Field of the Invention

The invention relates to a method for the production of low-medium glycaemic index (Gl) leavened breads with improved texture and appearance. More specifically the invention provides methods for the production of a leavened wheat bread supplemented with soluble dietary fibre, e.g. a (1 ,3), (1 ,4) mixed-linkage cereal betaglucan, that has a crumb and crust structure closely comparable to a typical wheat bread prepared in essentially the same way. Such supplemented breads may be prepared in the method of the invention in such a way that degradation of the soluble dietary fibre is reduced thereby maintaining the molecular weight of the soluble dietary fibre and associated health benefits. The invention further provides low-medium Gl leavened breads obtained from said methods and uses thereof in for maintaining or promoting health and well-being in a subject, and in the treatment or prevention of metabolic disorders associated with the over-consumption of starch/glucose and/or inappropriate metabolism of glucose.

Background

Carbohydrates represent the main source of energy in most Western countries, but the health effects of carbohydrate rich foods differ substantially due to their degree of processing, their glycaemic index (Gl), the content of whole grains and the content of dietary fibre. Cereal based foodstuffs, especially bread, are a particularly important food source in Western diets, but the most popular varieties typically have a high Gl.

The glycaemic index (Gl) describes the glycaemic potency of the available carbohydrates in a food and so is used to compare the carbohydrate quality of different foods. Several health benefits including reduced risk of cardiovascular disease (CVD), metabolic syndrome and type II diabetes (T2D) have been associated with low-GI diets, and a higher dietary Gl may be causally linked to an increased risk of T2D. Low Gl foods are considered to represent healthier choices. The prevalence of T2D has increased drastically over the last 35 years, and it is expected to continue to rise. T2D is a major risk factor for CVD, which in turn is the leading cause of morbidity and mortality among persons with T2D. Impaired glucose tolerance (IGT) and impaired fasting glycemia (IFG) are intermediate conditions between normal glucose metabolism and T2D and are often referred to as pre-diabetes. In 2012, the International Diabetes Federation estimated the global prevalence of pre-diabetes to 280 million and it is expected to rise to 400 million by 2030. Persons with pre-diabetes are at high risk of developing T2D, and it is estimated that 70% of those with pre-diabetes may develop T2D within 10 years.

The causes and aetiology of IGT and IFG are not fully understood but there is a strong link to obesity, age, ethnicity as well as a heredity component. Diet and nutrition also play a role in the development of pre-diabetes and progression towards T2D, with high whole grain and cereal fibre intake, but not refined grain, being consistently inversely associated with risk of T2D and coronary heart disease. In view of this, attempts to replace refined grains with fibre-rich whole grains in cereal-based foodstuffs are regarded as a major strategy to improve public health. However, although connected, whole grain, cereal fibre and glycaemic response provide complementary information regarding carbohydrate quality of importance to health. Indeed, many commonly consumed products rich in dietary fibre are characterized by a high Gl.

Thus, cereal-based foodstuffs that are both rich in whole grains and dietary fibre, but which also have a low to medium Gl should be advocated. Many foods that are naturally rich in soluble dietary fibre, e.g. (1 ,3), (1,4) mixed-linkage cereal betaglucan, galactomannan, pectin, and psyllium, meet these criteria. Oat- or barleybased foodstuffs are good examples of soluble beta-glucan enriched foodstuffs. Indeed, cereal-derived beta-glucan can improve postprandial blood glucose and can lower low density lipoprotein (LDL)-cholesterol. This has been endorsed through authorized health claims by the European Food Safety Authority (EFSA). In this regard, the European Commission (advised by the EFSA) permits products containing at least 4 g of oat or barley beta-glucan per 30 g available carbohydrate to claim that the product “reduces post-prandial glycaemic response”. However, previous attempts to produce leavened wheat bread products that are considered to be a low-medium Gl foodstuff, and in particular to meet the EFSA health claim criteria for reduction of postprandial glycaemic response, have resulted in suboptimal crust and crumb features that deter consumers from choosing the bread and so undermine widespread adoption by the population. These features include, inter alia, small loaf volume, overly firm texture, gummy texture, collapsed crumb with large holes/voids and/or detached upper crust, dense crumb structure with lower proportion of gas cells, high moisture content, darker crumb structure and a leathery instead of crispy crust. These features can all impart undesirable, or at least suboptimal, aesthetic and/or taste and mouth feel properties on the bread. Similar problems are experienced by leavened wheat bread products containing sufficient levels of other soluble dietary fibres to have a reduced post-prandial glycaemic response or be considered a low-medium Gl foodstuff. As such, at present, there are no such breads commercially available on the mass market. At present, a suboptimal balance must therefore be struck between the health benefits of soluble dietary fibre, e.g. (1 ,3), (1,4) mixed-linkage cereal beta-glucan, supplemented leavened bread and a texture that is perceived as desirable to the consumer.

Conventional bread making processes also lead to a partial degradation or depolymerisation of soluble dietary fibre by endogenous carbohydrate degrading enzymes present in wheat flour. This results in sticky doughs that are difficult to machine process and can reduce the potential of the bread to reduce a favourable glycaemic response.

It is therefore an object of the present invention to provide a new, scalable method for producing a soluble dietary fibre supplemented leavened wheat bread that: (i) has soluble dietary fibre levels sufficient to render the bread low-medium Gl or to reduce post-prandial glycaemic response in the consumer, in particular soluble beta-glucan levels which meet the above EFSA health claim criteria; and (ii) has a crust and a crumb that is closely comparable to a typical (non-supplemented) wheat bread prepared in essentially the same way, and preferably (iii) which imparts desirable taste and mouth feel which consumers are used to and like to consume. Such method may be performed in such a way that degradation of the soluble dietary fibre is reduced thereby maintaining the molecular weight of the soluble dietary fibre and associated health benefits.

It has now been found that a two stage dough forming process in combination with a vacuum cooling step as part of the production method has enabled a soluble dietary fibre supplemented wheat bread to be produced that meets these criteria and so is superior to similar breads prepared using methods available to date.

Thus, in a first aspect, the invention provides a method of producing a leavened wheat bread product comprising about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate, said method comprising the steps of:

(i) providing a wheat flour bread dough comprising a leavening agent;

(ii) combining said dough with a soluble dietary fibre so as to form a soluble dietary fibre supplemented wheat flour bread dough with said soluble dietary fibre distributed therein;

(iii) baking said soluble dietary fibre supplemented wheat flour bread dough at a temperature and for a time sufficient to produce a crust and crumb structure and raise the internal core temperature of the dough to about 90 to about 100 °C; and

(iv) cooling the baked dough so as to result in said leavened wheat bread product, wherein at least part of the cooling step is a vacuum cooling step.

As used herein, the term “bread” refers to a pliable cooked food staple prepared at least from flours derived from cereals, pulses or fractions thereof, and an amount of water or water-containing fluid, that is sufficient to create a dough when mixed with the flour component. Breads may be made to many recipes involving different flours, different water containing fluids, and the addition of further ingredients such as fats, eggs, sweetening agents, flavouring agents, and so on. A dough is a mixture of flours, water-containing fluid, and optionally other ingredients that is able to retain structural integrity on a flat, level surface without requiring external support or needing to be housed in a container when in an uncooked state.

In accordance with the first aspect of the invention, the bread produced by the method is a wheat bread. As such, at least about 30%, e.g. about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 of 95% w/w of the flour in the dough from which the bread product is created is wheat flour. In other words, at least about 30%, e.g. about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 of 95% w/w of the flour derived solids in the bread product is wheat flour.

Wheat flour is a powder-like substance produced by milling of wheat grain or fractions thereof. Wholegrain wheat flour is prepared by milling the entire grain, i.e. the endosperm, germ, and bran. Refined wheat flour (white wheat flour) is prepared by milling the endosperm only. The wheat flour of use in the invention may be any wheat flour suitable for bread making and so may be wholemeal, refined, bleached, enriched and so on. It may also be a mixture of fractions thereof, e.g. a mixture of wheat starch and wheat gluten. It may be advantageous to select a flour with a protein content (more specifically, a gluten protein content) which is appropriate for bread making. This will vary depending on the other ingredients in the recipe and the end result to be achieved, but in certain embodiments this will be a flour with a protein content of at least 9% by fresh weight, e.g. at least 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, or 14%. The skilled person would be able to select an appropriate wheat flour for use in the invention, but in certain embodiments the wheat flour will be a refined wheat flour with a protein content of at least 11%.

Bread may either be unleavened or leavened depending on whether or not a gas is generated in a dough before or during the cooking process. As used herein, the term “leavening” refers to the process of generating a gas in a dough before or during cooking so as to form voids in the finished bread and thus the three- dimensional structure of the “crumb”. The finished bread product is therefore referred to as leavened bread.

Leavening can be achieved by physical means and/or by use of a leavening agent. Physical means include aeration e.g. by using steam, air, carbon dioxide, and nitrous oxide. Chemical leavening agents include culinary alkalis such as metal carbonates or bicarbonates, e.g. sodium bicarbonate, ammonium bicarbonate and potassium bicarbonate which react with acids in the dough (endogenous or supplemented, e.g. potassium acid tartrate (cream of tartar), monocalcium phosphate, sodium acid pyrophosphate, sodium aluminium phosphate, dicalcium phosphate dehydrate, sodium aluminium sulfate, glucono delta-lactone, fumaric acid, dimagnesium phosphate, acetic acid, ascorbic acid, citric acid, lactic acid, vinegar, yoghurt, buttermilk) to generate gas. Microbiological leavening agents are yeast or bacteria which are added to the dough and allowed to ferment sugars in the dough and produce carbon dioxide Suitable yeasts for leavening include but are not limited to yeasts from the genera Dekkera/Brettanomyces baker’s yeast, for example, Saccharomyces (e.g. Saccharomyces cerevisiae, Saccharomyces exigus or Saccharomyces pastorianus), Dekkera/Brettanomyces (e.g. Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis, and Dekkera anomala) and Candida. Suitable bacteria include but are not limited to lactic acid bacteria, e.g. bacteria from the genera Lactobacillus (Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. casei , Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. plantarum, Lb. sanfranciscensis) Lactococcus (Lc. lactis, Lc. garvieae) and Leuconostoc (L. citreum, L. kimchi, L. mesenteroides). Microbiological leavening agents may be used in isolated form or as a combination. In particular the microbiological leavening agents may be provided in the form of a sourdough culture or a barm culture. Such cultures may comprise one or more of the above mentioned microorganisms.

