FIELD OF THE INVENTION

The present invention relates to a process for converting plant materials to plant extracts having antimicrobial activity. The invention also relates to plant extracts originating from plant materials, having antimicrobial activity. The invention further relates to the use of said extracts in cosmetics, hygiene products, nutraceuticals, food products and food supplements, feeds, packages and in pharmaceutical products.

BACKGROUND OF THE INVENTION

In the industry dealing with processing of plant materials, such as food industry processing berries and fruits, significant amounts of waste materials, side streams and by-products are formed. Food industry uses wild berries, cultured berries and fruits in the manufacture of wide range of products, such as pastes, beverages, alcohol products, jams, conserves, milk based products, sweets and the like. The use of berry and fruit fractions has also become very popular in cosmetic products and for example cloudberry seed oil, rich in polyunsaturated fatty acids, is regarded as a valuable component in cosmetic preparations.

Large volumes of waste are produced in the processing of plant materials, such as berries and fruit, particularly in the food industry, which waste material is utilized to a very small extent. Most of the waste material is currently discarded or transported to landfill or dumping area, thus increasing the environmental burden. Some of this waste material is subjected to drying followed by extraction of seed oils, however only a very small portion of the material is used.

Typically, in the processing, berries and fruit are mechanically cleaned, followed by removing of the juice, pulp or paste by suitable methods, such as pressing. The remaining waste material, such as pomace, berry cake, fruit cake or press cake comprises berry or fruit skins, peels, seeds and pith, which contain fibers, bioactive phenolic compounds, and other bioactive compounds.

FI 122664 B discloses a method for fractionating berries and separating nutrients from the fractions formed, in which method berry raw material is dried and ground lightly, so that the seeds of the berry separate from the fruit flesh and skin portion without breaking, followed by a second light grinding, which is carried out on the formed seedless fruit flesh and skin fraction, whereby a fine powder is formed, which is screened or classified. Seeds are not fractionated by this method. Seed are discarded and peels are further fractionated.

US 2013/0040005 Al relates to an antihypertensive agent comprising boysenberry seed extract as active ingredient and to a method for obtaining said agent. In said method boysenberry pomace is dried, crushed and sieved to separate the seed, followed by grinding the seed to fine powder, which is extracted with water or organic solvent, followed by contacting the extraction solution with a polyphenol absorbent and eluting with alcohol based solvent to obtain the target extract. Particularly, in the cosmetic products, skin care products, household products and hygiene products there is a need to replace the synthetic preservatives, such as parabens and triclosan with natural preservatives. It is expected that the use of currently used preservatives will be restricted in the future. Based on the above, it can be seen that there exists a need to provide safe natural preservatives for replacing currently used preservatives, and to provide a method for producing products having antimicrobial activity.

SUM MARY

In the present invention, it was found that plant extracts having antimicrobial activity can be obtained from plant materials with a simple and environmentally safe method.

The present invention is particularly based on studies on combined grinding and processing of plant material selected from the group consisting of seeds, berries, fruit, leaves, pomace, press cake, berry cake, fruit cake, side streams comprising leaves, sediments obtained from berries and/or fruit and/or oil plants, and combinations thereof, and the use of the obtained products. These materials are rich in dietary fibre and phenolic compounds having antioxidant and/or antimicrobial activity. The invention provides a very convenient and effective means particularly for utilizing seeds, leaves, waste materials, side streams and by-products from industry utilizing plant materials, such as berry and fruit and oil plant industry, where the plant material may comprise seeds, berries, fruit, leaves, pomace, press cake, berry cake and fruit cake, or sediment in producing plant extracts having particularly high antimicrobial activity, useful in cosmetics, skin care products, household products, hygiene products, food supplements, food products, feeds, packages and in pharmaceutical products, particularly as natural antimicrobials or natural preservatives.

An object of the invention is to provide a process for converting plant materials to plant extracts having antimicrobial activity.

A further object of the invention is to utilize seeds, berries, fruit, leaves, and waste materials, side streams and by-products from industry utilizing plant materials without the need to discard said materials. A further object of the invention is to provide plant extracts having antioxidant and/or antimicrobial activity from plant materials.

