure, crumb softness and resistance to staling.

Breads derived from the Chorleywood or other instant dough processes require the addition of improving agents to reach an acceptable quality with respect to volume, crumb structure, mouthfeel and staling characteristics but often 2 _Q lack acceptable sensory and olfactory parameters.

Recently, fermented flour and whey additives have become available, e.g., Empruv, Fermitech etc, which claim to cause an improvement in the quality of doughs to which they are added including sensory and olfactory parameters. 5 These manifestations are obvious to those persons with ordinary skill in the art. Coupled with our observations that the greater the contact time (from 2 to 24 hours) between the yeast and dough, sponge, brew or ferment system, the better leavening, texture, flavour and 0 resistance to staling in the bread or other yeast raised baked goods, led us to postulate that a metabolite or group • of metabolites formed during the fermentation acted upon the gluten and then on the physical properties of the dough in a beneficial manner. Historically, it has been known to those 5 skilled in the art that breads made with the liquors of microbial fermentations have a lighter, softer texture and are more resistant to staling than conventional breads (Tannahill R.. 'Food in History' Stein & Day 1972). We postulate that the causative metabolite or group of 0 metabolites is common to each of the described systems. The fact that such metabolites are biological in origin and are derived by natural processes upon food ingredients in day to day use in yeast raised baked goods is significant. There is a groundswell of public opinion and legislative action in 5 many countries, including Australia and the rnited Kingdom, to limit food additives to strictly defined substances,

which must be disclosed on labels ('Milling' June 1986). The potential exists therefore for the application of functional natural compounds in the food industry in general and in the production of yeast raised baked goods in particular.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide novel food quality improving agents for use in the production of bread and other yeast raised baked goods. o These improving agents are highly purified substances, including nicotinamide adenosine dinucleotide, flavin ononucleotide, flavin adenosine dinucleotide, the cytochromes, and the ubiguinones, derived from biological systems; the improving agents also include components and 5 derivatives of these substances, e.g., nicotinamide, riboflavin etc.

It is a further object of this invention to provide novel food improving agents for use in the production of bread and other yeast raised baked goods which 0 are concentrated extracts from spent fermentation residues. *

It is another object of this invention to provide novel food quality improving agents, which will improve the quality of bread and other yeast raised baked goods by complete replacement of, or reduction in, the levels of 5 chemical improving agents, such as potassium bromate, sodium metabisulfite, sodium stearoyl lactylate or the diacetyl tartaric esters of monoglycerides or distilled monoglycerides, which will permit a reduction in bulk fermentation time without the concomitant loss of quality 0 parameters of flavour, texture, resistance to staling etc.

It is yet a further object of this invention to provide novel food quality improving agents which will permit a reduction in the amount of yeast required for the manufacture of yeast raised baked goods. 5 It is yet another object of this invention to provide a process for recovery from fermented wastes or

.UBST1TUTE ^~ ϊl ^~ _

fermentation residues, e.g., from breweries, wineries, starch plants and dairies of highly concentrated natura novel improving agents for yeast raised baked goods.

These and other objects of the invention will b apparent from the following further disclosure of the invention.

According to one of its aspects the present invention provides food quality novel natural improving agents for yeast raised baked goods, including any one of, or any combination of, natural electron transfer biological oxido-reductants including oxidative chain phosphorylation components or derivatives thereof, including for example:- NAD nicotinamide adenosine dinucleotide NADP nicotinamide adenosine dinucleotide phosphate

FMN flavin mononucleotide FAD flavin adenosine dinucleotide Ubiguinones - i.e., the coenzymes 0 Cytochromes and other components of the electron transport chain.

According to another aspect of the invention there is provided a process for recovery of concentrated extracts of such oxido-reductants or electron passing compounds of the respiratory chain and intermediary metabolism from fermentation residues or "wastes", the process comprising subjecting such residues or wastes to treatment including cell disruption, heat treatment, clarification, purification, concentration, dehydration and stabilization of the extract. According to a further aspect the present invention relates to the use of such oxido-reductants or electron passing compounds, either singularly or in any combination as food quality improving agents, especially in the production of yeast raised baked goods as a full or partial replacement of, or in addition to, chemical improving agents.

