Field of the invention

The present invention relates to flour made from fermented grains, beans, legumes or the like. More particularly, the invention relates to a method of making flour by grinding or milling tempeh- fermented substrates that have been cooked, acid extracted and/or dried at low temperature.

Background of the invention

Tempeh is a traditional meat alternative produced in Indonesia since the 1700s. Tempeh is commonly made using solid-state fermentation of soybeans, peanuts, or other bean curds. Tempeh fermentation transforms raw materials such as beans, grains and legumes into a solid food with the help of fungi such as Rhizopus spp. The fermentation results in favourable properties as a food, for example meat-like texture, desirable unique taste, higher protein content, high protein and fat digestibility, and elimination of anti-nutrient phytate which can interfere with iron absorption in human body. By fermenting affordable ingredients like soybeans into tempeh, a tastier, more versatile, safer, and healthier protein source can be obtained sustainably.

Tempeh fermentation can increase the nutritional and sensory values of the food, for example by increasing the protein and fibre contents as well as decreasing the anti-nutritive content of the fermented substrates. Tempeh fermentation can also modify the emulsifying capacity and antioxidative activity of the substrate foods.

KR100907659B1 (Ha Mi Sook) describes a method for preparing bean paste using fermented barley powder, which enables the taste to be easily adjusted according to preference without the use of pigments and preservatives while showing high degree of nutrients. WO-A-98/56262 (Kikue) describes a brewed product prepared by adding malted rice to boiled beans and/or steamed beans followed by the fermentation and aging of the resultant mixture, and foods and beverages prepared therefrom. Leite et al (Food Sci. Technol, Campinas, 33(4): 796-800, Oct.- Dec. 2013) describes the use of tempeh flour as a substitute for soybean flour in coconut cookies.

However, the potential of the use of tempeh fermentation in the production of functional food ingredients has to date been limited by its bitterness and low solubility in water, especially if it is in powder form such as a flour.

There is a need to utilize the benefits of tempeh not only as a food itself, but as an ingredient in other foods. Summary of the invention

The present invention relates to flour produced from a foodstuff that has been fermented with a mycelium-producing microorganism, for example Rhizopus spp. The foodstuff to be fermented typically comprises grains, beans and/or legumes. The fermented foodstuff is typically tempeh.

Tempeh production typically comprises soaking, cooking, inoculating with a mycelium-producing microorganism such as Rhizopus spp., incubating, and drying fermentable food substrates such as grains, beans, and/or legumes. To produce flour, the product of this process is then crushed, chopped, ground, and/or milled.

The inventors have observed that tempeh flour can be improved by applying high temperature (i.e. cooking or blanching, or steaming) to the tempeh prior to milling. The inventors have also observed that performing acid extraction on the tempeh prior to milling improves the flour. Yet further, the inventors have observed that it is advantageous to dry the tempeh at low temperatures, typically between 0°C and 10°C, prior to milling. In some embodiments, the low temperature drying step comprises or consists of freeze-drying, also known as lyophilisation.

A first aspect of the invention provides a method of producing flour from fermented vegetable material, comprising the steps of: a. steaming, blanching or cooking the fermented vegetable material; and/or b. dissolving the fermented vegetable material at acidic pH, centrifuging the resulting solution and resuspending in water the solid phase that results from centrifugation; and/or c. drying the fermented vegetable material at a temperature above 0°C and below 10°C, or freeze-drying the fermented vegetable material; then d. grinding, crushing or milling the material that results from step (a), step (b) or step (c) to form flour.

The vegetable material is typically fermented using Rhizopus oryzae, R. oligosporus, and/or R. delemar. The fermented vegetable material is typically tempeh.

In some embodiments, step (b) increases the protein yield compared to the same process without step (b). In some embodiments, step (c) reduces or removes bitter flavours from the fermented vegetable material.

Step (a) may typically be carried out at 100°C or more for at least 30 seconds or at least two minutes, for example at least five minutes. In step (b), the acidic pH is typically around pH 4.5. The fermented vegetable material in step (b) is typically dissolved in lactic acid, citric acid or acetic acid.