A still further object of the invention is the use of said plant extracts having antioxidant and/or antimicrobial activity in cosmetics, skin care products, hygiene products, household products, nutraceuticals, food products, food supplements, feeds, petfood, packages and in pharmaceutical products.

The invention is directed to a process for converting plant material, which process comprises the steps, where in the first step at least one plant material is ground from 1 to 5 passes in the presence of a first medium or without a medium, to obtain particles having average particle size from 10 to 1000 μιη;

the second step the particles are mixed with a second medium in a ratio from 1 : 3 to 1 : 80 by weight, at a temperature from 10 to 120°C for 20 min to 20 hours to obtain a mixture comprising bioactive compounds, and the mixture is subjected to separation whereby a plant extract is separated from a plant residue.

The obtained plant extract may be blended with a cosmetic product, skin care products hygiene product, household product, nutraceutical, food product, food supplement, feed, petfood, package and pharmaceutical product, or with starting materials of said product during the manufacture of the product.

The antimicrobially active compounds particularly comprise phenolic compounds having antimicrobial activity, such as ellagic acid and ellagitannins. The invention is further directed to the use of the plant extracts in cosmetics, skin care products, hygiene products, household products, nutraceuticals, food products, food supplements, feeds, petfood, packages and in pharmaceutical products.

Accordingly, the present invention provides simple and economic means for utilizing plant materials, such as seeds, leaves, waste materials, side streams and by-products originating from industry processing plant materials, such as berry industry and fruit industry, in the manufacture of plant extracts having antimicrobial activity, for providing natural preservatives. The characteristic features of the invention are presented in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 1 shows the antimicrobial effect of plant extracts obtained with the process of the invention, against Staphylococcus aureus.

Figure 2 shows antimicrobial activity of plant extracts obtained with the process of the invention on Escherichia coli.

Figure 3 shows antimicrobial effect of plant extracts, obtained with the process of the invention, on Staphylococcus aureus.

Figure 4 shows antimicrobial effect of plant extracts, obtained with the process of the invention, on Pseudomonas aeruginosa.

Figure 5 shows the HPLC chromatogram of the hydrothermal extract of raspberry leaf. Figure 6 shows the specific spectra of the main compounds of the hydrothermal extract of raspberry leaf.

Figure 7 shows the antimicrobial effect of hydrothermal extracts of ground raspberry leaves against Staphylococcus aureus.

Figure 8 illustrates the HPLC chromatograms of the hydrothermal extraction residues.

DEFINITIONS

Unless otherwise specified, the terms, which are used in the specification and claims, have the meanings commonly used in the field of food industry and industry processing plant materials. Specifically, the following terms have the meanings indicated below.

The term "berry" is understood here to mean all edible, wild and cultivated berries comprising internal seeds, which berries belong to the genus Rubus, Sorbus, Empetrum, Rosa, Aronia, Vaccinium, Ribes, Fragaria, Juniperus or Hippophae, including all hybrid berries of these genera. Sea buckthorn is an example of the genus Hippophae. Raspberries, blackberries, arctic bramble, dewberries and cloudberries are examples of the Rubus species. As the Rubus species readily interbreed and are apomicts, the parentage of the hybrid plants is often highly complex, but it is generally agreed to include in the definition cultivars of blackberries and raspberries. Examples of said hybrid berries include loganberry, boysenberry, veitchberry, marionberry, silvanberry, tayberry, tummelberry and hildaberry. Bilberry, blueberry, lingonberry and cranberry are examples of the Vaccinium species. Blackcurrant, redcurrant, whitecurrant, greencurrant and gooseberry are examples of the Ribes species. An example of the Juniperus species is juniper (Juniperus communis). The term "fruit" is understood here to mean all wild and cultivated fruit belonging to the genus Vitis, Punica, Pyrus, Malus, Citrullus, Benincasa, Cucumis, Momordica, Olea and species Argania spinosa, including all hybrid fruit of these genera or species.

Fruit of the genus Vitis mean grapes comprising internal seeds, including all hybrid grapes, which are primarily crosses between V. vinifera and another grapevine. Grapes are used for making wine, jam, beverages, jelly, seed extract, raisins, vinegar, and grape seed oil. Vitis is a genus of about 60 vining plants in the family Vitaceae.