SUBSTITUTE SH ET

These compounds may be used either in a highly purified state or as components of concentrates and extracts contained in or derived from spent residues from the fermentations of yeasts (especially of the Saccharomyces genus) as for example from beer, wine and other alcoholic beverages, and also from the spent residues from fermentations of other micro-organisms, e.g., Acetobacter, Lactobacillus, Aspergillus, Leuconostoc and Streptococcus species. Also, starch plant residues and dairy residues. Generally, the concentrations of the electron-passing compounds employed are at least 0.001% by weight, normally expressed as a percentage of the weight of flour in the mixture.

NAD and NADP are natural oxido-reductant present in most biochemical systems. They are coenzymes for a wide variety of enzymes. The NAD dependent enzymes exhibit several modes of action, four of which are discussed herein. First, the NAD dependent dehydrogenases catalyse the oxidation of alcohols, aldehydes, c- and /B-hydroxy carboxylic acids and c-amino acids. The nucleotides themselves readily accept electrons from a reduced substrate or donate electrons to an oxidised substrate in a coupled reaction such as

1 ,3 diphoεphate

Secondly the nicotinamide nucleotides function in reduction o f the flavin coenzymes . These provide the link in the e le c tron transport chain between NAD an FMN or FAD . An example is the reduction u * oxidised glutathione by glutathione reductase .

, UBSTvnJ ^T T^i

NADH + H + oxi di sed «^=^- NAD + 2 reduced g lutathione g lutath ione .

Glutathione reductases are enzymes which contain FAD as a prosthetic group. The FAD reacts with the NADH as shown

NADH + H ⁺ + FAD ^= ^≤ = NAD ⁺ + FAD H FAD H ₊ oxidised FAD + ?. reduced glutathione glutathione

Thirdly, the nicotinamide nucleotides provide a source of electrons for the hydroxylation and desaturation of both aromatic and aliphatic compounds as for example in lipid metabolism. A further function of NAD is in the repair mechanism of DNA, catalysed by DNA-ϋgase.

As is indicated above, NAD is an end product of alcoholic fermentation. It is also one of the end products of intermediary metabolism. Historically, its function in yeast raised baked goods depends upon an interaction with the glutathione reductase system and the oxidation of reduced glutathione. While glutathione reductase has been reported to have specificity for NADP, NAD can collect H atoms from substrates acted upon NADP linked dehydrogenases.

2GSH ' ; 5* G-S-S-G glutathione reductase

NAD(P) r_ NAD(P)H ₂ and NADPH + NAD ⁺ -^a. NADP ⁺ + NADH. Traditionally, the rheological properties of doughs have been considered to be related to the total number of and distribution of disulfide bonds in gluten (Bloksma, 1972). The thiol/disulfide interchange represents a dynamically changing system dependent on the number, and distribution of these functional groups, i.e., either intermolecular or intramolecular, as well as the

SUBSTITUTE SHEET

oxidation-reduction parameters in the system.

However, the historical views of the complex processes occurring during dough fermentation are surprisingly restricted. While they encompass reactions which are likely to occur in a wheat flour/water dough where the glutathione and NADP sources are most likely the germ, they do not explain or appear to take into account reactions occurring between gluten and yeast during fermentation.

Yeast is a living organism. As such it requires nutrients during its logarithmic growth phase. _T hese are provi ^d ed from the constituents of the flour an _d /or by the ad ^d ition of yeast foods e.g., sugar, nitrogen sources and trace elements. Secondly, yeast cell membrane constituents ^' come into direct contact with the gluten in the dough. The possibility exists for interactions between the constituents o ^' f the yeast membrane and those of the gluten. _T hir _d ly, living cells have a finite life span. m longer fermentation doughs the percentage of damaged or _d ea _d yeast cells will _^ be higher than in _. short time _d oughs. Consequently there will be released into the fermenting ^d ough, intracellular enzymes, coenzymes and other metabolites.