Step (c) is typically carried out at about 4°C for 24 to 72 hours.

In some embodiments, only steps (a), (c), (d) are performed. In some embodiments, only steps (a), (c), (d) are performed in that order. In some embodiments, only steps (c), (a), (d) are performed. In some embodiments, only steps (c), (a), (d) are performed in that order. In one embodiment, only step (c) then step (d) is performed.

A second aspect of the invention provides flour obtained or obtainable by the method of the first aspect.

A third aspect of the invention provides a food comprising the flour according to the second aspect as an ingredient.

The flour that is produced according to the invention is typically less bitter than would be produced by simple milling of conventionally-produced tempeh.

The flour that is produced according to the invention is typically more soluble in water than would be produced by simple milling of conventionally-produced tempeh.

The flour that is produced according to the invention typically has increased emulsifying capacity than would be produced by simple milling of conventionally-produced tempeh.

The flour that is produced according to the invention typically has greater antioxidative activity than would be produced by simple milling of conventionally-produced tempeh.

The flour of the invention can be used to modify the nutritional value, sensory properties, and/or shelf-life of food products into which it is incorporated.

The invention typically relates to flour made from tempeh-fermented grains, legumes, and/or beans, wherein the flour has favourable bitterness and water-solubility.

Brief Description of the Drawings

Figure 1 shows the effect of chilled drying on protein yield.

Figure 2 shows the effect of acid extraction on sensorial perception: colour, aroma, taste and aftertaste.

Figure 3 shows the effect of chilled drying on protein content for market tempeh and homemade tempeh, and also the effect of blanching. Figure 4 shows the effect of centrifugation on protein content.

Detailed Description of the Invention

The inventors have surprisingly identified that tempeh flour can be improved by (i) cooking, blanching or steaming tempeh prior to milling; (ii) cold-drying tempeh prior to milling; and/or (iii) acid extraction of tempeh prior to milling. Each of these three steps individually provides an improvement in the resulting flour. These three steps can also be combined in any combination of two or three steps, and can be used in any order when combined. Typically, the cold-drying step is conveniently performed last out of these processing steps, so that the resulting dried product can be milled.

The invention is based on the investigation of tempeh, but can be applied to other fermented vegetable materials. Typically, the flour produced according to the invention has a neutral or pleasant taste and is therefore a versatile food ingredient. For example, flour produced according to the invention may avoid an undesirable bitter taste often associated with products that are heated prior to milling.

In one embodiment, a method to produce flour from vegetable material typically comprises (i) fermenting vegetable material using Rhizopus oryzae, R. oligosporus, and/or R. delemar; (ii) blanching or cooking, or steaming, the fermented vegetable material; and (iii) grinding or milling the blanched, cooked or steamed material to produce flour.

In another embodiment, a method of producing flour comprises drying fermented or unfermented vegetable material at low temperature. This cold-drying step has been observed to reduce or remove bitter flavours from the vegetable material. In some embodiments, the low temperature drying step comprises freeze-drying, also known as lyophilisation. Freeze-drying is well-known in the art.

In a further embodiment, a method of producing flour having increased protein yield comprises using pH adjustment and solid phase extraction. This is typically used in combination with at least one of the other embodiments of the invention. material

The vegetable material that is fermented typically comprises beans such as soybeans or lupin beans. In other embodiments, the food may comprise rice, wheat, grains, cereal, legumes or nuts. Other fermentable vegetable substrates such as coconut or cassava will be available to the skilled person. The food may comprise a combination of substrates.

In some embodiments, the fermented food that is produced and then milled is tempeh. Tempeh production is well known in the art, and any suitable technique can be used as will be apparent to the skilled person.

Tempeh is traditionally made by fermenting soybeans. Lupin beans are also relatively commonly used to make tempeh. However, tempeh can be made using any appropriate vegetable foodstuff. The fermentable vegetable foodstuff used in the invention is typically particulate vegetable material. Suitable particulate vegetable materials are beans, seeds, grains, legumes or nuts.

