Field of the invention

The present invention relates to foodstuffs containing galangin. In particular, the invention relates to the reduction of food spoilage by adding galangin to a foodstuff which is prone to spoilage.

Background of the invention

The Food and Agriculture Organization of the United Nations estimates that about a third of all food produced globally is lost or wasted, amounting to about 1 .3 billion tons per year (FAO, 201 1 ). The EU alone wastes over 100 million tons of food annually (2014 estimate). Without countermeasures, food waste is expected to rise to around 126 million tons by 2020. As the global population continues to rise, food security is a growing concern and thus the need to avoid food waste is considered to be of increasing importance.

Spoilage caused by a variety of microorganisms is one reason for food wastage. Examples of spoilage microorganisms include bacteria (e.g. various species and strains of Lactobacilli), moulds (e.g. various Aspergillus and Penicillium species and strains), and yeasts (e.g. various Zygosaccharomyces and Saccharomyces species and strains).

Spoilage is the process by which food deteriorates such that its edibility becomes reduced, ultimately resulting in the food becoming inedible to humans. Moulds and/or yeasts growing on or within the food matrix may be responsible for food spoilage. Bacteria can also be responsible for food spoilage. Acids and metabolites are typically created in the processes by which bacteria break down food - even if the bacteria themselves are not harmful, the waste products may be unpleasant to taste or may even be harmful to health.

Consumers increasingly want convenience food to stay fresh and have a prolonged shelf life. Addition of preservatives to foodstuffs (such as beverages, spreads, dressings, convenience food, and so forth) is common practice in the food industry. The market for preservatives is growing in line with consumer demand. Many countries have regulations that prohibit the use of certain food additives, including some preservatives, in foods and beverages. Although such regulations can vary widely, there is a clear trend for foods to contain fewer and lower levels of chemical preservatives, particularly synthetic ones.

Frequently used preservatives include benzoic acid, potassium sorbate and lactic acid. Unfortunately, the use of such preservatives can often impair the flavour of certain foodstuffs. Furthermore, some consumers view these preservatives as the sort of chemical additives they would rather avoid. Indeed, there is a growing consumer trend for so-called“Clean Label” food products.

However, it is difficult to replace existing preservatives with“Clean Label” alternatives. In particular the replacement of sorbates is challenging, since many of the“natural” alternatives do not have a sufficient degree of anti-fungal activity - especially in low fat foodstuffs.

Accordingly, there is a need for a“Clean Label” natural compound or extract which would provide the beneficial qualities of sorbates, particularly in terms of anti-fungal activity. Similarly, the formulation of foodstuffs that have low levels of synthetic preservatives would also be desirable.

Summary of the invention

The inventors have found that extracts of Alpinia officinarum have a strong effect against food spoilage microorganisms, particularly fungi such as moulds and yeasts, and have identified the main active anti-fungal compound to be galangin.

Thus, in one aspect the present invention relates to use of galangin as a food preservative, and in particular to use of galangin to reduce or prevent the proliferation of one or more fungal spoilage microorganisms in a foodstuff.

In another aspect, the invention relates to a foodstuff that contains a preservative system, wherein the preservative system comprises galangin, and wherein the galangin is present in the foodstuff at a concentration of 1 to 800 ppm. In a further aspect, the present invention relates to a method for preparing a preserved foodstuff, the method comprising adding a composition comprising galangin to the foodstuff such that the concentration of galangin in the preserved foodstuff is 1 to 800 ppm, preferably 10 to 500 ppm.

Detailed description of the invention

The present invention relates to a foodstuff that contains a preservative system, wherein the preservative system comprises galangin, and wherein the galangin is present in the foodstuff at a concentration of 1 to 800 ppm galangin, preferably 10 to 500 ppm.

Galangin is a flavonol that has been reported to have in vitro antibacterial activity against pathogenic bacteria (e.g. see T.P.T. Cushnie et al. “Aggregation of Staphylococcus aureus following treatment with the antibacterial flavonol galangin” J. Appl. Microbiol. 103 (2007) 1562-1567). However, the suitability of galangin as a food preservative has not previously been investigated, and there is no general correlation between a antibacterial activity of a compound and its potential activity against food spoilage organisms.