It has been surprisingly discovered by us, that the interactions between the individual components of the electron transport chain and the related compoun _d s of interme ^d iary metabolism with gluten and/or flour vary from, and are inconsistent with, the historical view. According to the present invention it can be demonstrated that there is also a ^d irect action of these substances on the gluten with does not appear to involve either glutathione reductase or thiol/disulfide interchange.

^A ccording to the present invention it can be shown that the spent yeast and micro-organisms of fermentations are a rich source of such metabolites. Fresh Baker's yeast is the usual source of commercial NAD and related metabolites, although patents describing its

SUBSTITUTE SHEET

production and purification from aerobic culturing of micro-organisms are extant (e.g., see USP 3,705,080; USP 3,705,081; USP 3,708,394 and USP 3,709,786) .

Yields of NAD and related metabolites from fresh baker's yeast are small, while those from the aerobic culturing of micro-organisms are higher. irrespective of method of production, costs of the final products are high.

Since NAD is produced as an end product of ethanolic and lactic fermentations: acetaidehyde + NADH ^a ?° ^~ ^ ethanol + NAD ⁺

2 dehydrogeπase

or pyruvic acid + NADH lactic acid ₊ NAD ⁺ it has now been surprisingly discovered according to the invention that its concentration in the end products of alcoholic and lactic fermentations and in the microbial residues from such fermentations is an order of magnitude greater than in baker's yeast, thus making its recovery commercially viable. NAD is estimated using the enzyme catalysed oxidation of ethanol to acetaidehyde at pH 9.0. The reaction mixture consists of lOOmM phosphate buffer pH 9.0, ethanol 170mM, alcohol dehydrogenase (Boehringer Mannheim)

20 units/mL in a total reaction mixutre of 2.5mL. The reaction is carried out at 37°C in a cuvette with a 1cm light path, and the change in absorbance at 340nm is used to calculate the concentration of NAD.

As is known to those skilled in the art of making yeast raised baked goods, those breads in particular which are made using beer or wine to replace some or all of the water have a different flavour, texture, mouthfeel and resistance to staling to those made using only water,

( 'Milling' , June 1986). Until the present invention, the components within these liquors which were responsible had not been identified. Further, inter ^r elationships between these active components and other improver ingredients.

.UBSTTTUTE SHΞΓΓ

e.g., or;-amylase, ascorbic acid etc., in breads and yeast raised baked goods had not previously been described or understood.

NAD and the related compounds according to the present invention act as strong oxidising agents on gluten, either alone or when in combination with flour, but at the same time increase extensibility of the dough. in the presence or ascorbic acid, the oxidising power of the NAD and related compounds is reduced without a concomitant o significant reduction in extensibility of the dough. In breads and yeast raised baked goods, the presence of ascorbic acid and cκ-amylase may be beneficial to produce maximum advantage with respect to volume? texture; mouthfeel and other physical parameters. According to the present 5 invention some embodiments of the improving agent may also comprise one or more additives selected from of-amylase , ascorbic acid and phospholipase A and/or phospholipase D, in an appropriate effective amount, usually at levels of not less than 0.0001% by weight. 0 Sources of NAD and Related Compounds.

Broadly speaking, natural sources of NAD, FAD, FMN, NADP, the ubiguinones are the end products of microbial fermentations. Examples of possible sources include:- (i) yeast residues from breweries 5 (ii) spent grain from breweries

(iii) yeast residues or lees from wineries (iv) residues from micro-organisms from lactic fermentations

(v) residue liquors from lactic fermentations, e.g.., 0 starch plants or dairy residues.