The bread produced in accordance with invention is leavened, at least partially, by a leavening agent. In certain embodiments the leavening agent comprises or consists of a yeast, e.g. S. cerevisiae. In other embodiments the leaving agent does not comprise, or is not part of, a sourdough or barm culture. Thus, in these embodiments the bread product, or the dough from which the bread product is created, does not comprise a sourdough or barm culture. In other words, in certain embodiments, the bread product is not a sourdough bread or a barm bread. These embodiments do not exclude embodiments in which the leavening agent may comprise or consist of a yeast, e.g. S. cerevisiae, but said yeast is not provided as part of a sourdough or barm culture.

The bread product of the invention can take any form. In some embodiments, the bread product of the present invention is in the form of a loaf, hard roll, soft roll, bap, baguette, muffin, or bun. It may have been panned or of a free-formed shape. In preferred embodiments, the bread of the present invention is in the form of a loaf. In some embodiments, the bread product is produced in a partially cooked state. In such embodiments it will be appreciated that the partially cooked bread product will need to be returned to an oven at a later time point to complete the cooking process prior to consumption. Alternatively, in other embodiments, the bread product is produced in a fully cooked state.

As used herein, “dietary fibre” means carbohydrate polymers with at least 3 residues which are not hydrolysed by the endogenous enzymes in the small intestine of humans. In the field of food science carbohydrate polymers which are considered to be dietary fibre are classed as “soluble” dietary fibre or “insoluble” dietary fibre. More specifically, soluble dietary fibre are carbohydrate polymers which are at least partially soluble or dispersible in water or other predominately aqueous (non-organic) liquids representative of the human digestive milieu. Viewed alternatively, such fibre may be considered too be those which may be extracted from foodstuffs when exposed to the conditions of the human digestive system. “At least partially soluble” in the context of the invention broadly encompasses a range of interactions between the carbohydrate polymer and water molecules from full dissolution of the polymer in water or the aqueous liquid, to interactions between the polymer and water molecules that are sufficiently intimate and persistent to create a hydration layer in and around the polymer or network/matrices formed therefrom. Molecules of soluble dietary fibre are not overall hydrophobic molecules and instead are generally hydrophilic, although this does not exclude some hydrophobic regions. Thus, the skilled person will appreciate that some soluble dietary fibre will solubilise readily in water or other aqueous liquids to form an essentially transparent solution, whereas others instead disperse in such liquids to form viscous mixtures and gels. Differences may be seen between isolated polymer chains and network/matrices formed therefrom. A carbohydrate molecule that may be insoluble in water or aqueous fluids when present in network/matrices formed therefrom, may still be considered “soluble” dietary fibre if isolated polymers are at least partially soluble in water or aqueous fluids. In such instances, reference to soluble dietary fibre encompasses the non-networked forms. Similarly, a type of carbohydrate that may be insoluble in water or aqueous fluids when in high molecular weight form, might still be considered “soluble” dietary fibre if smaller sized molecules are at least partially soluble in water or aqueous fluids. In such instances, reference to soluble dietary fibre encompasses the smaller sized molecules. Thus, in certain embodiments, the soluble dietary fibre of use in the invention may behave as an aqueous gel-forming hydrocolloid when dispersed in (combined with) water. This intrinsic ability may be determined by eye simply by combining the dietary fibre with water. Any method known in the art for determining (e.g. quantifying) the viscosity (e.g. the specific viscosity) of viscous mixtures or gels either directly or indirectly may be used to determine (e.g. quantify) the viscosity (e.g. the specific viscosity) of viscous mixtures or gels formed from aqueous mixtures of soluble dietary fibre. For example, viscosity of a viscous solution may be measured using a Physica MCR 301 rheometer (Anton Paar, Stuttgart, Germany) fitted with a double gap geometry (DG26.7), as described in Reider et al., 2017, Food Hydrocolloids 67, 74-84. As part of this method, the DG geometry is used with overfill (approximately 10 mL) to avoid undesirable surface tension and capillary effects. After a temperature equilibration of 60 s, apparent viscosity was measured at 37 °C in a shear rate range of 0.01-100 s' ¹ (highly viscous samples) or 0.5 to 500 s' ¹ (medium viscous samples) with seven measurement points per decade. The measurement point duration ranged from 20 to 0.1 s during the forward ramp and 0.1-20 s during the backward ramp. Data obtained can then be fitted to the Cross-equation (Cross, 1965) using Rheoplus software (Anton Paar) and the zero shear viscosity may then be calculated using data from the forward ramp. The specific viscosity of a polymer in solution is then given by the ratio of the viscosity of the dissolved polymer and the solvent viscosity.

Without wishing to be bound by any particular theory, viscous fibres thicken the contents of the intestinal tract and may attenuate the absorption of sugar, reduce sugar response after eating, and reduce lipid absorption (notably shown with cholesterol absorption).

In some embodiments, it may be advantageous to select soluble dietary fibres which in the bread product of the invention will provide a supernatant following in vitro digestion of the bread product which has a zero-shear specific viscosity above about 10 mPas at a total dilution of 1 :7 in water. Preferably in these embodiments the zero-shear specific viscosity is above about 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20 mPas. Preferably in these embodiments the zero-shear specific viscosity is less than about 11 , 12, 13, 14, 15, 16, 17, 18 , 19 or 20 mPas. Ranges with endpoints formed by the above values are expressly contemplated. The in vitro digestion procedure may be based on the Infogest protocol (Brodtkorb et al. (2019) “INFOGEST static in vitro simulation of gastrointestinal food digestion” Nature Protocols, volume 14, pages 991-1014).

Widely recognised and well-characterised examples of soluble (gel forming) dietary fibre include, but are not limited to, soluble beta-glucan, e.g. a (1 ,3)-(1 ,4) mixed linkage beta-glucan), galactomannan (guar gum, tara gum, locust bean gum, cassia gum), psyllium, pectin, xanthan gum, gellan gum, gum arabic, gum karaya, tragacanth, konjac gum, neem gum, alginate, polar or charged group modified cellulose (e.g. carboxymethyl cellulose, hydroxypropyl cellulose) carrageenan, and agar.

In certain embodiments the soluble dietary fibre of use in the invention is a soluble beta-glucan, e.g. a (1 ,3)-(1 ,4) mixed linkage beta-glucan. These occur naturally in the cell walls of cereals (e.g. barley, oat, wheat, rye, sorghum, millet, buckwheat, amaranth). All beta-glucan polysaccharides contain D-glucose units linked by glycosidic bonds, but may further comprise different structures with radically different properties. As an example, cereal derived beta-glucan is made up of D- glucose units comprising p-(1 ,4) glycosidic bonds arranged in a linear chain with interruptions of p-(1 ,3) glycosidic bonds every 3 or 4 glucose units. It is the occurrence of p-(1 ,3) glycosidic bonds in the predominant p-(1 ,4) glucose chain soluble (gel forming) in water and other aqueous liquids. In accordance with the invention, the soluble dietary fibre of use in the invention may be a derivative of a (1 ,3)-(1 ,4) mixed linkage beta-glucan which substantially retains the solubility characteristics (as defined above) of the unmodified (1 ,3)-(1 ,4) mixed linkage betaglucan. Common saccharide modifying groups would include acetyl, sulphate, amino, deoxy, alcohol, aldehyde, ketone, ester and anhydro groups, but modifications will typically be the addition of charged or polar side groups, e.g. to form carboxylated or carboxy methylated beta glucans.

The weight-average molecular weight of a soluble beta-glucan, e.g. (1 ,3)-(1 ,4) mixed linkage beta-glucan, extracts can vary between natural sources, extraction conditions and analytical methods. For example, previous studies have shown the molecular weight (MW) of soluble beta-glucan to be in the range of 21-1100 kDa, 31-2700 kDa, 65-3100 kDa and 209-487 kDa in the case of rye, barley, oat and wheat, respectively (A. Lazaridou et al., 2007 Molecular aspects of cereal p-glucan functionality: Physical properties, technological applications and physiological effects, Journal of Cereal Science, Volume 46, Issue 2, 101-118). The MW of soluble beta-glucan can be determined using any suitable means known in the art. For example, MW determination may be achieved using size exclusion chromatography (SEC) combined with multi-angle light scattering (MALS) or with calibrations against beta-glucan MW standards.