Galangin is a constituent of propolis (although this is not currently a commercially feasible source of galangin), and is present in several plants - including Alpinia officinarum, Castanea sativa, Lippia graveolens and Helichrysum aureonitens.

The galangin is preferably provided by a plant extract, more preferably by an ethanolic plant extract. Extracts are preferably obtained by extracting dried roots, although leaves could also be used. It is particularly preferred that the plant extract is an extract of the plant Alpinia officinarum, and is most preferably an extract of the root of Alpinia officinarum.

A successful preservative system inhibits spoilage microorganisms without interfering with the sensorial properties of the foodstuff. It is preferred that the preservative system comprises at least 5 ppm galangin, more preferably at least 10 ppm galangin, still more preferably at least 25 ppm galangin, and most preferably at least 50 ppm galangin. The preservative system preferably comprises no more than 700 ppm galangin, more preferably no more than 600 ppm, galangin, still more preferably no more than 500 ppm galangin, and most preferably no more than 250 ppm galangin. The specified concentrations relate to the level of galangin present in the end product as stored (i.e. the level found in the foodstuff). It should be noted that this will not necessarily be the level in the foodstuff as consumed, since some foodstuffs are sold in a concentrated format, and will be diluted prior to being consumed. Thus it is possible that the foodstuff as consumed may have a lower concentration of galangin than the foodstuff as stored.

The foodstuff of the present invention is a foodstuff that is sensitive to fungal spoilage. The foodstuff is preferably selected from group consisting of: beverages (including carbonated soft drinks, fruit drinks, beverage concentrates, energy drinks, flavoured water, nectars, sports drinks), sauces, dressings, marinades, ketchups, seasonings, bouillons, soups, and ice cream.

It is particularly preferred that the foodstuff is a beverage, especially a ready-to-drink tea beverage (such as ice tea). However, other beverages including fruit drinks and soft drinks can also be stabilised using the same preservation system. The beverage may be still or carbonated.

Where the beverage is a tea-based beverage it will contain tea solids. The tea solids can be provided by any suitable source, e.g. a tea extract, an expressed tea juice, etc. The skilled person knows how to obtain such source materials.

Tea is known to have certain antimicrobial properties in itself, however this property (i.e. suppression of the growth of yeasts and moulds) is only evident once the concentration of tea solids exceeds about 3%. At concentrations lower than this, which are typical for tea-based beverages, the tea acts as a nutrient that enhances the potential for microbial spoilage. The beverage preferably comprises 0.01 % to 3% tea solids by weight of the beverage, more preferably 0.05% to 3%, most preferably 0.1 % to 2%. As used herein the term “tea solids” refers to dry material extractable from the leaves of the plant Camellia sinensis var. sinensis and/or Camellia sinensis var. assamica.

Preferred formats of tea-based beverages are black tea beverages and green tea beverages. The health benefits of green tea are primarily attributed to its high catechin content. A positive correlation between catechin consumption and improved cardiovascular health has been established. Thus when the beverage is a green tea beverage, it preferably comprises catechins in an amount of at least 50 mg, more preferably at least 60 mg, and still more preferably at least 75 mg. In order to provide the health benefits associated with catechins in a convenient manner, it is particularly preferred that these catechin levels are delivered in a single serving of the beverage. The preferred mass of a single serving of the beverage is described below.

Caffeine is widely reported to increase mental alertness. However, high levels of caffeine are known to impart unwanted bitterness to the beverage. Furthermore, some consumers are concerned about the perceived health risks of a high caffeine intake. The beverage preferably comprises 0.001 to 0.1 % by weight caffeine, more preferably 0.005 to 0.05% by weight caffeine.

As used herein the term“beverage” refers to a substantially aqueous composition. The beverage may be in any format. For example, it may be in a read y-to-d rink format or a concentrated format. A“ready-to-drink beverage” refers to a drinkable composition suitable for direct human consumption, and preferably comprises at least 85% water by weight, more preferably at least 90%, and most preferably at least 95%. A“concentrated beverage” refers to a beverage composition which typically requires dilution with an aqueous liquid (e.g. water or milk) prior to consumption, as such this format will typically have a higher solids content (and thus a lower water content) than a ready-to-drink format. For example, a concentrated beverage may preferably comprise at least 25% water by weight, more preferably at least 50%, and most preferably at least 75%.

