PROCESS FOR BIOFORTIFICATION OF GRAINS

FIELD OF THE INVENTION

This invention relates to the biof ortification of grains and, in particular, to the biofortification of grains for use in brewing of beer.

BACKGROUND TO THE INVENTION

It is known that deficiencies in the human body of certain nutrients can lead to malnutrition and many other dysfunctions of the body. Such nutrients include iron, zinc and selenium. Deficiencies in selenium (Se), for example, can lead to thyroid dysfunction, cancer, severe viral diseases, cardiovascular disease, and various inflammatory conditions.

Selenium is naturally present in soils and is therefore part of the food chain that eventually becomes an element of the food consumed by humans. However, in many regions around the world, the level of selenium that exists in the soil is too low to ensure that a sufficient level migrates into foods grown in that soil to maintain good health in the relevant human population. While the population can be treated for the selenium deficiency by, for example, consumption of selenium-rich foods such as Brazil nuts, meat, fish and cereals, these foods can often be too expensive to form a "staple" component of the diet of a population. Another approach is to treat the selnium deficiency with "targeted" supplementation of selenium in direct organic form (e.g. by selenium tablets). However, while found to achieve considerable health benefits (e.g. cancer prevention, increased sperm motility, reduced spontaneous abortion and increased brain function including cognitive ability), again, this form of treatment can often be too expensive for much of a population to adopt.

Wheat and other grains are important carriers of selenium into the food chain and a very direct means for providing humans with selenium. It is known that the selenium content of wheat can be increased by what is known as "biofortification" by way of application of inorganic selenium (i.e. sodium selenate) to soils in which wheat is grown and by way of foliar application to the wheat at certain stages of its

growth, wherein the wheat plant transforms the inorganic selenium to a non-toxic organic form. However, while this biof ortif ication process can work well, it is expensive (due to the cost and transport of the large quantities of inorganic selenium required), and also can suffer from adverse weather conditions which can deplete the effective uptake of inorganic selenium. Alternatively, biof ortification of wheat can be achieved using an organic form of selenium (e.g. selenomethionine), however this involves the spreading of essentially unf easonable amounts of the organic selenium onto the crop area in order to achieve sufficient transference of selenium into the food product during its growth and later processing.

It is also known to add the inorganic form of selenium to the pitching yeast in the beer making process prior to fermentation so as to allow the yeast to bio-covert such that the resulting beer has higher levels of organic selenium than would otherwise be the case. However, adding selenium in this fashion may be interpreted as the addition of an ingredient, and this may have a negative connotation to food health authorities and beer consumers.

It is an aim of this invention to provide a solution to one or more of the problems discussed above or at least provide an alternative approach to the biofortification of food sources, in particular, beverages brewed from grain.

SUMMARY OF THE INVENTION

A process of biofortification of a grain with a nutrient comprising the step of adding an inorganic form of the nutrient to water used in the steeping and/ or germination stage(s) of the grain in a malting process.

Preferably, the inorganic form of the nutrient is added to the water used in the steeping and/ or germination stage(s) of the grain following chitting of at least 30 %, more preferably at least 50 %, most preferably at least 95%, of the grain.

More preferably, the inorganic form of the nutrient is added to the water used in the germination stage of the grain, particularly after the first day of the germination stage.

Preferably, the amount of the inorganic form of the nutrient that is added to the water is in the range of about 25 to 125 μg/g of water, more preferably about 50 to 100 μg/g of water.

Preferably, the nutrient is selected from iron, zinc and selenium.

Most preferably, the nutrient is selenium.

The inorganic form of selenium added to the water used in the steeping and/ or germination stage(s) of the grain in a malting process, may be sodium selenite or, more preferably, sodium selenate.

The process of the invention thereby involves subjecting the grain to a malting process, and adding an inorganic form of the nutrient to water used in the steeping and/ or germination stage(s) of the grain during the malting process in a manner whereby the grain absorbs at least some of the inorganic form of the nutrient, thereby biof ortifying the grain with the nutrient.

Preferably, the grain is selected from barley, wheat and rye. The biofortified malt produced from barley, wheat and rye biofortified in accordance with the process of the invention, may be used in the production of beverages, in particular, beer

(especially barley and wheat malts) and whisky/ whiskey (especially, wheat and rye malts).