Previous investigations have also revealed that a soluble beta-glucan, e.g. (1 ,3)- (1 ,4) mixed linkage beta-glucan, MW was negatively correlated with the Gl of the foods in which it is contained. Thus, foods containing a soluble beta-glucan with a higher MW have been shown to display a lower Gl, i.e. display a slower break down and cause a more gradual rise in blood glucose levels over time. It may therefore be advantageous that the soluble beta-glucan for incorporation into the bread products according to the present invention retains a high weight-average molecular weight in the final bread product for maximum physiological effect. Therefore, in some embodiments, the weight-average molecular weight of soluble beta-glucan in the bread product of the invention is at least 200 kDa, e.g. at least 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, 2050 kDa, 2100 kDa, 2150 kDa, 2200 kDa, 2250 kDa, 2300 kDa, 2350 kDa, 2400 kDa, 2450 kDa, 2500 kDa, 2550 kDa, 2600 kDa, 2650 kDa, 2700 kDa, 2750 kDa, 2800 kDa, 2850 kDa, 2900 kDa, 2950 kDa or at least 3000 kDa. In certain embodiments, the weight-average molecular weight of soluble beta-glucan in the bread product of the invention is equal to or less than 3000 kDa, e.g. equal to or less than 2950 kDa, 2900 kDa, 2850 kDa, 2800 kDa, 2750 kDa, 2700 kDa, 2650 kDa, 2600 kDa, 2550 kDa, 2500 kDa, 2450 kDa, 2400 kDa, 2350 kDa, 2300 kDa, 2250 kDa, 2200 kDa, 2150 kDa, 2100 kDa, 2050 kDa, 2000 kDa, 1950 kDa, 1900 kDa, 1850 kDa, 1800 kDa, 1750 kDa, 1700 kDa, 1650 kDa, 1600 kDa, 1550 kDa, 1500 kDa, 1450 kDa, 1400 kDa, 1350 kDa, 1300 kDa, 1250 kDa, 1200 kDa, 1150 kDa, 1100 kDa, 1050 kDa, 1000 kDa, 950 kDa, 900 kDa, 850 kDa, 800 kDa, 750 kDa, 700 kDa, 650 kDa, 600 kDa, 550 kDa, 500 kDa, 450 kDa, 400 kDa, 350 kDa, 300 kDa, or 250 kDa. Ranges with endpoints formed by the above values are expressly contemplated. In certain embodiment the weight-average molecular weight of soluble beta-glucan in the bread product of the invention is 250 to 950 kDa, e.g. 350 to 850 kDa, 450 to 750 kDa, 550 to 650 kDa or about 600 kDa, or 1000 kDa to 3000 kDa, e.g. 1250 to 2750 kDa, 1500 to 2500 kDa, 1750 to 2250 kDa, or about 2000 kDa .

It will be appreciated by those skilled in the art that soluble beta-glucans may be subject to degradation by enzyme catalysed hydrolysis reactions. For instance, mixed p-(1 ,3)-(1 ,4)-glucans may be selectively hydrolysed by the beta-glucanases. Such enzymes may be present in the ingredients of the bread product of the invention, e.g. the flour and microbial leavening agents, if used. Degradation may also take place as the cooked bread product ages. To allow for degradation of the soluble beta-glucan during the preparation of the bread product of the invention and after cooking, it may be advantageous to select a soluble beta-glucan ingredient for use in the method of the invention which has a weight-average molecular weight which is greater than the desired weight-average molecular weight for the soluble beta-glucan in the final bread product, e.g. is at the upper end of those recited above for incorporation into the dough of use in the method of the invention. Thus, in some embodiments, the soluble beta-glucan ingredient selected for use in the method of the invention may have a weight-average molecular weight of at least 500 kDa, e.g. at least 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, or 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2100 kDa, 2200 kDa, 2300 kDa, 2400 kDa, 2500 kDa, 2600 kDa, 2700 kDa, 2800 kDa, 2900 kDa, or at least 3000 kDa. In certain embodiments, the soluble betaglucan ingredient of use in the method of the invention may have a molecular weight of less than 3000 kDa, e.g. less than 2900 kDa, 2800 kDa, 2700 kDa, 2600 kDa, 2500 kDa, 2400 kDa, 2300 kDa, 2200 kDa, 2100 kDa, 2000 kDa, 1900 kDa, 1800 kDa, 1700 kDa, 1600 kDa, 1500 kDa, 1400 kDa, 1300 kDa, 1200 kDa, 1100 kDa, or 1000 kDa. Ranges with endpoints formed by the above values are expressly contemplated.

The present invention provides a method of producing a leavened wheat bread rich in a soluble dietary fibre, e.g. (1 ,3)-(1 ,4) mixed linkage beta-glucan. More specifically, the bread product comprises about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate. Although the wheat flour, and other ingredients such as yeast, which is/are contained in the bread may comprise soluble dietary fibre the amounts required by the invention require the bread product be supplemented with an exogenous source of soluble dietary fibre. As used herein in the context of the bread product of the invention, the term “supplemented” refers to an increased amount of soluble dietary fibre present in the final bread product as compared to a bread product which is prepared from the same wheat flour in essentially the same way.

The bread product contains about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate. In other embodiments the bread product contains about 6 g, e.g. 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g or 19g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate. In other embodiments the bread product contains about 5g to about 6g, e.g. 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g or 19g of a soluble dietary fibre per 100 g of available carbohydrate. Any ranges which may be formed from any of the above values are expressly contemplated.

Available carbohydrate (CHO) can be measured by any means in the art known to the skilled person. For example, available carbohydrate may be calculated using the methodology specified in Fig. 10 of Brouns et al (2005), Nutrition Research Reviews 18, 145-171. Such a method involves, if such information is not available, determining the amounts of the total starch, resistant starch, disaccharides, monosaccharides and non-digestible sugars in 100g of the food by appropriate analytical methods and calculating the content of available carbohydrate using formula I:

(Total starch x 1.1) - (resistant starch x 1.1) + (total disaccharide x 1.05) + (total monosaccharides) - (non-digestible sugars) = g available CHO/100 g food as eaten

There are standard methods known in the art for measuring total, soluble and insoluble dietary fibre content in a bread product. For example, the enzymatic- gravimetric method together with liquid chromatography may be used (as described in McCleary et al. 2012, Journal of AOAC International Vol. 95, No. 3, 824-844). The bread products to be tested should preferably first be prepared and baked before ca. 50 g of which is grinded in a grinding mill and passed through a 0.5 mm sieve. The baked material to be tested should preferably then subjected to shaking and inversion and stored in the presence of a desiccant. Determination of total, soluble and insoluble dietary fibre content may then be determined following the methods set out in McCleary et al. 2012.

Expressed differently in terms or wt% (%w/w) of available carbohydrate, the available carbohydrate of the bread product of the invention contains about 5% to about 20% w/w of a soluble dietary fibre. In other embodiments available carbohydrate of the bread product of the invention contains about 6 %, e.g. 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19% to about 20 % w/w of a soluble dietary fibre. In other embodiments the available carbohydrate of the bread product of the invention contains about 5% to about 6%, e.g. 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% or 19% w/w of a soluble dietary fibre. Any ranges which may be formed from any of the above values are expressly contemplated.

In certain embodiments the bread product of the invention contains at least 4.0 g, e.g. at least 4.5 g, 5.0 g, 5.5 g, or 6.0 g of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 30 g of available carbohydrate. Thus, in certain embodiments in which the bread product of the invention contains an oat or barley beta-glucan, the bread product meets the EFSA standard for claiming that the product “reduces post-prandial glycaemic response”.

Thus, in some embodiments, the bread product will contain at least 1 g of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 7.5 g of available carbohydrate, at least 2 g of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 15 g of available carbohydrate, 8 g of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 60 g of available carbohydrate, 16 g of a soluble dietary fibre, preferably a soluble beta- glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 120 g of available carbohydrate, or at least 32 g of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal beta-glucan, more specifically an oat or barley beta-glucan) per 240 g of available carbohydrate.

Expressed differently in terms of wt% (%w/w) of available carbohydrate, the available carbohydrate of the bread product of the invention contains at least 13% w/w, at least 14% w/w, at least 15% w/w, at least 16% w/w, at least 17% w/w, at least 18% w/w, at least 19% w/w, or at least 20% w/w of a soluble dietary fibre, preferably a soluble beta-glucan (e.g. a (1 ,3)-(1 ,4) mixed linkage cereal betaglucan, more specifically an oat or barley beta-glucan) .

It has been found that in the prepared bread product of the invention, a portion of the beta-glucan therein will be considered available for physiological extraction and a portion will be considered unavailable. The amount of extractable beta-glucan under simulated physiological conditions can be measured by subjecting the bread product to an in vitro digestion procedures e.g. based on the Infogest protocol (Brodtkorb et al. (2019) “INFOGEST static in vitro simulation of gastrointestinal food digestion” Nature Protocols, volume 14, pages 991-1014). Beta-glucan concentration in the supernatant of the in vitro digestion can be used to calculate the proportion of physiologically available beta-glucan in the total beta-glucan of the bread product. As described in the Examples, beta-glucan in foodstuffs (intact or following simulated physiological extraction) can be measured with commercially available assays, e.g. the Megazyme beta-glucan assay (Megazyme International, Bray, Ireland).

In some embodiments, it may be advantageous to select a soluble beta-glucan which in the bread product of the invention provides a proportion of physiologically available beta-glucan (as a % w/w of total beta-glucan in the bread product of the invention) of at least 30%, e.g. at least 40%, 50%, 60%, 70%, 80% or 90%. Preferably, the average total physiologically available beta-glucan (as a % of total beta-glucan) will be about 70%. The proportion of physiologically available betaglucan (as a % w/w of total beta-glucan in the bread product of the invention) may be determined by any convenient means, e.g. as described above. In other embodiments references to the content of a soluble beta-glucan in the bread product of the invention is the amount of beta-glucan considered available for physiological extraction as determined by the above described approaches.

The wheat flour bread dough comprising a leavening agent provided in step (i) of the method of invention may be prepared at least from a wheat flour, a leavening agent and a water-containing fluid, as described above.

The amount of wheat flour in the wheat flour bread dough will be sufficient to provide a final dough immediately prior to cooking in which at least about 30%, e.g. about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% w/w of the dry/solid ingredients (e.g. flour) in the final dough is wheat flour. In certain embodiments, at least about 75%, e.g. about 80, 85, 90, 95 or 99% w/w of the dry/solid ingredients (e.g. flour) in the wheat flour bread dough of step (i) is wheat flour. Refined wheat flour may be preferred.

The amount of leaving agent in the wheat flour bread dough of step (i) will be sufficient to cause the dough to rise prior to cooking, preferably immediately prior to cooking, in a time selected by the skilled person. Greater amounts of leavening agent may be required for shorter rising/proving times. In certain embodiments the leavening agent is a microorganism, e.g. a sourdough culture or a baker’s yeast, e.g. Saccharomyces cerevisiae. In other embodiments, a sourdough culture leavening agent is not used.

The water-containing fluid may be any aqueous liquid, e.g. water, carbonated water, milk, yoghurt-type fermented milk products, buttermilk, whey, nut milks, and mixtures thereof. In certain embodiments the water-containing fluid is water. The total amount of water-containing fluid will be sufficient to combine the dry ingredients into a pliable mass that retains its shape in an uncooked (raw) state.