Examples given relate to extensive tests and processing of yeast residues from breweries but also include those from wine lees. As supplied the yeast residue is a thick cake, still biologically active but with a high moisture and - ethanol content. The yeast cells contain large intracellular concentrations of NAD, in addition to other

components of the electron transport chain and intermediar metabolism as previously cited. FULL DESCRIPTION OF THE EMBODIMENTS

The preferred method for the processing of the residues from fermentation systems in order to obtain highl purified extracts and residues for use in yeast raised baked goods may comprise some of or all of the following processes in any combination or order:-

Cell disruption Heat treatment Clarification Purification Concentration Dehydration. Cell Disruption

High pressure homogenisation and freezing (or heating) of the fermented residue and residue products of fermentation are two of the methods which have been employed according to the invention to disrupt and/or weaken the cell walls of the yeast. Other methods may include for example, extrusion, colloid milling, microwaving, lysozymes, solvent extraction, high pressure spray drying, vibration ball milling, treatment with ultrasonics and grinding. Cell disruption may be followed by inactivation of deleterious enzyme and other catalytic systems by appropriate physical and chemical change. Heat Treatment

A number of techniques may be employed to inactivate enzyme systems, including elevating or depressing temperatures, adjustment of pH or combinations of these. The fermented microbial residues or the cell disrupted microbial residues are diluted with water to a viscosity approaching that of water. The diluted mixture is subjected to a temperature greater than 45°C but substantially less that 200°C for a period of not more that 30 minutes. This is of extreme importance not only as a consequence of

inactivating deleterious enzyme systems, but also because of the final yield and quality of the NAD and the other oxido-reductants and metabolites hitherto described. This type of process not only disrupts the cells in previously untreated microbial fermented residues but also permits the release into the medium of intracellular pools of the NAD and other oxido-reductants. It will be appreciated that inactivation of enzyme systems and cell disruption may be achieved by temperature reduction to below freezing point followed by thawing. Moreover, a combination of adjustments of pH and temperature is to be preferred. NAD is unstable in alkaline solutions and furthermore is unstable at low pH ^■ and high temperatures. Preferred working conditions for maximum recovery of NAD are within narrow pH limits known to those skilled in the art.

Clarification and Filtration

Clarification of the cell disrupted enzyme inactivated fermented waste to remove cell debris from the extract containing the NAD and other metabolites is successfully achieved by centrifugation of filtration, e.g., the use of Sharpies decanter or starch bed filtration.

Other means of clarification will occur to those skilled in the art in the light of the present disclosure, as for example the use of filter aids such as diatomaceous earth, vacuum drum filtration flocculation, flotation bubbles, pressure candles, enzymes or membranes. Dehydration and Concentration

The present invention relates to a process for producing a highly concentrated extract from one of a group of fermented residues and "waste" residues of fermentations including brewery residues, winery residues, lactic fermentation residues containing the natural oxido-reductant NAD and related compounds previously described. It will be understood by those skilled in the art that several dehydration and concen ^t ration techniques may be applied to obtain the highly concentrated dehydrated extract include

^~ ~ ^~ xvr~~~~ ^~ ^l

freeze drying, spray drying, drying on carriers, e.g., starch or microwave drying. A highly concentrated liquid extract may be obtained by any or all of a variety of concentration processes including utrafiltration, reverse osmosis, falling film evaporators, and the like.

Each of these methods of dehydration and concentration can be used on the crude ferment residues or on the extract containing the active principles of the coenzymes, metabolites and oxido-reductants. Freeze drying of the heat treated residue, the heat treated extract and the residual cell debris, have provided free-flowing products. Dehydration of both the heat-treated residue and extract provided products which, when baked, results in significant improvement in volume, crumb texture and ■ retardation of staling in yeast raised baked goods. Addition of the crude residue or the heat treated extract to starch in a 1:1 ratio permitted the dehydration to be carried out using a fludised bed drier. The final product required grinding but provides increases in NAD and in volume and improved textural characteristics in bread and yeast raised baked goods.

Notwithstanding- the fact that fermented yeast and microbial residues contain high concentrations of endogenous non-protein, non-starch hydrophilic colloids and carbohydrates, the products herein described can be successfully obtained by spray drying. The preferred method uses an inlet temperature into the spray drier of between about 180°C and 300°C and an outlet temperature of between 70°C and 120°C. Advantages of lower inlet and outlet temperatures on the performance of the final spray dried concentrate in yeast raised baked goods will be obvious to those skilled in the art in view of the present disclosure.