The beverage of the invention is preferably packaged. Non-limiting examples of suitable packages include bottles, cans, cartons, pouches and sachets.

The beverage is preferably sanitised, e.g. by pasteurisation or sterilisation. The purpose of using a preservative in a beverage is mostly related to situations where sanitisation by heat is not sufficient to assure quality of the final product. In particular, ordinary filling operations can be unreliable and the alternative of aseptic filling too expensive or unavailable. It may be desirable to avoid hot filling because some packaging materials (particularly thin and lightweight packaging materials) may be sensitive to heat.

For convenience of transport and handling, it is preferred that the mass of the beverage in the package is no more than 2100g, preferably 1600g, more preferably no more than 1 10Og. It is particularly preferred that the beverage is packaged in an amount that constitutes a single serving. A single serving of the beverage in a ready-to-drink format preferably has a mass of less than 600g, more preferably less than 550g, and most preferably from 100g to 350g. For concentrated beverages, the beverage may be packaged in an amount that will give a single serving on dilution. If this is the case, the concentrated beverage preferably has a mass of less than 20g, more preferably less than 15g, and most preferably from 1 g to 10g.

It is preferred that the beverage has an acidic pH (i.e a pH of less than 7). In particular the pH (at 20°C) is preferably less than 5, more preferably less than 4.5 and most preferably from 2 to 4.

In order to achieve an acidic pH, the beverage preferably comprises one or more acidulant. Suitable acidulants include organic acids such as citric acid, malic acid, lactic acid, tartaric acid, ascorbic acid, phosphoric acid, and salts thereof. Mixtures of one or more of these acidulants are also suitable. A particularly well-balance flavour may be provided when the acidulant comprises malic acid and/or a salt thereof. Mixtures of citric acid (and/or its salt), malic acid (and/or its salt), and ascorbic acid (and/or its salt) also provide good flavour. Typically, the concentration of the acidulant in the beverage will be from 0.001 to 1 % by weight, more preferably from 0.01 to 0.5% by weight.

It is particularly preferred that the beverage is a flavoured beverage, more preferably a fruit-flavoured beverage, and most preferably a fruit-flavoured tea beverage. Suitable flavours include natural or synthetic fruit flavours, and/or natural or synthetic herb flavours. Examples of fruit flavours include: apple, apricot, blackcurrant, cherry, cranberry, grape, grapefruit, guava, kiwi, lemon, lime, lychee, mandarin, mango, nectarine, orange, peach, pear, pineapple, plum, passion fruit, raspberry, and strawberry. Examples of herb flavours include: chamomile, chrysanthemum, elderflower, hawthorn, hibiscus, jasmine, mint, osmanthus, rose, and verbena. Consumers prefer beverages with a sweet taste. Therefore, the beverage preferably comprises nutritive sweetener, non-nutritive sweetener, or mixtures thereof.

Non-nutritive sweeteners allow beverages to be formulated that have a low energy content, and yet still taste pleasantly sweet. Health-conscious consumers often prefer such beverages. Preferred examples of non-nutritive sweeteners include aspartame, saccharin, acesulfame K, glycyrrhizin, stevia-derived sweetening agents (for example: stevioside, rebaudioside A, rebaudioside C, ducloside A; preferred examples being stevioside and/or rebaudioside), sucralose, and mixtures thereof. Owing to their well- rounded flavour, the most preferred non-nutritive sweeteners are acesulfame K, aspartame, sucralose, rebaudioside A, or mixtures thereof. The concentration of non- nutritive sweetener will depend on the relative sweetness of the sweetener, and the composition of the beverage. Typically, the beverage will comprise non-nutritive sweetener in an amount of 0.00001 to 10% by weight of the beverage, more preferably 0.001 to 1 % by weight and most preferably 0.01 to 0.1 % by weight.

On the other hand, consumers may prefer the perceived naturalness of nutritive sweeteners. Examples of nutritive sweeteners include glucose, sucrose, fructose, and mixtures thereof. A particularly preferred example of a natural nutritive sweetener is honey.

The beverage may have a high calorie content (e.g. have an energy content of more than 100 kCal per 100 g of the beverage, preferably between 150 and 1000 kCal). Such beverages preferably comprise one or more nutritive sweetener(s), optionally in combination with one or more non-nutritive sweetener(s).