Most preferably, the grain is barley.

The process of the invention is preferably applied to the biof ortification of barley during the production of malt for use in beer production. As such, it is preferable that the amount of the inorganic form of the nutrient, in particular sodium selenate, is selected so that:

(i) the colour of the biofortified barley is approximately 5 E.B.C Units or less; and/ or

(ii) the diastatic power (DP) of the biofortified barley is above approximately 250 WK.

In other aspects of the invention, there is provided a biofortified malt produced by a malting process applied to a grain, the malting process comprising the step of adding an inorganic form of a nutrient to water used in the steeping and/ or germination stage(s), thereby biof ortifying the grain with the nutrient, and a beverage produced from said biofortified malt, particularly a beer brewed from said biofortified malt.

It has been found that beer produced from a biofortified malt in accordance with the invention may comprise a concentration of selenium in the range of about 10 to 40 parts per billion (ppb), which compares favourably with the amounts of selenium found in typical beer of about 1 to 4 ppb (e.g. Australian beer typically comprises about 3 ppb selenium, and European beer typically comprises about 1.4 ppb selenium).

Thus, the invention further extends to a beer produced from a biofortified malt, wherein said beer comprises a concentration of selenium in the range of about 10 to 40 ppb.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic flow diagram of a conventional malting process;

Figure 2 is a schematic flow diagram of the steps involved in using the process of the invention for a steeping step in the malting process; and

Figure 3 is a schematic flow diagram of the steps involved in using the process of the invention for a germination step in the malting process.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The process of malting grain is most commonly associated with barley and in this specification, the grain used by example will be barley (in particular, "New Season's Gairdner" variety of barley). However, other grains which can undergo a malting process are within the scope of the present invention. Further, by way of example, the barley grain as described hereinafter is biof ortified with the nutrient selenium, however, the process of the invention is also applicable for grain biof ortified with other nutrients, for example, iron or zinc.

Barley is an annual cereal plant (Hordeum vulgare and sometimes other species) of the family Gramineae (grass family). It is a valuable stock feed, often as a corn substitute, and is used for malting when the grain is of high quality. The malt can be used to produce beverages and foods, for example, malt can be used in the production of beer. Indeed, its use for brewing has been dated back to about 300 BC. Barley is generally used for malting because it contains around 65% starch. The barley husk protects the developing shoot and embryo during germination, and also has a firm internal consistency, both of which mean that barley is more resistant to damage during handling than other grains.

MALTING PROCESS

The malting process transforms high-grade barley into malt. Malt is a rich source of minerals, amino acids, vitamins, carbohydrate and protein content that makes it a valuable human nutrient. When malt is used to brew beer, the malt primarily supports the fermentation process of beer production. The malting process assists the conversion of cereal starches to sugars by enzymes, chiefly diastatic enzymes.

Malts also provide varying flavour, colour, head and body to beer depending on the style of malt being used because different barley varieties malt differently. Moreover, malts differ depending upon grain type and quality, steeping profiles, germination rates and the choice of temperatures during the germination step of the malting process. Consequently, typically only one variety of barley is malted in the malting apparatus at a time.

The stages of the malting process include steeping, germination, kilning and sometimes roasting. Figure 1 provides a shematic flow diagram of a conventional malting process including the optional preparation of the raw barley in a mill (10), which is designed to adequately breach the seed coat (husk) to expose the seed embryo (this process step is typically no longer carried out in large scale malting operations, as the barley is now grown to have suitable characteristics obviating this processing step). The milled, or in most cases, the unmilled barley is placed in a tank (20) for steeping. In a conventional malting process, the next step requires the barley to be placed in an environmentally controlled vessel (30) for germination. Finally, the partially germinated barley is transferred to a kiln (40) for drying or partial cooking. Each step is described in greater detail below.

Steeping

Barley is immersed in tanks of aerated water so that the barley can absorb moisture. The water content rises in the barley from about 10% to between 38% and 40% so as to kick start the germination process. When barley is wetted, moisture rapidly distributes itself throughout the surface layers. Water gains entry to the internal tissues at, or near, the embryo through the micropyle region. After approximately 24 hours, the first visible sign of germination is the appearance of the root as a white "chit". Once the grains have chitted, they are moved to a germination vessel for the germination stage of the malting process. Depending upon the grain, the steeping stage can involve submersion of the grain in the steeping tank, shown in Figure 1 at (20), for five to eight hours at a time, with water being drained to allow for air-rests in between the submersions.