The wheat flour bread dough may comprise other non-aqueous ingredients, e.g. flours and brans, in particular refined flours, from other cereals (barley, oat, wheat, rye, sorghum, millet, rice, triticale, maize), non-cereal flours (chickpea, lentil, pea, bean, tuber (potato, cassava, yam), fats (butter, lard, vegetable oils (rapeseed, corn, olive, sunflower seed, groundnut, palm, coconut, almond, sesame), sodium chloride, culinary preservatives (sorbic acid, propionic acid, benzoic acid, citric acid) and/or flavouring or aromatising agents (fruits, nuts, vegetables, chocolate, herbs, spices, non-sugar sweeteners, artificial flavours and artificial aromas). In certain embodiments wheat flour bread dough provided in step (i) does not comprise barley or oat bran.

In one embodiment, a wheat flour bread dough comprising a leavening agent of use in the invention comprises, e.g. consists of, the following ingredients and preferably the relative amounts thereof as shown in Table 1.

Table 1 - Ingredients and amounts thereof (in g) in an embodiment of a wheat flour bread dough comprising a leavening agent of use in the invention.

Ingredient Amount (g)

Refined (white) wheat flour 1000

Water 660

Rapeseed oil 33

Salt 45

Yeast 30

The wheat flour bread dough provided in step (i) may be fully or partially kneaded or unkneaded. As used herein the term “kneading” refers to a process in the production of bread dough that is used to combine the dough ingredients into an essentially uniform mass and give strength to the dough such that it retains its shape in raw form and is capable of producing a crumb once baked. Kneading may be achieved by hand or mechanically, e.g. in a dough mixer (commercial or domestic) equipped with a suitable mixing attachment such as a dough hook, paddle(s), baffle(s), spiral(s), and the like or any combination of these methods. The amount of kneading required for a particular dough will vary depending on the types of ingredients of the dough, the amount of dough, the water absorption capacity of the flour and other dry ingredients, the gluten strength of the wheat flour, the environmental conditions (water temperature, desired end dough temperature) at the time and the strength of kneading (i.e. mechanical energy input from e.g. a dough mixer). Kneading of the wheat flour bread dough of use in the invention by hand may take up to about 30 mins (e.g. at least about 10 to about 15 mins), whereas mechanical kneading may take up to about 10 mins, (e.g. at least about 5 to about 8 mins). The skilled person would be able to judge if the wheat flour bread dough has undergone optimal kneading and will readily appreciate by the skilled person that too much or too little kneading will result in an inferior bread product.

In some embodiments the method may further comprise a step in which the dough provided in step (i) is kneaded prior to combining with the soluble dietary fibre, e.g. in accordance with the conditions recited above.

The wheat flour bread dough provided in step (i) may be fully or partially rested or unrested. As used herein the term “resting” refers to a step in the preparation of a bread dough in which the sufficiently (preferably optimally) kneaded dough is allowed to stand untouched. This allows the macromolecular matrix of the dough to relax and equilibrate and, if a leavening agent, in particular a microbial leavening agent, is present for that agent to produce gas and thus cause the dough to rise. In some instances a resting step may be termed a rising or fermentation step. Where a microbial leavening agent is present, resting typically takes place at a temperature which is optimal for the microorganism growth and respiration state. As an example, this may be about 25 to about 35 °C, e.g. about 26, 27, 28, 29, 30, 31 , 32, 33, 34 to about 35 °C or about 28 °C. Resting preferably takes place at a humidity that prevents drying of the dough but not wetting and/or which is optimal for any microbial leaving agent in the dough. As an example, this may be about 50 % to 80% humidity, e.g. about 55 % to 75%, 60% to 70% or about 60%.

In other embodiments, in particular where a chemical leavening agent is present, resting may take place at chilled conditions to allow the dough to relax without rising. As an example, this may be about 2 to about 10 °C, e.g. about 3, 4, 5, 6, 7, 8, or 9 to about 10 °C or about 4 °C

In some embodiments, the resting time of the wheat flour bread dough is less than about 90 mins, e.g. less than about 80, 70, 60, 55, 50, or 45 mins. In other embodiments, the resting time of the wheat flour bread dough is at least 10 minutes, e.g. at least 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Preferably, the resting time of the wheat flour bread dough is about 45 minutes. The skilled person would be able to judge if the dough has undergone sufficient, preferably optimal, resting/fermentation.

It is an advantage of the invention to perform the method such that the wheat flour bread dough is sufficiently, preferably optimally, rested/fermented because this will result in a final bread product with desirable physical properties and limited degradation (size-reduction) of the soluble dietary fibre. The skilled person would be able to ascertain what resting/fermentation conditions are optimal without undue burden.

In some embodiments the method may further comprise a step in which the dough provided in step (i) is rested prior to combining with the soluble dietary fibre, e.g. in accordance with the conditions recited above.

In some embodiments, the method of the invention comprises a step in which the wheat flour bread dough comprising a leavening agent is prepared from the basic ingredients described above. More specifically the method comprises a step in which the ingredients are provided, combined into a dough, and kneaded and rested sufficiently.

In accordance with the method of the invention, following any kneading and/or resting periods deemed to be required or desired, the wheat flour bread dough comprising a leavening agent is combined with a soluble dietary fibre, preferably a soluble beta glucan. The step of combining the soluble dietary fibre with the wheat flour bread dough may be performed by any convenient means which is able to achieve a soluble dietary fibre supplemented wheat flour bread dough with said soluble dietary fibre distributed therein.

The amount of soluble dietary fibre combined with the wheat flour bread dough will be sufficient to result in a soluble dietary fibre supplemented wheat flour bread dough having about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate. The requisite amount of soluble dietary fibre may be combined as a single measure, or as portions thereof. The soluble dietary fibre may be provided as an isolated, e.g. essentially pure, compound. In other embodiments it may be provided as a partially purified preparation (e.g. a concentrate) from a natural source thereof. In certain embodiments, the soluble dietary fibre, or preparation thereof, is provided in a powder form, e.g. a flour. If provided in a powder form, it may be necessary to combine water, or water containing fluid, e.g. those described above, with the wheat flour bread dough and soluble dietary fibre at substantially the same time. In other embodiments, the soluble dietary fibre supplemented wheat flour bread dough may be combined with water, or water containing fluid, to alter its consistency. In still further embodiments the soluble dietary fibre may be wetted (pre-hydrated) prior to being combined with the wheat flour bread dough. In such embodiments, the soluble dietary fibre, or preparation thereof, may be in the form of a batter, paste, slurry or dough. Two or more of the above approaches may be combined. The skilled person would be able to judge the amount of water, or water containing fluid, required and how best to incorporate it into a soluble dietary fibre supplemented wheat flour bread dough in order to optimise the final bread product.

In some embodiments where a preservative is an ingredient of the bread of the invention, the preservative is added to the water, or water-containing fluid, prior to it being combined with the soluble dietary fibre and/or the wheat flour bread dough and/or soluble dietary fibre supplemented wheat flour bread dough. As discussed above, any suitable culinary preservative could be used including, but not limited to, sorbic acid, propionic acid, benzoic acid, etc.

In certain embodiments, the combining of the soluble dietary fibre with the wheat flour bread dough comprises kneading the wheat flour bread dough in the presence of the soluble dietary fibre, or preparation thereof, until said soluble dietary fibre is distributed in the dough, e.g. reasonably uniformly, although it might not be possible to obtain a homogenous blend. This may be by hand or by mechanical means.

In certain embodiments the soluble dietary fibre is a beta glucan from a cereal, e.g. as discussed above, in particular oat or barley beta glucan. In such embodiments, the beta glucan may be provided as a preparation from cereal grain. Conveniently, this preparation may be a flour or flakes prepared by the milling and fractionation of said grains, preferably fractions thereof which contain the most beta glucan, e.g. the bran (the aleurone, pericarp, and seed coat (testa)). Such grains which undergo milling and fractionation may be heat tempered or non-heat tempered (i.e. kilned or non-kilned) grains or may be ethanol or hexane treated grains.

Thus, in certain embodiments the beta glucan is provided as whole grain oat and/or barley flour or flakes. In other embodiments the beta glucan is provided as oat and/or barley bran flour or flakes. Many oat and/or barley bran flour or flakes are available commercially. Any oat and/or barley bran flour or flakes, concentrates or isolates that contain sufficient concentration of physiologically extractable betaglucan may be used. In certain embodiments SWEOAT™ Brans may be used as the source of beta-glucan. Any of SWEOAT’s standard products may be used, for example, SWEOAT Bran BG14, SWEOAT Bran BG14 Bakery, SWEOAT Bran BG22 or SWEOAT Bran BG28. Other sources of beta-glucan include, but are not limited to, PromOat™ (Lantmannen), Cerebeta™ (GrainFrac Inc.), Glucagel™, and Aurora Oat Beta-glucan™ 20 and Aurora Oat Beta-glucan 10 (Frazer, Finland).

In certain embodiments an amount of beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, sufficient to supplement the wheat flour bread dough to about 5 g to about 20 g of a soluble beta-glucan per 100 g of available carbohydrate, is combined with the wheat flour bread dough in pre-hydrated form, e.g. in the form of a batter, paste, slurry or dough. In such embodiments, prehydration of the beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, involves combining the beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, with the desired amount of water, or water-containing fluid, or a portion thereof (or vice versa) and mixing the combination to a substantially uniform consistency. This may be by hand or mechanically. In certain embodiments the mixing will proceed for at least 1 minute, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In other embodiments, the mixing will proceed for is no more than 60 minutes, e.g. no more than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 minutes. Mechanical mixing typically takes less time than mixing by hand. Any ranges which may be formed from any of the above values are expressly contemplated. Depending on the relative proportion of water, or water-containing fluid, to the betaglucan containing ingredient, e.g. oat and/or barley flour or flakes, this mixing may be considered whisking, whipping and/or kneading and may involving two or more of these mixing procedures depending on the consistency of the mixture as it is mixed and/or as additional water, or water-containing fluid, or the beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, are added. In certain embodiments, the mixing is mechanical and incorporates a grinding action to reduce the size of any larger particles or flakes in the mixture. In other embodiments the beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, are ground to a smaller particle size prior to pre-hydration.