The present invention relates to the novel use of naturally occurring oxido-reductants, metabolites and coenzymes in bread and other yeast raised baked goods as in adjunct to currently available "chemical improvers", or as a

" ^~ ^Λ

Mix ing 3.75 minutes for white loaves and minutes for wholemeal

Bulk fermentation 20 minutes at 30°C Bench time 10 minutes Mould ing 400g per loaf proof ing 45 minutes at 38°C and 70% relativ humidity

Baking 210oC. for 25 minutes The raw materials used for each loaf are set forth in Tables la and lb. Table la Raw Materials (part by weight)

V8 .

Ingredients White Control White Natural

Supreme ++ Flour 1000 1000

Salt 20 20

Soya Flour 10 -

Yeast* 12 12

Improver V8 2 . 5 -

Improver Natural** - 23

Table ^' lb Formulation (parts by weight)

Ingredients Wholemeal Control WhlOlemeal Natural

Supreme++ Flour 820 820

Harvest* Meal 180 180

Salt 20 20

Gluten 40 40

Yeast' 14 14

V8* 2. ,5 -

Natural improver** - 23

Harvest is a registered trademark of George Weston

Foods Ltd, Sydney, N.S.W. Australia.

Yeast is Mauripan Active Dried Yeast produced by Mauri

Foods, Camellia, N.S.W. Australia.

** Natural improver is produced by Cereform, Wetherill

Park, N.S.W. Australia, containing soya flour, soya lecithin, of-amylase (cereal) and ascorbic acid. ++ Supreme flour is a high protein baker's flour produced by N.B. Love, Enfield, N.S.W. Australia. V8 is a chemical based improver containing potassium bromate, sodium etabisulfite, but no emulsifiers.

Following baking, the loaves were cooled to room temperature, volumes were measured using displacement of rapeseed, sliced, sealed in polyethylene bags and stored at room temperature for several days. Staling measurements were performed using an Instron 4301 Universal Testing Machine. Measurements were carried out on minimum 30cm slices of the loaf, at six sites across the loaf, excluding the heel. Mean values of force per unit area to compress the centre of individual slices of bread to a depth of 10mm were calculated. Results for volume and staling are shown in Table 2.

Table 2♦ Effect of Example 1 product on Loaf volume.

Type of Loaf Volume Staling

(ml.) (Newtons)

White Control 5430 5.015^0.245 White Control + 0.05% product 5740 4.49 +0.260* from Example 1 on a flour weight basis

White Natural 5220 5.582+0.226

White Natural + 0.05% product 5440 4.841+0.278* from Example 1

Wholemeal Control 5500 4.447+0.419

Wholemeal Control + 0.05% product 5760 3.971+0.245 from Example 1

Wholemeal Natural 4960 4.859+0.214 Wholemeal Natural + 0.05% product 5510 3.971+0.194 from Example 1

SUBSTITUTE SHEET

Representative volumes for 3 loaves baked on th same day are given. values which differ from controls b more than 100 units are significant. * Significant at the p<0.05 level. The lower th value the softer the bread.

It can be seen from the representative results o Table 2 that replacement of chemical improvers such as V with natural improvers results in a significant reduction i loaf volume. Addition of the product from Example 1 at the preferred usage rate of 0.05% based on the flour or flour/meal content of the system provided an increase in volume back to volumes obtained with chemical improvers alone. When the product from Example 1 was added to systems containing chemical improvers, it provided a significant increase in the loaf volume. In addition to the improvement in loaf volume, addition of product from Example 1 at the usage rate of 0.05% based on the flour or flour/meal content of the system, provided a retardation in the staling or firming characteristics of the loaves. Crumb structure and texture in loaves containing the product of Example 1 were markedly different from those containing only V8 or natural improver. The crumb structure is much finer, cells are small and elliptical in shape. The crumb is more resilient and possesses an iridescent sheen. Example 3.