Germination

The grain (in this case, barley) with absorbed water is placed in an environment controlled germination vessel so as to allow for the activation of enzymes in the moist barley. In the controlled environment, the grain begins to grow and further develops the root and shoots. The temperature and humidity of the barley is controlled in the germination vessel by heating and the spraying of water on the barley. The endosperm of the grain (i.e. the energy source of the grain during early growth), changes in a process called "modification". Manipulating the humidity and temperature in the environment of the germination vessel controls diastatic enzyme production. The germination stage aims to maximise grain modification and minimise root and shoot growth that would otherwise lower the resultant extraction of malt product. The grain remains in the germination vessel as shown in Figure 1 at (30) for three to four days.

Kilning

Once the grains have reached a required level of biological change (i.e. when the shoot is about three quarters the length of the grain after about four days in the germination vessel) they are dried, terminating further growth and curing the grain for storage. During the kilning stage, malt develops its colour and flavour. Low temperatures are used to ensure the survival of diastatic enzymes required for the subsequent brewing process, then a progressive increase in temperature to effect the selected degree of flavour and colour changes in the malt. The kilning stage takes place in the kiln shown as (40) in Figure 1.

Following the malting process, the malt product is then packaged for sale to the brewery and used in the beer making process.

Beer is not generally considered a healthy beverage and indeed, like anything that is not consumed in moderation, there can be adverse health consequences to consuming too much beer. For example, it is known that damage to the liver can

occur, as can many other ailments including depleting the body's supply of essential nutrients including selenium. It is hoped that by providing beer having a supplemented level of selenium, some of the adverse effects of beer drinking can be balanced, and in some cases the selenium added to the consumption of the beer drinker will have beneficial effects on the health of the individual.

BEER BREWING

The brewing of beer starts with malt that is ground into a coarse powder, known as grist. The grist is mixed with heated water in a vat called a "mash tun" for a process known as "mashing". During this process, enzymes within the malt, including the diastatic enzymes mentioned above, break down much of the starches into sugars, which play a vital part in the fermentation process. After the mashing, the resulting liquid is strained from the grains in a process known as lautering. At this point, the liquid is known as wort. The wort is moved into a large tank known as a "copper" or kettle, where it is boiled with hops and sometimes other ingredients such as herbs or sugars. The boiling process serves to terminate enzymatic processes, precipitate proteins, isomerise hop resins and concentrate and sterilise the wort. The wort is then moved into a "fermentation vessel" where yeast is added or "pitched" with it. The yeast converts the sugars from the malt into alcohol, carbon dioxide and other components through a process known as glycolysis. After a week or so, the fresh (or "green") beer is run off into conditioning tanks. After conditioning for a week or longer, the beer is often filtered to remove yeast and particulates. The "bright beer" is then ready for serving or packaging.

So as to make a consistent quality beer, there are three measures of malted barley used by maltsters and brewers.

First, the "malt extract" measures the amount of fermentable sugars in the grain, and determines the amount of alcohol that can be made from a tonne of grain. The higher the extract levels, the more alcohol that can be made. The malt extract value is determined by measuring the amount of soluble sugar (like glucose and maltose) in the wort. The measure is presented as a percentage (dry basis), 80% being one

example of malt extract value. This is usually achieved with a four-day malt. This target cannot be met if maltsters use poor quality grain. It is the duty of a maltster to meet the specification set by a brewer planning to purchase the malt extract.

Second, the "diastatic power" (DP) measures the amount of diastatic enzymes (e.g. α- amylase, β-amylase and limit-dextrinase). As mentioned above, diastatic enzymes convert the starches of the grain into soluble sugars (or malt extract). The levels of the various diastase enzymes are important in achieving the quality standard required by a buyer of malt. For example, low diastase levels are associated with a low potential for malt extract. Diastatic power is measured by the enzymic activity in the malt. The measure is presented in units of Windich Kolbach (WK). As the diastatic enzymes are proteins, their level is directly related to protein concentration in the grain.