The pre-hydrated beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, e.g. a batter, slurry, paste or dough containing oat and/or barley flour or flakes, may be rested following mixing. As an example, resting may take place at about 25 to about 35 °C, e.g. about 26, 27, 28, 29, 30, 31 , 32, 33, 34 to about 35 °C or about 28 °C. Resting preferably takes place at a humidity that prevents drying but not further wetting. As an example, this may be about 50 % to 80% humidity, e.g. about 55 % to 75%, 60% to 70% or about 60%. In some embodiments, the resting time of the pre-hydrated beta-glucan containing ingredient is at least 10 minutes, e.g. at least 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In other embodiments, the resting time of the pre-hydrated beta-glucan containing ingredient is less than about 90 mins, e.g. less than about 80, 70, 60, 55, 50, or 45 mins. Preferably, the resting time of the wheat flour bread dough is about 30 to 50 minutes, e.g. about 35 minutes or about 45 minutes. The skilled person would be able to judge if the pre-hydrated beta-glucan containing ingredient has undergone sufficient, preferably optimal, resting. Any ranges which may be formed from any of the above values are expressly contemplated.

Combining the wheat flour bread dough with the pre-hydrated beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, for example in the form of a batter, slurry, paste or dough containing the beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, may be by any convenient means. In certain embodiments, the combining comprises kneading the wheat flour bread dough in the presence of the pre-hydrated beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, until the pre-hydrated beta-glucan containing ingredient, e.g. oat and/or barley flour or flakes, more specifically the soluble beta-glucan contained therein, is distributed in the dough. This may be by hand or by mechanical means.

In some embodiments, the kneading time for the step of combining the wheat flour bread dough with the soluble dietary fibre, e.g. pre-hydrated oat and/or barley flour or flakes, is at least 1 minute, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes. In other embodiments, the kneading is no more than 15, e.g. no more than 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 minutes. Any ranges which may be formed from any of the above values are expressly contemplated. Care may need to be taken not to knead the resultant dough for so long that the macromolecular matrix of the dough begins to be undermined and the dough becomes gluey and/or limp, because this will result in suboptimal physical properties in the baked product.

Once the soluble dietary fibre has been combined with wheat flour bread dough, e.g. through kneading, the resultant soluble dietary fibre supplemented wheat flour bread dough may, in certain embodiments be allowed to rest/rise at least once prior to cooking. The final period of resting/rising prior to baking is usually termed “proving”. Prior to the proving step, the dough may be shaped ready for cooking. In certain embodiments, this may comprise placing the dough into one or more baking pans.

As discussed above, in order to retain the maximum physiological effect of soluble dietary fibre, in particular soluble beta-glucan, it is important to minimize degradation during food processing. As such, without wishing to be bound by any particular theory, the two-stage bread making process described herein is potentially advantageous because it reduces the time the soluble dietary fibre, e.g. the soluble beta-glucan, is in contact with other components of the dough and so hydrolysis of the soluble dietary fibre, e.g. soluble beta-glucan, by endogenous enzymes present in other components of the dough (e.g. the flour or yeast) is reduced.

Thus, the skilled person will appreciate that given the possibility of soluble dietary fibre, e.g. soluble beta-glucan, hydrolysis by endogenous enzymes of other components of the dough (e.g. flour or yeast) the resting/proving time of the soluble dietary fibre supplemented wheat flour bread dough should be kept to the minimum required for an adequate rise to give an acceptable crumb. In some embodiments, the resting/proving time of the soluble dietary fibre supplemented wheat flour bread dough is less than about 90 minutes, e.g. less than about 80, 70, 60, 55, 50, or 45 minutes. In other embodiments, the resting/proving time of the soluble dietary fibre supplemented wheat flour bread dough is at least 10 minutes, e.g. at least 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. Any ranges which may be formed from any of the above values are expressly contemplated. Preferably, the resting/proving time of the soluble dietary fibre supplemented wheat flour bread dough is about 45 minutes.

The proving step may be performed about 28 to about 35 °C, e.g. about 29, 30, 31, 32, 33, or 34 to about 35 °C or about 32 °C. Resting preferably takes place at a humidity that prevents drying of the dough but not wetting and/or which is optimal for any microbial leaving agent in the dough. As an example, this may be about 60 % to 85% humidity, e.g. about 65 % to 80 % or about 72%.

In accordance with the invention the soluble dietary fibre supplemented wheat flour bread dough, following any resting/proving periods, is baked at a sufficient temperature and time to produce a crust and a crumb and to raise the internal core temperature to 90-100 °C during cooking. As used herein, the term “crust” refers to the outside surface/outer layer of the bread and the term “crumb” refers to the internal substance of the bread below the crust. The two structures merge with one another. Typically, however, the crumb will be softer than the crust and the crumb will comprise small air pockets which are absent from the crust, and so the crust will have a thickness to it and is visibly distinct from the crumb. The final bread product after cooking will therefore comprise a crust and a crumb.

As used herein, the term “cooking” refers to the process of applying heat, dry or humid, to a foodstuff, or precursor thereof, for a time period sufficient to irreversibly transform the foodstuff. In accordance with the invention the cooking step transforms raw dough into bread.

In accordance with the invention, the term “cooking” extends to baking. In certain embodiments, the baking of the soluble dietary fibre supplemented wheat flour bread dough will comprise baking under dry or semi dry conditions. In some embodiments, the method of the invention may comprise an injection of saturated steam or liquid water at the start of the baking process. Preferably the soluble dietary fibre supplemented wheat flour bread dough is baked, e.g. in an oven. As used herein, the term “oven” is used to refer to a hollow, enclosed chamber with a means of heating said chamber, preferably in a controlled way, or which may be heated by an external heat source. The skilled person will readily be able to select a suitable oven for cooking the dough which allows heating of the oven to a desired temperature. The fuel powering the oven or providing heat to the oven may be electric, gas, oil, wood, peat, or biomass. Preferably, the oven will have good air circulation to enable fast heat transfer and increased removal of water from the dough. It is also preferred that the oven permits rotation of the dough as it cooks. A suitable example of an oven of this type is, for example, a rotating hearth oven (Revent type 626 G EL IAC, Revent international, Vasby, Sweden). Optimal bread quality may be achieved in such ovens because it enables fast heat transfer and removes more water from the dough which gives a better crumb structure (less sticky).

The baking process of the present method will be performed at a temperature and for a time sufficient to raise the internal core temperature of the dough to a temperature of at least about 90 to about 100 °C, e.g. a temperature of at least about 91 , 92, 93, 94, 95, 96, 97, 98 or 99 to about 100 °C, or about 90 to about 91 , 92, 93, 94, 95, 96, 97, 98 or 99 °C. Any ranges which may be formed from any of the above values are expressly contemplated. In other embodiments the internal core temperature of the dough is raised to a temperature of about 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C or 100 °C, during cooking. Preferably, the internal core temperature is raised to about 96 °C during cooking. These temperatures may be mean average temperatures of several different locations from the interior of the baked dough.

Any suitable method known in the art may be used to measure the internal temperature of the dough continuously during cooking or at intervals, for example, a food thermometer or in situ temperature probe may be used.

In some embodiments, the cooking temperature in the oven may be, for example, about 150 to about 300 °C, e.g. about 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290°C to about 300 °C, or about 150 to about 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290°C. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. 160-170 °C, 170-180 °C, 180-190 °C, 190-200 °C, 200-210 °C, 210-220 °C, 220-230 °C, 230-240 °C, 240-250 °C, 250-260 °C, 260-270 °C, 270-280 °C, 280-290 °C, 290- 300 °C. In some embodiments, the cooking temperature is 210-290 °C, preferably 240-280 °C and, more preferably, 250-270 °C.

The cooking time may be, for example, about 20 to about 240 minutes, e.g. about 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 210, 220 or 230 to about 240 minutes, or about 20 to about 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 210, 220 or 230 minutes. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. 30-40 minutes, 40- 50 minutes, 50-60 minutes, 60-70 minutes, 70-80 minutes, 80-90 minutes, 100-110 minutes, 120-130 minutes, 130-140 minutes, 150-160 minutes, 160-170 minutes, 170-180 minutes, 180-190 minutes, 190-200 minutes, 200-210 minutes, 210-220 minutes, 220-230 minutes, 230-240 minutes.

It will be readily appreciated by the skilled person that any appropriate combination of temperature and time for cooking may be selected to produce a desired baked dough product with the desired internal core temperature. For instance, the cooking time might be reduced if baking takes places at a higher temperature, and vice versa. The skilled person would be able to judge or determine what a suitable temperature and time would be for the bread product being produced. Since the cooking time is dependent on the weight of the dough portions that are baked, it will also be readily appreciated that the cooking times may need adjusting based on the weight of the dough portions that are to be baked.

In accordance with the method of the invention the baked dough is cooled to ambient temperature and the cooled product is a leavened wheat bread product comprising about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate. This cooling period/step comprises at least one vacuum cooling step. Thus in some embodiments the baked dough may also be allowed to reduce in temperature without vacuum cooling for a period time in addition to the cooling which occurs under vacuum. Other cooling techniques, e.g. fan assisted, refrigeration, or chilled gas streams, might be used. In certain embodiments substantially, e.g. essentially, all of the cooling period is under vacuum, i.e. the cooling period is entirely a vacuum cooling step. In other embodiments the vacuum cooling portion of the cooling period/step commences as soon as practical after the baking of the dough is completed. In certain embodiments the baked dough may be subjected to vacuum cooling in the same vessel as baking has taken place. More typically, the baked dough may be removed from where it has been baked and transferred to a vacuum cooling apparatus and subjected to vacuum cooling. It will be appreciated by the skilled person that in such embodiments, “as soon as practical” allows for a short period of non-vacuum cooling between the completion of baking and the commencement of vacuum cooling.