While the vegetable material to be fermented is typically seeds, beans, grains, pulses or legumes, the use of flowers, fruits, stems, nuts, leaves, pods, roots, rhizomes or tubers is also provided. Depending on the form of the vegetable material, it may need to be chopped or otherwise mechanically processed to allow for effective fermentation. Mechanically-processed versions of vegetable materials may therefore also be used, for example chopped, ground, grated, milled or crushed material that can be fermented to form tempeh.

In further embodiments, the vegetable material that is fermented is or comprises one or more of chickpeas, lentils, white beans, black beans, broad beans, black gram, green gram, yam-beans, velvet beans, rice bran, barley, pigeon peas, quinoa, oats, millet, cowpeas, koro benguk (Mucuna pruriens), buckwheat kernels, red sorghum, wheat, fava beans, peas, koro kratok bean (Phaseolus lunatus), jack bean (Canavalia enisformis), okara (filtration residue of soymilk production), finger millet (Eleusine coracana), cottonseed kernels and corn grits.

The fermentation is typically solid-state fermentation. The fermentation is typically carried out by one or more mycelium-producing fungus, for example food-safe mycelium-promoting organisms such as Rhizopus spp. In some embodiments, a non-fungal mycelium-promoting microorganism may be used instead of or in combination with a fungus. The non-fungal mycelium-promoting microorganism may be a fungus-like bacteria. The mycelium-producing inoculated microorganism may also comprise one or more microbes, such as bacteria, that do not form mycelium. These may be present to add nutrients such as a B vitamin.

The mycelium-producing microorganism typically comprises Rhizopus spp. These are typically selected from Rhizopus oligosporus, Rhizopus oryzae, and/or Rhizopus delemar

In some embodiments, the mycelium producing microorganism used to produce the fermented food , e.g. tempeh, comprises a single fungus for example Rhizopus oligosporus as the sole fungi. Other suitable single Fungi include Rhizopus oryzae and Rhizopus delemar. In some embodiments, two or more Fungi are used to inoculate the foodtsuff. In certain embodiments the two or more Fungi may comprises two or more of Rhizopus oligosporus Rhizopus oryzae and Rhizopus delemar.

One exemplary embodiment uses Rhizopus oligosporus:Rhizopus oryzae at a ratio of 4:1.

The inoculation of the vegetable material with the microorganism may typically comprise inoculating between 1 E+5 and 1 E+8 CFU of mycelium-forming fungus inoculant per 100 g of total ingredients.

An exemplary method of producing tempeh is described below.

Dried soybeans are soaked overnight with water to rehydrate and soften the soybeans. The rehydrated soybeans are then dehulled mechanically using a dehuller. If dehulled and cracked dried soybeans are used, then there is no dehulling step necessary after the soaking step. Next, the soybeans are boiled in water for an hour to cook, sterilize, and soften the beans even more to allow the fermenter microorganisms to digest the substrate. After the boiled beans are allowed to dry and cool to room temperature (ranging around 25-35°C), the beans are inoculated with the starter culture, and incubated at room temperature, at around 20°C, at around 25°C or at around 30-37°C. In some embodiments, the beans are inoculated with the starter culture and incubated at around 30-37°C. After 18-24 hours of fermentation, the white mycelium of the grown fungi tightly packs the beans into solid tempeh cakes.

High heat prior to milling

In one embodiment of the invention, the fermented vegetable material (e.g. tempeh) is subjected to high heat prior to milling. This is typically referred to as cooking, blanching or steaming the fermented vegetable material. In some embodiments, the high heat step is cooking or blanching. In some embodiments, the high heat step is steaming.

Typically, this step comprises exposing the fermented vegetable material to a temperature of at least 100°C, more typically at least 120°C, for example 150°C or more. The period of exposure at this temperature is typically at least 30 seconds, at least two minutes, or at least five minutes. Typically, this period is less than about ten minutes.