Fruit of the genus Citrullus, Benincasa, Cucumis, Momordica mean melons comprising internal seeds, including all hybrid melons.

Fruit of the genus Olea means olives, particularly Olea europaea.

Fruits refers also to fruits of fruit trees, such as pomegranate (Punica granatum), pear trees (Pyrus family, such as Pyrus communis) and apple trees (Malus family) and all hybrids thereof.

The term "oil plant" is understood to mean here plants comprising seeds rich in plant oil, said plants being selected from turnip rape (Brassica rapa), sunflower, flax, hemp (Cannabis sativa) and rape (Brassica napus).

The terms "berry industry" and "fruit industry" and "oil plant industry" refer to industry dealing with the development, refining and manufacture of products and processes relating to wild and cultivated berries and fruit and oil plants. The term "antimicrobially active" compound refers here to compounds being able to kill microorganisms or inhibit their growth. Examples of these compounds are phenolic compounds, such as ellagic acid and ellagitannins. In addition to antimicrobial and preservative activity, these compounds often have other biological activities, particularly antioxidant activity.

The term "hydrothermal" processing means here processing in the presence of water at elevated temperatures.

Hygiene products include here particularly diapers, female hygiene pads, tampons, incontinence pads and products, and the like.

Household products refer here to dishwashing liquids, household cleaning agents, liquid soaps, shower gels and the like. Machine parts refer here to membranes and filters in industrial and household washing machines and dishwashing machines.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a convenient process for converting plant materials to plant extracts having antimicrobial activity.

In the present invention, it was found that plant extracts obtained by the process show particularly strong antimicrobial activity for example against the human pathogen Staphylococcus aureus.

Recent studies have shown that phenolic compounds, such as ellagic acid and ellagitannins possess many interesting biological activities and thus they may play preventive role in disease prevention. Remarkable amounts of biologically active phenolic compounds, particularly ellagic acid and/or ellagitannins and their derivatives are contained plant materials. For example, said compounds remain in the waste material, which is currently not utilized. Phenolic compounds, such as ellagic acid and ellagitannins are natural antioxidants and preservatives having strong antimicrobial effect, thus useful as natural preservatives and antioxidants in various applications in the field of cosmetics, hygiene products, food industry and feed industry, as well as in packaging industry and pharmaceutical industry. In cosmetics, skin care and hygiene products the invention may be utilized to provide plant extracts that stabilize, balance and protect healthy skin microbiome, as these plant extracts exhibit antimicrobial activity, which inhibit the growth of pathogenic microbes without effecting the growth of beneficial microbes. With the process of the invention plant material is ground to obtain finely divided particles as dry powder or as a suspension, and the particles are subjected to extraction followed by separation, whereby a plant extract having high antimicrobial activity and a plant residue are obtained. Particularly, the invention is directed to a process for converting plant materials, which process comprises the steps, where in the first step at least one plant material, is ground from 1 to 5 passes in the presence of a first medium or without a medium, to obtain particles having average particle size from 10 to 1000 μιη;

in the second step the particles are mixed with a second medium in a ratio from 1 : 3 to 1 : 80 by weight, at a temperature from 10 to 120°C for 20 min to 20 hours to obtain a mixture comprising bioactive compounds, and the mixture is subjected to separation whereby a plant extract is separated from a plant residue.

The plant residue separated after the extraction, from the plant extract may be used particularly in nutraceuticals, food products, food supplements, petfood and feeds because of high contents of ellagic acid, C18 fatty acids, lignin and fibres, and in cosmetic products, particularly in masks and peeling products because of the solid particles contained in the residue. Ellagic acid is more easily absorbed in the GI tract than ellagitannins typically present in the natural berries and fruit.

Plant material

The plant material is selected from seeds, berries, fruits, leaves, pomace, press cake, berry cake, fruit cake, and from waste materials, sediments, side streams and by-products originating from industry processing plant materials, and any combinations thereof.

In an embodiment, the plant material is selected from berry material, fruit material, oil plant material and combinations thereof.