In still further embodiments a final short period of non-vacuum cooling occurs after the baked dough has been cooled under vacuum. This might occur in embodiments where the vacuum cooling takes the temperature of the baked dough to slightly above ambient temperature. In other embodiments this might occur where the vacuum cooling takes the temperature of the baked dough to ambient temperature and the non-vacuum cooling step chills the baked dough to below ambient temperature.

Vacuum cooling is a well-established food processing technique widely adopted in perishable produce packaging lines, e.g. fruits vegetable and salad, but not in the production of baked goods. The technique cools water-containing items, potentially rapidly, by reducing, typically significantly, the atmospheric pressure surrounding the item and thereby causing the moisture present in the item to evaporate and thermal energy to be lost. As well as efficient cooling, the vacuum cooling step can therefore additionally aid the evaporation of (excess) water from the item, e.g. the baked dough as described herein.

The vacuum cooling step of the invention can take place in any suitable appliance comprising an airtight chamber of a size suitable to accept the baked dough operatively connected to a vacuum pump. The vacuum pump will act to remove air from the inside of the chamber to reduce air pressure in the chamber, e.g. create a vacuum. Such apparatus are commercially available from, for example, Cetravac AG, Vacuum Cooling & Baking Solutions, Huaxian Fresh Care Technology and WEBER cooling. In preferred embodiments, the vacuum cooler is from Cetravac AG and, more preferably, is the LAB-5x600x400 - 200 K1-G21 model.

In some embodiments, at the beginning of the vacuum cooling step, the pressure in the chamber may be in the region of 900-1100 millibar (mbar), i.e. about standard atmospheric pressure. The pressure in the chamber, i.e. the pressure to which the cooked dough is exposed during the vacuum cooling step, is then lowered, typically gradually, e.g. by the action of the vacuum pump. In some embodiments, during the vacuum cooling step the pressure is lowered to (i.e. the baked dough is exposed to a pressure of) about 600 mbar or less, e.g. to about 500 mbar, 400 mbar, 300 mbar, 200 mbar, 100 mbar, or 50 mbar or less. In other embodiments the pressure is lowered to no less than about 50 mbar, e.g. no less than about 100 mbar, 200 mbar, 300 mbar, 400 mbar, 500 mbar, or 600 mbar. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. about 100 mbar to about 300 mbar, e.g. about 200 mbar, or about 50 mbar to about 100 mbar, e.g. about 80 mbar.

In some embodiments, at the beginning of the vacuum cooling step, the pressure in the chamber may be in the region of 90-110 Pascal (kPa), i.e. about standard atmospheric pressure. The pressure in the chamber, i.e. the pressure to which the cooked dough is exposed during the vacuum cooling step, is then lowered, typically gradually, e.g. by the action of the vacuum pump. In some embodiments, during the vacuum cooling step the pressure is lowered to (i.e. the baked dough is exposed to a pressure of) about 60 kPa or less, e.g. to about 50 kPa, 40 kPa, 30 kPa, 20 kPa, 10 kPa, or 5 kPa or less. In other embodiments the pressure is lowered to no less than about 5 kPa, e.g. no less than about 10 kPa, 20 kPa, 30 kPa, 40 kPa, 50 kPa, or 60 kPa. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. about 10 kPa to about 30 kPa, e.g. about 20 kPa, or about 5 kPa to about 10 kPa, e.g. about 8 kPa..

The reduction in pressure to the above values may be a continuous and constant rate of change. In other embodiments the pressure may be reduced continuously to said values but at varying rates. In other embodiments the pressure may be held at a certain level for distinct periods of time, e.g. levels which differ by about 20 to about 100 mbar (about 2 to about 10 kPa). In some embodiments, the pressure difference is about 20 to about 100 mbar in distinct steps of 10-120 seconds, i.e. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 seconds, preferably in distinct steps of 20 or 30 seconds. The individual time steps may be of the same length or may be of differing lengths. For example, the time steps may be a combination of 20 second steps and 30 second steps, etc.

In certain embodiments the above mentioned pressures are the pressure to which the cooked dough is exposed immediately prior to full release of the vacuum so created. In other embodiments, the above mentioned pressures are the minimum (lowest) pressure to which the cooked dough is exposed, but exposure to such minimum pressures might be transient during the vacuum cooling step. The terms “during the vacuum cooling step”, ’’over the course of the vacuum cooling step”, and “for the duration of the vacuum cooling step” should be construed accordingly.

In accordance with the invention, the internal core temperature of the dough after baking will be 90-100 °C, i.e. 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C or 100 °C, thus the temperature of the baked dough at the start of the vacuum cooling step may, in certain embodiments, be such temperatures, or lower. The temperature of the baked dough may be lowered by at least about 10 °C, e.g. at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 95 °C over the course of the vacuum cooling step. In certain embodiments the internal core temperature of the baked dough at the cessation of the vacuum cooling step may be, for example, less than 70°C, e.g. less than about 50 °C, 40 °C, 30 °C, 20 °C, or 10 °C. In certain embodiments the internal temperature of the baked dough at the cessation of the vacuum cooling step may be, for example, greater than about 5 °C, e.g. greater than about 10 °C, 20 °C, 30 °C, 40 °C, 50 °C or 60 °C. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. about 20 to about 50 °C, about 30 to about 45 °C, about 40 to about 45 °C and about 42 °C. These temperatures may be mean average temperatures of several different locations from the interior of the baked dough.

It will be readily appreciated by the skilled person that the time required for the vacuum cooling step will vary depending on the starting pressure, the pressure reduction rate/steps to give rise to the final or minimum desired pressure in the chamber and the starting temperature and desired end temperature of the product after the vacuum cooling step. Other factors such as the power of the vacuum pump, size of the chamber, volume of air in the chamber, size of the pump, dead volume, final temperature and water content of the bread will also influence the time required for the vacuum cooling step.

In certain embodiments, the time required for the vacuum cooling step will be at least about 1 minute, e.g. at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50 or 60 minutes. In certain embodiments, the time required for the vacuum cooling step will be no greater than about 2 minutes, e.g. no greater than about 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50 or 60 minutes. Any ranges which may be formed from any of the above values are expressly contemplated, e.g. about 1 to about 60 minutes, about 1 to about 30 minutes, about 5 to about 15 minutes, about 6 to about 12 minutes or about 6 minutes.

In a further aspect, the invention also provides a leavened wheat bread product comprising about 5 g to about 20 g of a soluble dietary fibre per 100 g of available carbohydrate obtained or obtainable by the method disclosed herein.

The soluble dietary fibre supplemented leavened wheat bread products of the invention have a crumb and crust structure which is closely comparable to a typical (non-supplemented) wheat bread product prepared in essentially the same way (i.e. without the step of supplementation and/or without the vacuum cooling step). More specifically one or more of loaf volume, slice area, crumb texture, crumb structure, proportion of gas cells, moisture content, crumb colour, crust texture and crust structure of the soluble dietary fibre supplemented leavened wheat bread product of the invention is comparable to a typical (non-supplemented) wheat bread product prepared in essentially the same way, or is at least improved as compared to leavened wheat bread products that are considered to be low-medium Gl foodstuffs, and in particular to meet the EFSA health claim criteria for reduction of postprandial glycaemic response.

In certain embodiments the leavened wheat bread product of the invention may have a specific loaf volume of at least 1.85 ml/g, e.g. at least 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 3.00, 3.50 or at least 4.00, preferably as measured by laser topography, e.g. with a BVM 6630 vol meter (Perten Instruments, Hagersten, Sweden). For tin baked products a specific loaf volume of at least 1.85 ml/g, e.g. at least 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.05, 3.10, 3.15, or 3.20 may be preferred.

In certain embodiments the leavened wheat bread product of the invention may have a crumb firmness, expressed as the force in grams at 25% compression of 1 x 25 mm slice of bread, of less than 1050g, e.g. less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250 or 200 g, preferably as measured according to the AACC 74-09 method (AACC Approved Methods of Analysis, 1999) using a TA XT Plus Texture Analyzer (Stable Micro Systems Ltd, Godaiming, UK) fitted with a 5 kg load cell and a 35 mm diameter aluminium probe.

In certain embodiments the leavened wheat bread product of the invention may have a moisture content of less than 53 wt %, e.g. less than 52, 51, 50, 49, 48, 47, 46,45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30 wt %, preferably as measured by weighing before and after infrared drying.

In certain embodiments the leavened wheat bread product of the invention may have a slice brightness (crumb colour) on the monochrome 256 greyscale of greater than 102, e.g. greater than 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140 or 150 as measured by a C-cell imagining system (Calibre Control International Ltd., Warrington, UK) in slices of 25 mm thickness.

In certain embodiments the leavened wheat bread product of the invention may have bread slice crumb structure having a gas cell area of at least 48.5 %, e.g. at least 48.6, 48.7, 48.8, 48.9, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5, 52.0 , 52.5, or 53.0 % preferably as measured by a C-cell imagining system in slices of 25 mm thickness.

In other embodiments the soluble dietary fibre supplemented leavened wheat bread products of the invention have a crumb and crust structure which is improved as compared to a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step. More specifically one or more of loaf volume, slice area, crumb texture, crumb structure, proportion of gas cells, moisture content, crumb colour, crust texture and crust structure of the soluble dietary fibre supplemented leavened wheat bread product of the invention may be improved as compared to a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step.

In these embodiments, the soluble dietary fibre supplemented leavened wheat bread product of the invention may have a specific loaf volume which is increased by at least 5%, e.g. at least 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15%, as compared to that of a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step.

In these embodiments, the soluble dietary fibre supplemented leavened wheat bread product of the invention may have a crumb firmness, expressed as the force in grams at 25% compression of 1 x 25 mm slice of bread, which is less than 70%, e.g. less than 65, 60, 55, 50, 45, 40, 35, or 30% of that of a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step.