Third, "wort viscosity" measures the gumminess of wort relative to water. It is essentially a measure of the amount of stress a plant has undergone during grain filling. Genotype (i.e. hereditary constitution) also has a major influence on wort viscosity. Wort viscosity is determined by measuring the amount of β-glucan (or cell wall material) in the wort. The measure is presented in units of centipoises (cP). Barley grain with low viscosity, germinates more evenly than grain with high cell wall material. This cell wall material can also restrict the conversion of starches into malt extract. Highly viscous malt slows down the separation of the sugar-rich wort from husks during brewing. This slows the amount of beer processed in a brewery each day and increases production costs.

None of these measures appear to be affected by, or adversely affect, the biof ortif ication of barley malt with a nutrient such as selenium.

BIOFORTIFICATION Figure 2 is a schematic representation of the steps involved in using the process of the invention for a steeping stage in the malting process. In this embodiment of the invention, sodium selenate (50), an inorganic form of selenium, is added to the water

(52) to be used for steeping the barley grain. It has been found, however, that the concentration of selenium in the grain following steeping is relatively low. While not wishing to be bound by theory, it is considered that this is due to the fact that during steeping, most of the grains do not yet have a developed ion transport mechanism to facilitate the absorption of the sodium selenate into the grain. Accordingly, while sodium selenate (50) can be added to the steeping water (52), it is generally not preferred, due to the likely wastage of the sodium selenate that will ensue.

During the steeping stage (with or without sodium selenate), the water (52) is added to the grain while it resides in a tank (20) (see Figure 1) so as to submerge the grain. The grain typically remains in the tank water for five to eight hours and, if the total steeping process is not over as illustrated by the "NO" arrow (57) located at the base of the decision diamond (55), then the tank water is removed and a predetermined time is lapsed before re-filling the tank with the water. The predetermined time is judged by the maltster and would not be affected by the added step of adding sodium selenate to the water. The tank may be re-filled with new water (52) that may or may not have sodium selenate added to it.

When the steeping process is over as illustrated by the "YES" arrow (61) located to the side of the decision diamond (55), the water is removed from the tank. The steeping process is over when >95% of the barley grains have chitted which is the first visible sign of germination. The barley, with absorbed water, is then transferred (62) to a germination vessel as shown by the continuation marker "A" which can be found again in Figure 3.

The germination vessel (66), in Figure 3, provides a controlled environment, as mentioned above, that is heated and sprayed with water (70) as required to control the germination of the grain in the vessel as determined by the maltster. The maltster controls the setting of temperature and humidity (69) and the time (67) and (68) of the germination process. In some instances, these decisions may be programmable and thus automatically controlled.

Also, as mentioned above, it is possible to germinate the previously described enhanced selenium content grain without further inorganic selenium addition via water sprayed on the grains during the germination process, however, the selenium content of the grains will be low (perhaps too low to provide a commercial advantage in the final beer product). Alternatively, it is possible that the grain received for germination has not previously been processed to have enhanced selenium content. That is, once the grains are transferred to the germination bed, most if not all of the grains will have chitted. As there seems to be a correlation between chitting of the grain and the development of an ion transport mechanism that may facilitate the absorption of the selenium into the grain, it is, accordingly, preferable to add sodium selenate to the water used during the germination stage of the malting process. In this case, the sodium selenate is added (72) to the water that is used for spraying the germinating grain in the germination vessel.

The addition of the sodium selenate during the germination stage can occur over predetermined periods of time in one or more of the first and subsequent days of germination of the grain. Alternatively, the sodium selenate can be added on the second day of the germination process to ensure maximum selenium uptake since on the second day, the grain is ready and able to efficiently absorb more water. The maltster or an automated process control determines the times, and on what days, the sodium selenate is added.

It is during the germination stage of the malting process that, in each grain, there is a transformation of the inorganic form of selenium into the organic form of selenium which then remains within the grain during the further malting process. The organic form is predominantly selenomethionine which is more bioactive and bioavailable than the inorganic sodium selenate.

Following the required germination in the germination vessel, the grain is transferred to a kiln (74) for kilning (and completion of the malting process).