The berry material is selected from seeds, berries, leaves, pomace, press cake, berry cake, and from waste materials, sediments, side streams and by-products, originating from industry processing berry materials, and any combinations thereof. The fruit material is selected from seeds, fruits, leaves, pomace, press cake, fruit cake, and from waste materials, sediments, side streams and by-products, originating from industry processing fruit materials, and any combinations thereof.

The oil plant material is selected from seeds, fruits, leaves, pomace, press cake, fruit cake, and from waste materials, sediments, side streams and by-products, originating from industry processing oil plant materials, and any combinations thereof.

Berries

In the present invention, species of all edible wild berries, cultivated berries and all hybrid berries of the genus Rubus, Sorbus, Empetrum, Rosa, Aronia, Vaccinium, Ribes, Fragaria, Juniperus and Hippophae and any combinations thereof may be used. Raspberries, blackberries, arctic bramble (synonym arctic raspberries), dewberries and cloudberries, and hybrid berries including loganberry and boysenberry are examples of the Rubus species suitable for the invention. Rowanberry is an example of Sorbus species, crowberry of Empetrum species, rose hip and dog rose of Rosa species, chokeberry of Aronia species and sea buckthorn berry of Hippophae species suitable for the invention. Bilberry, blueberry, lingonberry and cranberry are examples of the Vaccinium species suitable for the invention. Blackcurrant, redcurrant, whitecurrant, greencurrant and gooseberry are examples of the Ribes species suitable for the invention. An example of the Juniperus species suitable for the invention is juniper (Juniperus communis).

Cloudberries are valuable wild berries having high aroma content and they contain also valuable seed oil. Cloudberries are used in food, liqueur and cosmetic industry, however only the seed oil is presently utilized from the waste material remaining after pressing the berries. Ellagic acid content found in cloudberry is in the berry fruit (fruit + seeds) 0.6 mg/g dry weight. Ellagitannin content found in cloudberry is in the berry fruit 24,6 mg/g dry weight.

Arctic brambles contain very high ellagic acid and ellagitannin contents and thus they are also particularly suitable as raw material for the process of the invention. From the ecological point of view, wild berries, such as cloudberries and arctic bramble which have grown without any manmade fertilizers or pesticides, are particularly suitable.

Fruits

Species of the fruits of the genus Vitis, Punica, Pyrus, Malus, Citrullus, Benincasa, Cucumis, Momordica, Olea, and of species Argania spinosa are suitable for the process of the invention. All grapes and all hybrid grapes, containing seeds and belonging to the genus Vitis may be used in the present invention. Typically, huge amounts of waste material are obtained from processing of grapevines, such as from pressing grapevines and thus grapes offer also a particularly suitable raw material source for the present invention. Fruits of fruit tree, such as pomegranate trees (such as Punica granatum) , pear trees (such as Pyrus communis) and apple trees (Malus family), including all hybrids thereof are also suitable raw material source. Remarkable amounts of waste material are obtained during processing of these fruits. Oil plants

Species of the genus selected from turnip rape (Brassica rapa), sunflower, flax, hemp (Cannabis sativa) and rape (Brassica napus), including all hybrids thereof are suitable for the process of the invention. The berry materials, fruit materials and oil plant materials suitable for the process of the invention may be selected from seeds, leaves, berries, fruits, and from by-products, side streams and waste materials, originating from processing of berries, fruit or oil plants. Examples of such by-products, side streams and waste materials are press cakes, pomaces, berry cakes, fruit cakes and fractionating residues. Said by-products, side streams and waste material typically comprise berry or fruit skins or peels, seeds, pulp, leaves, arbors and conifer needles, depending also how well the berries or fruit or seeds are cleaned mechanically before processing.

Processing of berries, fruit or oil plants may be carried out for example at a facility carrying out processing or refining or fractionating of berries, fruit or oil plants, or at a facility in the food or feed processing industry, from the manufacture of beverages, pastes, purees, wines, jams, conserves, sweets and the like. Particularly preferably by-products, side streams and waste materials are used in the present invention. Typically, in a juicing line, berries or fruit are pressed and the remaining press cake is frozen and stored at approx. -20°C for further use, or alternatively it may be dried.