In one preferred embodiment, the beverage is a low-calorie beverage (e.g. having an energy content of less than 100 kCal per 100g of the beverage). It is particularly preferred that a single serving of the beverage has a total energy content of less than 10 kCal, more preferably less than 5 kCal, most preferably less than 1 kCal. The mass of the beverage in a ready-to-drink format which preferably constitutes a single serving is set out above. Low calorie beverages preferably comprise one or more non-nutritive sweetener(s). In designing foodstuffs which are protected against spoilage, some of the more commonly used preservatives are sorbic acid, and salts thereof (hereinafter referred to as“sorbates”). Sodium sorbate and potassium salts are most commonly used. The sorbates are particularly effective anti-fungal agents, and finding“natural” replacements has been a challenge. However, we have found that a preservative system comprising galangin can be used to replace sorbates in foodstuffs. Alternatively, the presence of galangin and/or plant extracts containing galangin in the foodstuff according to this invention, can allow the use of a reduced amount of sorbate, and still achieve the same anti-spoilage effect as the conventional full dosage of sorbate. A typical amount of potassium sorbate in foodstuffs is from 250 ppm to 1000 ppm. Thus, the foodstuff according to the present invention preferably comprises less than 200 ppm sorbates, more preferably less than 100 ppm sorbates, still more preferably less than 50 ppm sorbates, and most preferably less than 10 ppm sorbates.

Other food preservatives are used in the industry, including: benzoates, sulphites, nitrates, nitrites, propionates, natamycin, nisin, dimethyl dicarbonate, ethyl lauroyl arginate, and lysozyme. It has been found that galangin or plant extracts containing galangin can be used to replace or reduce the levels of the aforementioned preservatives. Typical use levels of these food preservatives are known in the art. Thus, this invention relates to foodstuffs containing a combination of galangin and/or a plant extract containing galangin and less than typical use level of a preservative selected from the group consisting of: benzoates, sulphites, nitrates, nitrites, propionates, natamycin, nisin, dimethyl dicarbonate, ethyl lauroyl arginate, and lysozyme. It is particularly preferred that the foodstuff according to the present invention comprises less than 200 ppm, more preferably less than 100 ppm, still more preferably less than 50 ppm, and most preferably less than 10 ppm of a preservative selected from the aforementioned group.

It is preferred that the foodstuff has an acidic pH (i.e a pH of less than 7). In particular the pH (at 20°C) is preferably less than 6, more preferably less than 5, and most preferably from 2 to 4.6.

In order to achieve an acidic pH, the foodstuff preferably comprises one or more acidulant. Suitable acidulants include organic acids such as citric acid, malic acid, lactic acid, tartaric acid, ascorbic acid, phosphoric acid, and salts thereof. Mixtures of one or more of these acidulants are also suitable. Typically, the concentration of the acidulant in the foodstuff will be from 0.001 to 1 % by weight, more preferably from 0.01 to 0.5% by weight.

The foodstuff of the invention is preferably packaged. Non-limiting examples of suitable packages include bottles, cans, cartons, pouches and sachets.

The foodstuff is preferably sanitised, e.g. by pasteurisation or sterilisation.

For convenience of transport and handling, it is preferred that the mass of the foodstuff in the package is no more than 2100g, preferably 1600g, more preferably no more than 1 10Og. The mass of the foodstuff in the package is preferably at least 100g, more preferably at least 150g, and most preferably at least 200g. Where the package is a can, bottle, carton or pouch, it is preferred that the mass of the foodstuff in the package is from 100g to 600g, more preferably from 150g to 550g, most preferably from 200g to 500g. Where the package is a sachet, it is preferred that the mass of the foodstuff is from 1 g to 20g, more preferably from 2g to 15g, most preferably from 5g to 12g.

The present invention relates to a method for preparing a preserved foodstuff comprising adding a composition comprising galangin to the foodstuff such that the concentration of galangin in the preserved foodstuff is 1 to 800 ppm, preferably 10 to 500 ppm.

It is preferred that the composition comprising galangin is a plant extract, more preferably an ethanolic plant extract. It is particularly preferred that the plant extract is an extract of the plant Alpinia officinarum, and is most preferably an extract of the root of Alpinia officinarum.