The following study was undertaken to determine the effect of addition of pure NAD ( Boehringer-Mannheim lithium salt 100% pure or sodium salt 98%) into a variety of breads made using a range of Harvest and Cereform improvers. Loaves of bread are made by mixing raw materials set out in Table 1 and following the same breadmaking process as outlined in Example 2 , with the addition of low concentrations of pure NAD in lieu of the processed highly concentrated extract produced in Exa ^m ple 1. The volumes of the loaves results are shown in Table 3.

Table 3. Effect of NAD on volume

Type of Loaf Volume Staling

(ml.) (Newtons)

White Control 5430 5, .015+0.245

White Control + 0.005% NAD 5690 4, .56 +O.312*

White Natural 5220 5, .582+0.226

White Natural + 0.005% NAD 5460 4, .903+0.188*

Wholemeal Control 5500 4, .447+0.419

Wholemeal Control + 0.005% NAD 5730 3. .715+0.276*

Wholemeal Natural 4960 4, .859+0.214

Wholemeal Natural + 0.005% NAD 5450 3, .827+0.255*

* Significant at the p<0.05 level.

As can be seen from the above representative sample, the addition of pure NAD provided an increase in volume as well as a retardation in the onset of staling. Crumb structure and texture replicated that seen in the loaves baked using the highly concentrated extract of Example 1. Example 4.

In order to determine the interaction between the product of Example 1 or pure NAD with ascorbic acid, loaves of bread were made by mixing the raw materials as set out in Table 1 and using the same breadmaking process of Example 2 , using varying levels of: a) the extract obtained from the process of Example 1 and ascorbic acid, b) Pure NAD and ascorbic acid. Representative results for wholemeal are shown in Table 4.

SUBSTITUTE SI ΞET

Table 4 ■ Interaction between NAD and Ascorbic Acid.

Loaf Volume % Ascorbic Acid 0 0.0035% 0.007% 0.01 Wholemeal 6390 6430 6730 6960

Wholemeal + 0.005% NAD 6150 6640 6930 7150

Wholemeal + 0.05% product of Example 1 6140 6670 6970 7180

Loaf volumes guoted are representative only and vary from day to day. Results in Table 4 show the NAD and the product from Example 1 interact with ascorbic acid in the dough. Unless ascorbic acid is present the NAD and product from Example 1 exert such a strong effect on the dough, that there is a resultant reduction in loaf volume.

Example 5.

The following example demonstrates the interaction between c-C-amylase and the products described in Examples 3 of the present invention. Loaves of bread were baked using the raw materials as set out in Table 1 and the breadmaking process of Example 2. The source of CK-amylase was cereal, and the activity was determined using the phadebas (TM) (Pharmacia) amylase activity test. Representative results are shown in Table 5 for wholemeal loaves. Table 5. Effect of o<-amylase on loaf volume.

Loaf Volume

* o-amylase addition 0 0.25% 0.5% 0.75% Wholemeal + Natural Improver 4800 4910 4930 5230

Wholemeal + NAD 0.005% + Natural Improver 4880 5060 5220 5430

* c^-amylase added as diastatic malt flour containing not more than 600 SKB units per g. % representation of the flour.

SUBSTITUTE SHEET

The above clearly illustrates the desirability of using oc-amylase in conjunction with the products claimed in the present invention, in improvers.

Example 6

The following example illustrates the effects of flavin adenosine dinucleotide (FAD) as an improving agent with similar properties to those of NAD and the highly concentrated extract of Example 1. FAD was added to the loaves at levels up to 0.005%, the loaves were baked according to the scheme in Example 2, volumes were measured as described in Example 2. Results from this example indicate that FAD, when added to bread doughs at the level of 0.005%, causes an increase in loaf volume of 3% (significant at the p 0.05 level) and crumb structure and texture similar to that of the product from Examples 2 and 3.