The high temperature may be provided by hot air e.g. baking in an oven, or by boiling water (e.g. the process commonly known as blanching). The high temperature may be provided by steam, for example by placing the fermented vegetable material in contact with steam, for example in proximity to (typically above) boiling water. In some embodiments, the period of exposure to high temperature may be stopped rapidly by exposing the material to much lower temperature, for example by plunging the material into cold or iced water. In some embodiments, this rapid exposure to lower temperatures is not performed, for example to preserve the sterile nature of the high-heat-treated product.

The fermented vegetable material is typically chopped, diced, sliced or crushed into pieces prior to this high heat step. The use of pieces of the material that are smaller than the typical tempeh “cake” that results from fermentation, aids the rapid, even and effective heating of the product through the increase in surface area. In one embodiment, tempeh is sliced into pieces having a thickness of 0.2-0.8 cm thickness prior to heating. In other embodiments tempeh may be roughly diced, for example into rough cubes having length, width and depth from about 0.1cm to about 3cm, for example about 0.2 to about 0.8cm, or about roughly 1cm ³ .

Without wishing to be bound by theory, the inventors believe that the high heat “cooking” step breaks the food matrix in the fermented vegetable material. For example, this step may break or degrade dietary fibres that encapsulate and limit the extractability of proteins, emulsifiers, and antioxidants. Accordingly, performing this step prior to milling may increase the protein yield.

Acid Extraction prior to milling

In one embodiment, the fermented vegetable material (e.g. tempeh) is dissolved in a solution at acidic pH, e.g. pH 4.5. The pH may be any acidic pH sufficient to dissolve the material. Typically this will be between pH 2 and pH 5, or between pH 3 and pH 5, or between pH 4 and pH 5. This is typically achieved using food-grade acids, for example lactic acid, citric acid or acetic acid.

The dissolution of the tempeh can be aided by chopping, dicing, slicing, crushing or grinding the tempeh prior to its exposure to the acid. In one embodiment, tempeh is sliced into pieces having a thickness of 0.2-0.8 cm thickness prior to acid dissolution. In other embodiments tempeh may be roughly diced, for example into rough cubes having length, width and depth from about 0.1cm to about 3cm, for example about 0.2 to about 0.8cm, or about roughly 1cm ³ . In other embodiments, the tempeh may be ground into a coarse powder or fine powder prior to dissolution. Once the tempeh is dissolved, the acid-tempeh solution is centrifuged under conditions in which a solid pellet is precipitated. The liquid phase is then removed and the pellet re-suspended in water or aqueous solution. The resulting solution is then dried (e.g. water evaporated) to leave the product of interest. This drying step may be spray drying, or using an oven. This drying step may alternatively be at low temperature, according the cold-drying embodiment of the present invention. Without wishing to be bound by theory, the inventors consider that the exposure to low pH such as pH 4.5 increases the protein yield because pH 4.5 is the isoelectric point of proteins in tempeh and therefore an optimum precipitation point.

Chilled drying prior to milling

In one embodiment of the invention, the fermented vegetable material e.g. tempeh is dried after the fermentation step and prior to milling, and this drying step is carried out at low temperature. Typically, the low temperature is between 0°C and 10°C, for example between 1°C and 8°C, or at about 2°C, about 3°C, about 4°C, about 5°C, about 6°C or about 7°C.

Figures 1 and 3 show the advantages provided by cold drying at 4°C, in particular an improved protein yield.

Drying at these low temperatures is typically aided by air circulation, for example a constant or intermittent flow or exchange of air. In some embodiments, the volume of air in which the drying occurs is exchanged at least once an hour.

The cold drying step typically takes at least 12 hours, for example at least 24 hours. The cold drying step may take up to a week, but is typically complete with about three days. In some embodiments, cold drying lasts for about 24 hours to about 72 hours.

The cold drying step can be aided by chopping, dicing, slicing, crushing or grinding the tempeh prior to its exposure to the cold. In one embodiment, tempeh is sliced into pieces having a thickness of 0.2-0.8 cm thickness prior to cold drying. In other embodiments tempeh may be roughly diced prior to chilled drying, for example into rough cubes having length, width and depth from about 0.1cm to about 3cm, for example about 0.2 to about 0.8cm, , or about roughly 1cm ³ .