The method preferably comprises the step of packaging the preserved foodstuff, most preferably in a sealed container. Any suitable container may be used. Several non- limiting examples of suitable packaging formats have already been described above.

As set out above, the foodstuff is preferably sanitised. Thus the method preferably comprises the step of pasteurising or sterilising the foodstuff. For example, the preserved foodstuff may be pasteurised or sterilised and then packaged in a heat stable container. This is known as“hot filling”.

The method is preferably used to prepare the foodstuff described above, and consequently the preferred technical features described for the foodstuff also apply mutatis mutandis to the method.

The invention also relates to the use of galangin as a food preservative, and in particular to use of galangin to reduce or prevent the proliferation of one or more spoilage microorganisms in a foodstuff. It is particularly preferred that the invention relates to the use of galangin to reduce or prevent the proliferation of one or more fungal spoilage microorganism(s) in a foodstuff. Preferably the use of galangin reduces or prevents the proliferation of one or more fungal microorganism(s) selected from the group consisting of: Paecilomyces variotti, Aspergillus niger, Trichoderma harzanium, Trichoderma virens, Penicillium verrucosum, Neosartorya pseudofischeri, Byssochlamys nivea, Candida parapsilosis, Aureobasidium pullulans, Zygosaccharomyces bailii, Saccharomoyces cerevisiae, Candida valida, Candida magnoliae, Candida versatilis, Zygosaccharomyces rouxii, Eurotium herbariorum, Eurotium amstelodami, Penicillium roqueforti, Cladosporium cladosporioides, Penicillium brevicompactum, Aspergillus tamarii, Pichia membranaefaciens, issatchenkia orientalis, Penicillium expansum, Neosartorya fischeri, and Trichoderma atroviride.

As used herein the term“comprising” encompasses the terms“consisting essentially of” and“consisting of”. Where the term“comprising” is used, the listed steps or options need not be exhaustive. Except in the examples and comparative experiments, or where otherwise explicitly indicated, all numbers are to be understood as modified by the word “about”. As used herein, the indefinite article“a” or“an” and its corresponding definite article“the” means at least one, or one or more, unless specified otherwise.

Unless otherwise specified, numerical ranges expressed in the format "from x to y" are understood to include x and y. In specifying any range of values or amounts, any particular upper value or amount can be associated with any particular lower value or amount. All percentages and ratios contained herein are calculated by weight unless otherwise indicated. The various features of the present invention referred to in individual sections above apply, as appropriate, to other sections mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections as appropriate. Any section headings are added for convenience only, and are not intended to limit the disclosure in any way.

The following examples are intended to illustrate the invention and are not intended to limit the invention to those examples per se.

Examples

Example 1 : Determination of qalanqin levels in various extracts

A first Alpinia officinarum extract (E1 ) was obtained from a contract laboratory. This extract was obtained from dried and milled roots using methyl tert-butyl ether (MTBE) and methanol.

The content of galangin in the first extract was determined by HPLC-UV according to the procedure set out below, and was found to be 7.4% (w/w) galangin.

A second Alpinia officinarum extract (E2) was prepared as follows: Dried and milled roots of Alpinia officinarum (4.03 g) were extracted using an accelerated solvent extraction device (ASE 300, Dionex/Thermo) with ethanol as the extraction solvent. The extraction temperature was 60°C, the pressure in the extraction cell was 100 bar, and the extraction time was 10 minutes for each of the 3 cycles. The extraction was evaporated to dryness, giving a yield of 278 mg extract. The content of galangin in the second extract was determined by HPLC-UV according to the procedure set out below, and was found to be 8.4% (w/w) galangin. The content of galangin in the dried roots was thus calculated to be 0.58% (w/w).

A third Alpinia officinarum extract (E3) was obtained by extracting the dried, chopped roots of Alpinia officinarum with ethanol. The extraction was carried out overnight at a temperature of 25°C. The content of galangin in the third extract was determined by HPLC-UV according to the procedure set out below. The dried extract had a galangin content of 8.3% (w/w). A fourth Alpinia officinarum extract (E4) was a commercially available extract obtained from Basic Nutrition, China. This extract was described as an ethanol/water“bulb” extract (product name: galangal extract; batch number 20151016; extract ratio 10:1 ). The content of galangin in the third extract was determined by HPLC-UV according to the procedure set out below, and determined to be 0.02% (w/w).