The amount of sodium selenate added to the water during the malting process is determined by the amount of selenium desired in the final malt product. A predetermined amount of sodium selenate can be added to the water to ensure optimal biof ortification. Experiments have shown that too much of the inorganic form of selenium can have undesirable effects upon the grain (see Example 1 hereinafter). For example, if too much selenium is provided, the grains can experience some level of toxicity and have a much lower level of root growth. Other undesirable effects include the development of high colour (pink tone) in the grain which is undesirable for beer making since the final beer product will exhibit the high colour. Preferably, the amount of selenium added to the grain is selected or adjusted so that the colour of the biofortified grain is approximately 5 E.B.C or less (E.B.C. is a unit of the European Brewing Corporation used to measure the colour of beer. It is noted that in the "control" of Example 1 (i.e. where no selenium is added), the untreated grain had a colour of 4.5 EBC units.

The preferred amount of selenium added to the water used during the malting process is between approximately 50 to 100 μg/g. At levels higher than 100 μg/g (e.g. 150 μg/g and higher), the grains subjected to the experiment of Example 1 gave off high levels of irritating selenium fumes and were considered a health and safety risk.

Preferably, the amount of selenium added to the grain is selected or adjusted such that the DP of the grains remains above a preferred threshold. As mentioned above, DP is a measure of the amount of diastatic enzymes in the grain (i.e. α-amylase, β- amylase and limit-dextrinase). Since diastatic enzymes convert the starches of the grain into soluble sugars, a low level of enzymes is undesirable. The preferred DP of the selenium fortified grain is from about 250 W.K. and above. Again, selenium added at to the water used during the malting process at a concentration between approximately 50 to 100 μg/g is suitable for maintaining the desired DP. Once selenium levels are increased, the DP of the grain decreases undesirably.

Using water having between 50 to 100 μg/g of sodium selenate results in a grain having approximately, 25% of the original applied concentration (e.g.25 μg/g for 100 μg/ g application). In the case of beer making, it has been found that beer brewed from biof ortified malted barley retains a considerable proportion of the selenium (approximately 40%) that was supplied during the malting process. The balance of the selenium from the selenium biof ortified malt is lost in the spent grains (approximately 50%) and yeast (approximately 10%). Since the selenium is not added during the pitching of the yeast, it is not considered to be an ingredient of the beer which is an advantage from a consumer and regulatory perspective.

It has been found that beer produced from a malt of barley biof ortified in accordance with the invention may comprise a concentration of selenium in the range of about 10 to 40 parts per billion (ppb), which compares favourably with the amounts of selenium found in typical beer of about 1 to 4.

The potential benefits of biof ortified beer are reduced liver damage and lower prostate cancer in the consumer, assuming moderation in its consumption.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non- limiting examples.

EXAMPLES

Example 1 Selenium biof ortification of malt

Methods and Materials

1 kg can size malt preparations were produced in accordance with the process of the present invention, using New Season's Gairdner barley, standard micro-malt

sodium selenate tested were: 0, 50, 100, 150, 200, 300, 400 and 500 μg/g of water. The selenium is added to the water to achieve the required concentration directly prior to spraying of the grain during germination.

Results

The results of the malting trials are shown in Table 1 ("observations"), Table 2 ("grain analysis results") and Table 3 ("screening results").

Discussion The difference between the control and even the 50 μg/g concentration of Selenium was noticeable; the grain was darker and there was significantly less root growth (see Table 1). The difference in colour between the control and the 50 and 100 μg/g samples were not significant analytically (as can be seen in the Table 2 below). However, the difference in colour was significant for the remaining concentrations. The majority of the other analytical results were quite similar to the control, with the exception of KI (Kolbach Index) for the 200, 300 and 400 μg/g concentrations and the DP (Diastatic Power) for the 300 and 400 μg/g concentrations. KI is an indication of how well modified the grain is. For some reason, the samples with higher concentrations of selenium seem to have over-modified. Also, the higher concentrations have lower levels of diastatic enzymes, which are important for the brewing process. Table 3 clearly shows the decrease in root growth with increasing selenium concentration, indicating that the higher concentrations were killing the grain.

Table 1

Table 2

M = moisture (%) CO = colour (E.B.C. units)

FG = extract (%) TP = total protein (%)

SP = soluble protein (%) KI = Kolbach Index

V = viscosity (cP) WBG = wort beta glucan (ppm)

AA = alpha amylase (DU) BG = beta glucanase (U/kg)

DP = diastatic power (WK)

Table 3

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/ or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.