Without wishing to be bound by theory, the inventors consider that the chilled drying step has at least one or more of the following benefits:

1) Reduction of bitterness due to less protein and amino acid denaturation and folding, especially those that promote bitter flavours in tempeh e.g. tryptophan.

2) Reduction of the denaturation of emulsifiers: soy lecithin as well as Rhizopus cell wall constituents chitin and chitosan.

3) Reduction of antioxidant degradation, which is mainly promoted by light-mediated oxidation and high temperature.

In some embodiments, the low temperature drying step comprises freeze-drying. As is known in the art, freeze-drying typically comprises steps of freezing, primary drying and optional secondary drying. Typically, during the freezing stage the material is cooled below its triple point, which is the lowest temperature at which the solid, liquid and gas phases of the material can coexist. During the primary drying phase, the pressure is typically lowered (e.g. to the range of a few millibars), and enough heat is supplied to the material for the ice to sublime. The amount of heat necessary can be calculated using the sublimating molecules' latent heat of sublimation. In this phase, pressure may be controlled through the application of partial vacuum. The partial vacuum can speed up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plate can be used to provide a surface onto which the water vapour can re-liquify and solidify. When used, the secondary drying phase aims to remove unfrozen water molecules. This can be achieved, for example, by raising the temperature higher than in the primary drying phase, and can even be above 0°C (32 °F). At the end of the freeze- drying process, the final residual water content in the product is typically extremely low, for example under 5% or around 1% to 4% by weight.

Combinations of high heat, acid extraction and cold drying

The high heat, acid extraction and cold drying steps can be combined in any combination and any order. Typically, the cold drying step is the last step before the milling step to create flour.

Different orders of cooking/blanching and chilled drying, in particular, can produce different flavour profiles. In general, chilled-drying without a cooking step has been observed to produce the least bitter powder. Adopting a cooking step towards the end of the production flow has also been observed to produce an agreeable aroma.

Milling

As used herein, the term “milling” is used to mean any technique used to create flour from vegetable material. It typically involves grinding the material to create a powder.

The production of flour is very well known and typically involves the physical grinding or crushing of vegetable material to form a powder. In some embodiments, the flour has a particle size of 80-120 mesh, or at least 100 mesh.

The preparation of tempeh flours and mixed flours is known in the art, for example as described in Leite et al, Food Science and Technology 33(4):796.

Uses of the flour

The flour of the invention can be used as an ingredient in other foods. In some embodiments, the flour of the invention is incorporated into a mixed flour comprising at least one other type of flour, for example cornflour or wheat flour.

Example An exemplary and non-limiting process according to the invention is summarised in Table 1 , below. One or more of these steps can be omitted, and one or more steps that are not in this table can be added, as will be apparent to the skilled person. Each step can be varied within practical constraints, as described herein and as will be apparent to the skilled person. Table 1

A selection of experimental results for various method steps according to the invention are shown in Figures 1 to 4. These include results for protein content and sensorial perception, for methods involving chilled drying, pH adjustment (acid extraction), high heat (blanching), and centrifugation

Figures 1 and 3 show the improvement protein yield provided by chilled drying of tempeh prior to milling. Figure 3 also shows an improved protein content in a method comprising blanching.

Interestingly sensory tests performed by the inventors indicate that different orders of cooking/blanching and chilled drying can produce different flavour profiles. In general, chilled- drying without a cooking step produces the least bitter powder. Once, adopting a cooking step towards the end of the production flow produced aroma that was more preferred by the panellists.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise.

The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e. , meaning “including, but not limited to,”) unless otherwise noted. Where appropriate, the open-ended terms such as “comprising” may be replaced with the closed-ended term “consisting of” when a narrower definition is required.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

New methods for creating a food are discussed herein. In the description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.

The present disclosure is to be considered as an exemplification of the invention, and is not intended to limit the invention to the specific embodiments illustrated by the figures, description and Examples.

All citations and referenced documents are incorporated by reference in their entirety, as if each individual disclosure had been separately and expressly incorporated.