In these embodiments, the soluble dietary fibre supplemented leavened wheat bread product of the invention may have a moisture content which is less than 96%, e.g. less than 95, 94, 93, 92, 91 , or 90% of that of a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step.

In these embodiments, the soluble dietary fibre supplemented leavened wheat bread product of the invention may have a slice brightness (crumb colour) on the monochrome 256 greyscale which is increased by at least 2%, e.g. at least 3, 4, 5, 6, 7, 8, 9, or 10%, as compared to that of a soluble dietary fibre supplemented wheat bread product of the same recipe which has been prepared in the same way but without the vacuum cooling step.

The bread of the present invention is designed to have an advantageous glycaemic profile, e.g. a peak blood glucose rise, an incremental area under the blood glucose response curve above fasting baseline (iAUC) and/or a Gl which is significantly lower than that of white bread, and/or promote a reduced post-prandial glycaemic response. It is routine for the skilled person to determine these parameters. In some embodiments the iAUC may be calculated using the standard trapezoid geometric method (Wolever, T.M.S. 2006, The Glycaemic Index: A Physiological Classification of Dietary Carbohydrate. CABI Publishing: Wallingford, UK, pp 120- 123). Peak blood glucose rise (PBGR) may then be calculated as the difference between a subject’s fasting glucose value and the peak value follow consumption of the test foodstuff. Gl may be calculated by expressing the iAUC for a test food in a subject as a percentage of the same subject’s mean reference (glucose) iAUC (Wolever, T.M.S. 2013, European Journal of Clinical Nutrition, 67, 522-531).

In certain embodiments the leavened wheat bread product of the invention is low Gl, e.g. has a Gl of less than 55. In other embodiments the leavened wheat bread product of the invention has a medium Gl (55-70). Low-medium Gl thus means a Gl or less than 70.

Complex metabolic conditions associated with the over-consumption of available carbohydrate e.g. starch, glucose and/or sucrose and/or inappropriate metabolism of glucose, e.g. metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis are well known, however successful treatment thereof has remained elusive. It has now surprisingly been found that simply reducing a subject’s dietary intake of available carbohydrate does not effectively treat these conditions but that a more fruitful approach is to provide these subjects with a food-substitute having a more favourable glycaemic profile. Given the fact that bread is a widely consumed food staple, the ability to produce breads with favourable glycaemic profiles, e.g. a leavened wheat bread product of the invention, can be of great benefit to public health.

The leavened wheat bread product of the invention is therefore useful as part of a healthy diet in healthy subjects, as well as in treating subjects with, or at risk of developing, complex metabolic disorders associated with the over-consumption of glucose and/or sucrose and/or inappropriate metabolism of glucose including metabolic syndrome, diabetes, obesity, dyslipidaemia, insulin resistance, hypertension and liver steatosis. Thus, in a further aspect the invention provides a method of assisting in maintaining the health and well-being of a subject or for maintaining or promoting health and well-being in a subject, said method comprising consuming a leavened wheat bread product of the invention. The use of the leavened wheat bread products of the invention in such methods is contemplated as is the use of the leavened wheat bread products of the invention in the manufacture of a nutraceutical or foodsubstitute for use in such methods.

In a further aspect, the invention provides a method for the treatment or prevention of a disease or condition associated with the over-consumption of glucose or starch and/or inappropriate metabolism of glucose, said method comprising administering a leavened wheat bread product of the invention to a subject on a calorie-controlled diet.

Expressed differently, the invention provides a leavened wheat bread product of the invention for use in the treatment or prevention of a disease or condition associated with the over-consumption of glucose or starch and/or inappropriate metabolism of glucose in a subject on a calorie-controlled diet.

Expressed differently, the invention provides for the use of a leavened wheat bread product of the invention in the manufacture of a medicinal product for use in the treatment or prevention of a disease or condition associated with the over- consumption of glucose or starch and/or inappropriate metabolism of glucose in a subject on a calorie-controlled diet.

A calorie-controlled diet is a diet which permits a subject to consume a defined number of calories per day, typically this will be a calorie-restricted diet that permits the subject to consume a number of calories per day that is fewer than the number the subject consumed before adopting the diet. This may be fewer than the number of calories recommended by the skilled practitioner for the average subject or a subject of equivalent body proportions. Preferably the diet will be sugar- controlled/sugar-restricted, in particular will be glucose- and/or sucrose- controlled/restricted, which terms should be interpreted as for calorie-controlled and calorie restricted. The disease or condition associated with the over-consumption of glucose and/or inappropriate metabolism of glucose may be selected from metabolic syndrome, diabetes mellitus type II, obesity, coronary heart disease, abdominal obesity, dyslipidaemia, insulin resistance, hyperinsulinemia, impaired glucose metabolism, hypertension, liver steatosis, steatohepatitis, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, pancreatitis, neurodegenerative disease, retinopathy, nephropathy and neuropathy.

"Treatment" when used in relation to the treatment of a medical condition in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect on the condition or in relation to the condition. Thus, not only included is eradication or elimination of the condition, or cure of the subject, but also any improvement in the condition. Thus included for example, is an improvement in any symptom or sign of the condition, or in any clinically accepted indicator of the condition (for example, an improvement in the metabolism of glucose). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed condition, i.e. a reactionary treatment.

"Prevention" as used herein refers to any prophylactic or preventative effect. It thus includes delaying, limiting, reducing or preventing the condition or the onset of the condition, or one or more symptoms or indications thereof, for example relative to the condition or symptom or indication prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom or indication thereof, and any delay in the onset or development of the condition or symptom or indication, or reduction or limitation on the development or progression of the condition or symptom or indication.

The subject may be any human or non-human animal subject, e.g. a non-human mammal. Preferably the subject is a human, especially a human suffering from or at risk of developing a disease or conditions recited herein, in other words a human subject in need of the treatments disclosed herein. The invention will be further described with reference to the following non-limiting Examples in which:

Figure 1 shows A) the real time output of an internal temperature probe which monitored the internal temperature (°C) of six bread loaves prepared as described in Example 1 (three cooled with vacuum cooling and three cooled without vacuum cooling) against time (seconds); and B) the vacuum cooling program used for cooling where pressure (mbar) is plotted against time (s).

EXAMPLES

Example 1 -Recipe for Beta-glucan enriched (Oatbran 14) Bread

Table 2 - Dough ingredients and weights thereof (in g) for Oatbran 14 Bread

As a first step a first dough (i.e. the main dough) was prepared as below.

Water temp: 14-18 °C

Flour temp: 20-23 °C

Kneading time: 4min at speed 1 then until dough reaches 26.5 °C at speed 2

Rest time: 45min, 28 °C and 60% humidity

All ingredients were mixed together. For the smaller batch (using 3kg wheat flour - column to the left in the above table), a Diosna sp 12 spiral mixer was used. For this the low speed (speed 1) was at 30 Hz, while the high speed (speed 2) corresponded to 40 Hz. The dough was first mixed at low speed for 4 min and then mixed again at speed 2 (40Hz) until the dough temperature reached 26.5 °C. The second mixing step lasted for approximately 10 min and the final energy input into the dough was around 230 kJ.

The larger batch of wheat flour dough (approx. 70kg in total - column to the right in the above table), was prepared in a commercial bakery and a control of energy input was not possible. Instead the final dough temperature was used to achieve equivalent mixing and energy input.

A second “dough” (which comprises an oat bran mix) was prepared as below. The dough referred to here was a very loose dough and may also be described as a paste, batter or slurry.

When preparing the second dough, the preservative (sorbic acid) is added to the additional water prior to mixing in the oat mix (Oatbran 14).

Water temp: 24 °C

Flour temp: 20 °C

Kneading time: 2min using a beater in a planetary mixer at speed 2

Rest time: ca. 35min

A Bear Planetary Varimixer was used for the kneading step at speed 2 (where speed 2 corresponds to this speed setting on the Bear Planetary Varimixer, which is 40 Hz).

The second dough is rested (as described above) concurrently with the first dough’s resting phase and under the same conditions. The two doughs are kept separate at this point. The consistency of the second dough changes during resting and becomes more plastic.

After resting the first and second doughs are combined and kneaded until a uniform dough is obtained. This resultant combined dough is then divided into 850 g pieces and formed (either by hand or by machine) and then allowed rise in steel loaf pans. The combined dough is allowed to rise/prove for 45 min at 32 °C and at 72% humidity.

Following the rising period the dough is baked according to a standard method or vacuum method as discussed below.

Baking - oven (standard method) For the standard method, the risen dough was baked in an oven set at 240 °C which was immediately reduced to 220 °C when the dough was put into the oven. The dough was then baked for 45 minutes with steam (0.5 L of water) being injected during the first 10s. A rotating hearth oven with fan-assisted air circulation, specifically the Revent type 626 G EL IAC model (Revent International, Vasby, Sweden), was used.

Baking - oven followed by vacuum cooler step (vacuum method)

For the vacuum method, the resultant dough was baked in a rotating hearth oven with fan-assisted air circulation oven (Revent type 626 G EL IAC model) set at 270 °C which was immediately reduced to 250 °C when the dough was put into the oven. The dough was then baked for 30 minutes with steam (0.5 L of water) being injected during the first 10s. The internal core temperature of the bread was raised to 96 °C during cooking. The internal core temperature was taken upon removal of the bread from the oven. The baking time was chosen to achieve the desired internal temperature of 96 °C during the cooking process.

Vacuum cooling

After completion of the baking period, the bread was immediately subjected to a cooling step using a vacuum cooler (Cetravac AG, model LAB-5x600x400 - 200 K1- G21). The cooked bread was transferred immediately to the vacuum cooling apparatus and the pressure in the vacuum cooler starting at atmospheric pressure (not measured, but around 1000 mbar) was reduced to a pressure of 80 mbar in 5 minutes. The cooling program as shown in Table 3 was used.