This example shows that commercially available extracts of Alpinia officinarum have only a low amount of galangin. Furthermore, such extracts do not have a clear activity (see the data for extract E4 in Example 3 below).

HPLC procedure:

The HPLC system was controlled with the MassLynx 4.1 software, and consisted of an iCIass Binary Solvent Manager, an iCIass FTN Sample Manager, an iCIass Column Manager, a PDA eLambda Detector (all from Waters Corporation, Mildford, USA). The separations were performed with an HSS T3 column (100 A, 1.8 pm particle size, 2.1 mm x 100 mm), with an HSS T3 (100 A, 1 .8 pm particle size, 2.1 mm x 5 mm) guard column, operated at a temperature of 45°C. The diode array detector was set at an acquisition range of 210 to 600 nm at a spectral sampling rate of 20 points per second with a resolution of 1.2 nm. The mobile phase consisted of 1000/0.05 (v/v) water/formic acid as eluent A and 1000/0.05 (v/v) acetonitrile/formic acid as eluent B using a linear gradient as follows: 0% eluent B for 1 min, 0% to 35% eluent B in 5 min, 35% to 98% eluent B in 5 min, 98% eluent B for 7 min, 98% to 0% eluent B in 0.1 min, 0% eluent B for 3.9 min. The flow rate was 0.6 ml/min and the total run time was 22 min. The injection volumes were between 0.2 pi and 2 pi, and the extracts were dissolved to around 1 mg extract per ml of DMSO. The retention time of galangin was 7.93 min (± 0.05 min). The concentration of galangin was calculated using a calibration curve generated by measurements of a commercial reference sample of galangin at different concentrations. The peak areas were integrated at a wavelength of 358 nm.

Example 2: Fungal growth inhibition by Alpinia officinarum extracts

Several different Alpinia officinarum extracts were tested for growth inhibition of some common spoilage microorganisms. A panel of 4 yeasts ( Zygosaccharomyces bailii, Candida parapsilosis, Candida valida, Saccharomyces cerevisiae) and 2 moulds ( Aspergillus niger, Paecilomyces variotii) was used. In a typical experiment, cell suspensions of the appropriate microorganisms were incubated in 96 well microtiter plates in the presence of the Alpinia officinarum extracts. Stock solutions of these extracts were prepared in DMSO, water or ethanol. Different amounts of the stock solutions were dosed into the culture medium to achieve the concentrations that were tested.

The culture medium for the incubations was either YPD broth (20 g/L bacteriological peptone, 10 g/L yeast extract, 20 g/L glucose) or ice tea (non-sparkling, non-preserved peach flavour Lipton Ice Tea was used). Different pH conditions were applied (between pH 3 and pH 7), and the cultivation period was between 1 and 20 days with the temperature controlled so as to be 25°C. Growth of the spoilage microorganisms was determined visually at the given time point. Concentrations of galangin which were sufficient to inhibit spoilage microorganisms were represented by wells where the culture medium remained clear and free from discrete fungal colonies.

The experimental data below illustrates the growth inhibitory effect of various Alpinia officinarum extract preparations against food spoilage microorganisms. In each case, the conditions used are specified alongside the results.

Sample 1: pure galangin (CAS No. 548 83-4)

Galangin was found to be active against a panel of food spoilage microorganisms consisting of 6 yeasts and moulds grown in either YPD medium (pH 6.5 to 6.8) or Ice Tea medium (peach flavour Lipton Ice Tea, pH 3.2).