Example 7 The following example illustrates the effects of flavin mononucleotide (FMN) as an improving agent with similar properties to those of NAD and the highly concentrated extract of Example 1. FMN was added to the loaves at levels up to 0.005%. The results from these tests show in Table 6 that the 0.005% level of addition of FMN is preferred. Crumb structure and texture was similar to that of loaves baked using the products from Example 1 and Example 3. Table 6 Effect of FMN addition on Loaf volume. Loaf volume

White + Natural Improver 5160 White + Natural Improver 5010

+ 0.001% FMN White + Natural Improver 5340 + 0.005% FMN

Example 8

This study was undertaken to determine the effect of binding free sulfhydryl groups in flour with FAD and then adding NAD into the improver system. It is known that FAD binds to free sulfhydryl groups and that in the Supreme Flour used in these Examples the concentration of these groups is approximately 3 mg %. Accordingly the FAD was added at 0.003% and NAD added to the ingredients listed in Table 1 at the rate of 0.0025% on a flour basis. From the results in Table 8 it is obvious that having removed the free sulfhydryl groups, the NAD is free to interact at other parts of the gluten in the flour and thus provide an increase in loaf volume and concomitant improvement in crumb structure and texture. Example 9

This example describes a preferred process for the recovery of a highly purified extract from either bentonite wine lees or wine lees. Bentonite is added to wine lees as a filter aid at the rate of 5%. The lees are autoclaved for 10 minutes at 120°C, cooled rapidly to 4°C and filtered under vacuum. The filtrate is frozen to -20 C and freeze dried. The final dried product is ground and mixed with an eguivalent mass of wheaten starch. Example 10 This example pertains to the use of the product from Example 9 as an improving agent in yeast raised baked goods. Loaves of bread were formulated using the raw ingredients of Table 1 and baked according to the description of Example 2. volumes of the loaves were measured by rapeseed displacement and recorded. Crumb structure and texture were noted.

Table 7.

Effect of product from Example 9 on Loaf volume.

SUBSTITUTE S .EST

Volume

-White + Nat. Improver 5140

-White + Nat. Improver 5280

+ 0.025% Product Example 9

-White + Nat. Improver 5440

+ 0.05% Product Example 9

The results from this study indicate that the product obtained in Example 9 which is a highly concentrated extract from wine lees provides an increase in loaf volume and crumb structure and texture similar to that obtained with pure NAD, FAD, FMN or the product of Example 1.

Table 8 Summary of Representative Values for Loaf Volume for Examples 2-9 in White Natural Improver Loaves. Type of Loaf Additive Loaf Volume

- 5160

0-005% NAD - 5460

0.05% Product of Example 1 5400

White + 0.003% FAD 5230 Natural 0.005% ^" FAD 5280

0.003% FAD + 0.0025% NAD 5380

0.001% FMN 5010

0.005% FMN 5340

0.05% Product of Example 9 5440

Example 11 The following example illustrates the effect of the addition of product from Example 1 to proprietary chemical improvers containing emulsifiers. White and wholemeal loaves were baked as hitherto described using s.uch improvers. Representative values for vol ^m e and staling measurements for both white and wholemeal are shown in Table

9 . The results ind icate that the presence of the product o Ex amp le 1 and emu ls i f ie rs in a chem i ca l improve r g i ve s ignif icant increase in volume .

Table 9

Effect of Emulsifiers

White Wholemeal

Volume Staling Volume Staling

V8 5180 6.855 4690 6.307

V8 + 0.05% product of Example 1 5380* 5.842* 4980* 5.728*

TN80 5430 4.523 5250 4.538

TN80 + 0.05% product of Example 1 5540* 4.645 5430 ^: 4.508

NAF 5410 5.241 5430 4.364 NAF + 0.05% product of Example 1 5600* 5.003 5550* 4.508

* Significant at P<0.05 level.

NAF and TN80 are proprietary improvers produced by Cereform, Wetherill Park, New South Wales, Australia, containing potassium bromate, sodium metabisulfite, ascorbic acid, L-cysteine and emulsifiers such as sodium stearoyl lactylate. The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention. All such modifications as would be obvious to one skilled in the art are intended t be included herein.

i i — ~"T ITUTE SHΣ:t