Table 3 - Cooling program used for vacuum cooling (1 mbar = 0.1 kPa) Upon completion of the 6 minute cooling period pressure had returned to atmospheric. The temperature of the bread immediately following the 6 minute cooling period was measured as 42 °C using a hand held temperature probe following removal from the cooling apparatus. The internal core temperatures during baking and cooling can also be monitored in real time using temperature probes/loggers placed inside the bread. The results of other baking experiments using such methods are shown in Figure 1 where vacuum cooling results in more rapid cooling compared to cooling without using a vacuum. Example 2 - Recipe for Beta-glucan enriched (PromOat) Bread

Dough ingredients

The first dough was prepared as described in Example 1 for the Oatbran 14 bread.

When preparing the second dough, the preservative (sorbic acid) is added to the flour prior to mixing with PromOat. The water is added to the bottom of the mixing bowl first before the addition of PromOat.

Water temp: 24 °C

Flour temp: 20 °C

Kneading time: 2min using a beater in a planetary mixer at speed 2

Rest time: ca. 45min

A Bear Planetary Varimixer was used for the kneading step at speed 2.

The second dough is rested (as described above) concurrently with the first dough’s resting phase and under the same conditions. The two doughs are kept separate at this point. After resting the two doughs are combined and kneaded into a uniform dough. All further steps were identical to Example 1 for the Oatbran 14 bread. Example 3 - Comparison of breads produced by standard method vs vacuum method

For the comparison studies of the Oatbran 14 breads baking was done twice for both the standard method and the vacuum method on two randomised days. For the comparison studies of the PromOat breads, baking was only performed once on the same day and the two breads measured for both the vacuum and the standard method are from the same dough batch.

Various parameters were then assessed in the bread products resulting from either the standard method or the vacuum method and the results from each method were compared.

All data presented below are from breads prepared with 3kg wheat flour (as described in Example 1 and 2) using the Diosna sp 12 spiral mixer for the first dough (wheat flour dough) and a Bear planetary Varimixer for the second dough (Oatbran 14 dough or PromOat dough).

Specific volume

The day after baking the test bread products, the volume of two (PromOat) or three (Oatbran 14) replicate loaves were determined by laser topography using a BVM 6630 vol meter (Perten Instruments, Hagersten, Sweden). Weight was determined on weighing scales. The specific volume (ml/g) was calculated from the weight and volume measurements. Analysis of mean difference by analysis of variance at a set 95% significance level. The greater the volume, the better product quality.

Table 4 - Specific volume (in ml/g) results for bread produced by the standard and vacuum methods.

Oatbran specific PromOat ^s P ^ec 'f' ^c

14 volume volume bread (ml/g)

N average st.dev. N average st.dev.

Standard method 6 1.84 0.04 2 2.84 0.02

Vacuum method 6 2.06 0.05 2 3.15 0.07 The loaf volume is significantly greater for vacuum method compared to the standard method for Oatbran 14 bread (p<0.001) and PromOat bread (p<0.05).

Crumb firmness

Bread crumb firmness, expressed as the force in grams at 25% compression of 1 x 25 mm slice of bread, was measured according to the AACC 74-09 method (AACC Approved Methods of Analysis, 1999) using a TA XT Plus Texture Analyzer (Stable Micro Systems Ltd, Godaiming, UK) fitted with a 5 kg load cell and a 35 mm diameter aluminium probe. Firmness may be taken as a measure of freshness and quality. The softer the product the better the quality.

Table 5 - Crumb firmness (in g) results for bread produced by the standard and vacuum methods.

„ .. Crumb „ . Crumb

Oatbran 14 ,. PromOat ,.

. . firmness . . firmness bread , . bread , .

(g) (g)

N average st.dev. N average st.dev.

Vacuum method

Standard _{6 10} 84 _{143 9} 9 _{6 413 52} method

The bread crumb firmness was significantly (P<0.001) lower (softer) for the vacuum cooled bread than the standard method for both Oatbran 14 and PromOat.

Moisture content

For the Oatbran 14 bread, moisture content (Wt. %) was determined by freeze drying followed by infrared drying and weighing before and after drying. For infrared drying, breadcrumbs were prepared using a standard kitchen food processor using a knife attachment and 5 g thereof were added to an aluminium tray and this was then placed inside an infrared dryer (Sartorius Moisture Analyser YTC01 L (Satorius AG, Gottingen, Germany)). The total weight was then recorded at time zero and the dryer was switched on. The dryer proceeded to dry the breadcrumbs whilst measuring the change in weight in real time. The percentage moisture loss during the drying process was calculated by the dryer from these data (see Table 6A). The moisture content values for the Oatbran 14 bread products are the sum of moisture content as determined from the freeze-drying and infrared drying steps.

For the PromOat bread, the moisture content was determined by freeze-drying 4 times 3 slices of bread and the moisture is expressed as weight loss during freeze- drying (see Table 6B). The moisture content values given for the PromOat bread products are based on a freeze-drying step only.

Table 6A - Moisture content (in Wt. %) results for Oatbran 14 bread produced by the standard and vacuum methods.

Oatbran Moisture

14 (Wt. %)

N average st.dev.

Vacuum method 4 50.4 1.1

Standard method 4 53.7 1.7

Table 6B - Moisture content (in Wt. %) results for PromOat bread produced by the standard and vacuum methods.

_. - . Moisture

^PromOat (Wt. %)

N average st.dev.

^V m ^a e ^C _t thh ^U oHd ⁴ 49.3* ^{0 3}

Standard . _

.. . 4 51.5* 0.5 _c method

*these values do not take into account the bound moisture that remains after freeze drying (Wt. % moisture values may therefore be 5-10% higher than measured).

Vacuum cooling results in significantly less moisture (P<0.006) in the breadcrumbs and hence in the intact bread products for both the Oatbran 14 and PromOat. This is explained by an increase in the removal of water by vacuum cooling compared to standard oven treatment. Extractable beta-glucan as a percentage of total beta-glucan

The test bread products were subjected to a simulated static in vitro digestion as described by Brodtkorb et al. (2019) “INFOGEST static in vitro simulation of gastrointestinal food digestion” Nature Protocols, volume 14, pages 991-1014. A total amount of 2 g bread crumbs per tube were digested in duplicate as previously described (Rieder, Knutsen, & Ballance, 2017, Food Hydrocolloids 67, 74-84). After digestion, enzymes were inactivated in a boiling water bath for 10 min. Tubes were cooled and centrifuged at 4000 x g for 10 min. The supernatants were aliquoted for determination of extractable beta-glucan content via the Megazyme beta-glucan assay (Megazyme Mixed-Link Beta-Glucan Assay Procedure - AACC Method 32- 23.01 AOAC Method 995.16 EBC Methods 3.10.1 , 4.16.1 and 8.13.1 ICC Standard Method No. 166 Codex Type II Method). Total beta glucan in the starting bread product is also measured using the Megazyme beta-glucan assay. A greater proportion of extractable beta-glucan (in vitro simulation of physiological extraction) can be associated with its increased physiological efficacy in the small intestine.

Table 7 - Results for extractable beta-glucan level (expressed as % total betaglucan) for Oatbran 14 bread produced by the standard and vacuum methods.

Extractable beta-glucan (% total betaglucan)

N average st.dev.

Vacuum method 4 70.7 4.5

Standard method 4 62.1 1.75

Vacuum cooling results in a significantly (p<0-018) greater percentage of soluble beta-glucan in the Oatbran 14 bread as evaluated by simulated static in vitro digestion than compared to the standard baking process.

Slice area

The crumb structure of bread slices (25 mm) was analysed by using a C-cell imaging system (Calibre Control International Ltd., Warrington, UK). The larger the slice area (mm ² ), the better product quality. Table 8 - Slice area (in mm ² ) results for bread produced by the standard and vacuum methods.

Oatbran Slice Slice

14 Area(mm ² ) Area(mm ² )

N average st.dev. N average st.dev.

^Vac u ^u m ₅ 7664.0 50.3 4 11082.8 387 method s ^tan d ^a rd ₇ 7098.0 108.90 7 10375.3 410 method

Vacuum cooling results in a significantly larger slice area for Oatbran 14 (p<0.001) and PromOat (p<0.05) breads compared to the respective standard method.

Slice brightness

Crumb brightness of bread slices (25 mm), as measured on the monochrome 256 greyscale, was analysed by using a C-cell imaging system (Calibre Control International Ltd., Warrington, UK). The brighter the slice, the better product quality.

Table 9 - Slice brightness results for bread produced by the standard and vacuum methods.

Oatbran Slice Slice

14 Brightness Brightness st.dev st.d

N average N average _ev

^V m ^a ethho ^U IdT ^{5 105 8 1 9 4 124 6 1 7}

Standard _{7 101 2 1 30 7 1 17 9 3 5} method

Vacuum cooling results in a significantly (p<0.001) brighter slice compared to the standard method for both the Oatbran 14 and PromOat breads.

Area of cells and cell/cm ² of bread slice Crumb structure of bread slices (25 mm) was analysed by using a C-cell imaging system (Calibre Control International Ltd., Warrington, UK). The larger the % cell area, the better product quality. Table 10 - % cell area results for bread produced by the standard and vacuum methods.

Area of Area of

Oatbran 14 cells PromOat cells

(%) (%)

N average st.dev. N average st.dev.

^V m ^a ethho ^U IdT ^{5 49 9 0 5 4 52 7 0 7 sta} ndard _{7 48 3 0} .44 7 54.5 1.2 method

For Oatbran 14 breads, vacuum cooling results in a significantly (p<0.001) greater percentage area occupied by cells compared to the standard method. For PromOat breads, this was not the case and instead a greater percentage area was occupied by cells for breads prepared by the standard method. This is due to a difference in crumb structure. The PromOat breads were much airier and more porous compared to the Oatbran 14 breads. Thus, vacuum cooling of the PromOat breads lead to a greater number of smaller gas cells compared to the standard method.

Oatbran 14 Cells/cm ² PromOat Cells/cm ²

N average st.dev. N average st.dev.

^V m ^a ethho ^U IdT ^{5 56 9 3 4 47 6 4 6}

Standard _{7 60 5 2} .5 7 39.1 2.5 method