Table 1 : Anti-fungal activity of sample 1

Sample 2: commercially available extract

This sample is the commercially available extract E4 from Example 1 (0.02% w/w galangin). Its anti-fungal activity could not be determined, most likely because the galangin content is too low to be effective. Based on the MIC of galangin (Sample 1 ), the extract would need to be added to the culture medium at 20 to 50% in order to inhibit growth of yeast. Although a sample containing 10% DMSO and 22.8 pg/ml extract E4 (i.e. 0.05 pg/ml galangin) was found to inhibit both Z. bailii and C. parapsilosis (Ice Tea medium, pH 3.2), this was also the case for a control sample containing 10% DMSO (and no extract). Thus the inhibition observed with extract E4 could not be distinguished from that seem with DMSO alone. A further control sample containing 5% DMSO (and no extract) did not inhibit the growth of Z. bailii and C. parapsilosis (Ice Tea medium, pH 3.2). However, neither did a sample containing 5% DMSO and 1 1.4 pg/ml extract E4 (i.e. 0.025 pg/ml galangin). Sample 3

A fifth Alpinia officinarum extract (E5) was obtained from dried and milled roots using MTBE/methanol following the general protocol described for E2 (see Example 1 above). The content of galangin was determined to be 5.4% (w/w). The extract was found to have anti-fungal activity (see Table 2).

Sample 4: E2 from Example 1

The preparation of this ethanolic extract (8.4% w/w galangin) is described in Example 1. The extract was found to have anti-fungal activity (see Table 2). Sample 5

A sixth Alpinia officinarum extract (E6) was obtained from dried and milled roots using ethanol following the general protocol described for E2 (see Example 1 above). The content of galangin was determined to be 7.5% (w/w). The extract was found to have anti-fungal activity (see Table 2). Table 2: Anti-fungal activity of Alpinia officinarum extracts

^† Contained 0.5% acetic acid (undissociated)

* ND = not determined

Sample 6

A seventh Alpinia officinarum extract (E7) was obtained by extracting roots with food- grade ethanol following the general protocol described for E3 (see Example 1 above). The content of galangin was determined to be 8.2% (w/w). The extract was found to have anti-fungal activity (see Table 2).

Example 3: Effect of heat stress on anti-fungal activity of galangin

The heat sensitivity of galangin was evaluated by testing the inhibition of Zygosaccharomyces bailii growth after exposure to different heat regimes. YPD broth (pH 6.5) containing different concentrations of galangin was heated to 95°C for 30 seconds, 95°C for 30 minutes, or 80°C for 30 minutes. When assessed after 4 days, the MIC of galangin was 50 pg/ml for both heat treated and untreated samples. When assessed after 1 1 days, the MIC of galangin was 100 pg/ml for samples heated to 95°C, and 50 pg/ml for the sample treated at 80°C and the untreated sample. This demonstrates that galangin is not sensitive to heat, and can thus resist food-processing steps involving heat treatments. Example 4: Spectrum of anti-fungal activity

An eighth Alpinia officinarum extract (E8) was obtained by extracting roots with food- grade ethanol following the general protocol described for E3 (see Example 1 above). The content of galangin was determined to be 15.4% (w/w). The extract was tested against two panels of moulds: one panel representative of spoilage strains for spreads and one panel representative of spoilage strains for beverages.

Anti-fungal activity against representative spoilage strains for foodstuffs

A panel of moulds ( Penicillium roqueforti, Paecilomyces variotti, Aspergillus niger, Trichoderma harzianum) were cultivated in YPD Broth (pH 6.8) for 14 days in the presence of Alpinia officinarum extract. The concentration of galangin in the medium was from 0 pg/ml to 300 pg/ml.

The ethanolic Alpinia officinarum extract had anti-fungal activity against most of the different mould strains investigated, with a MIC between 25 pg/ml and 300 pg/ml galangin (depending on the strain). However, growth of Aspergillus niger was not found to be inhibited by the concentration range of galangin that was tested.

Anti-fungal activity against a broader range of representative spoilage strains for foodstuffs

A panel of moulds ( Penicillium verrucosum, Penicillium expansum, Aspergillus niger, Neosartorya pseudofischeri, Neosartorya fischeri, Paecilomyces variotti, Trichoderma harzianum, Trichoderma atroviride, Trichoderma virens, Byssochlamys nivea) were cultivated in Iced Tea medium (pH 3.2) for 14 days in the presence of Alpinia officinarum extract. The concentration of galangin in the medium was from 0 pg/ml to 300 pg/ml.

The ethanolic Alpinia officinarum extract had anti-fungal activity against most of the different mould strains investigated, with a MIC between 12.5 pg/ml and 300 pg/ml galangin (depending on the strain). However, growth of Aspergillus niger was not found to be inhibited by the concentration range of galangin that was tested.