Berry and fruit pastes and purees are obtained for example by squeezing berries or fruit through a sieve and the remaining berry cake or fruit cake is frozen and stored at approx. -20°C, or alternatively it may be dried. The obtained frozen pomace, berry cake or fruit cake may contain from 40 to 70 % by weight of water, typically from 50 to 60 % by weight of water.

The by-products, side streams, waste material obtained from the berries of the genus Rubus or grapes from the genus Vitis, such as press cake or pomace contains predominantly seeds, skins or peels and some pulp.

Seeds may comprise small amounts of any of skin, peel and pulp.

Process

The present invention is directed to a process for converting plant materials, which process comprises the steps, where in the first step at least one plant material is ground from 1 to 5 passes in the presence of a first medium or without a medium, to obtain particles having average particle size from 10 to 1000 μιη;

the second step the particles are mixed with a second medium in a ratio from 1 : 3 to 1 : 80 by weight, at a temperature from 10 to 120°C for 20 min to 20 hours to obtain a mixture comprising bioactive compounds, and the mixture is subjected to separation whereby a plant extract is separated from a plant residue.

The present invention is particularly directed to a process for converting plant materials to plant extracts having antimicrobial activity.

Preferably the plant material is selected from seeds, leaves, fruit, pomace, press cake, sediment and combinations thereof, originating from berries or fruits or oil plants selected from the genus Rubus, Sorbus, Rosa, Empetrum, Aronia and Hippophae and from combinations thereof, and the fruit are selected from the genus Vitis, Punica, Pyrus, Malus, Citrullus, Benincasa, Cucumis, Momordica, Olea and from species Argania spinosa, and combinations thereof. Particularly preferably plant materials originating from raspberry are used. First step

In the first step, at least one plant material is ground from 1 to 5 passes in the presence of a first medium or without a medium, to obtain particles having average particle size from 10 to 1000 μιη. The grinding may be carried out in the presence of a first medium or without a medium. In an embodiment, at least the first pass may be carried out without the first medium. The first medium may be selected from water, ethanol, ethyl acetate, acetone, methylethyl ketone, glycerol and combinations thereof. When food grade product is produced preferably water or ethanol is used.

The grinding (or milling) in the presence of a first medium is carried out in a stone mill, such as a colloid mill or a supermasscolloider. Said grinding is carried out from 1 to 5 passes in the presence of a medium, preferably from 2 to 4 passes, whereby a milled suspension is obtained. In an embodiment, the grinding is carried out from 2 to 3 passes. Particularly preferably water is used as the first medium (wet grinding). Optionally, the plant material may be preground using a homogenizer, twin screw refiner or a coarse mill. When pregrinding is used, often one pass with the stone mill is sufficient.

In an embodiment, the grinding (or milling) without a medium (dry grinding) is carried out in a mill or grinder selected from the group consisting of fine impact mill, jet mill, mortar grinder, disc mill, rotor mill, hammer mill, ultra-centrifugal mill, turborotor mill, classifier mill and a combination of these.

Examples of these mills are Masuko Ceren-miller DAU, Netzsch CGS Fluidized Bed Jet Mill, Hosokawa Alpine Fine Impact Mill Ultraplex UPZ. Materials can be preground using e.g. cutting mill, hammer mill or disc mill. Also, drying can be done simultaneously with grinding using e.g. Gorgens Turborotor mill.

The dry grinding is carried out from 1 to 5 passes and with varying rotor speeds. The grinding parameters are set according to the instructions provided by the mill suppliers. In the grinding in the presence of a first medium, the medium, suitably water, is added to the plant material to obtain dry matter content of 3 - 30 % by weight, preferably 5 - 25 % by weight in the milled suspension. The plant material may also contain the first medium (water) in an amount whereby no additional medium is needed. The first medium provides for effective grinding. The grinding apparatus is suitably adjusted according to the instructions provided by the mill supplier.

From the grinding in the presence of a first medium a homogeneous gel or suspension comprising particles is obtained, said particles having an average particle size from 10 to 1000 μιη. Suitably the average particle size distribution by laser diffraction is D50 = 10- 200 μιη and D90= 100-1000 μιη. The obtained ground suspension comprising the particles stays homogeneous, even at heating. Thus, after grinding the suspension can be frozen or used immediately in the second step without the risk of phase separation.

Second step

In the second step the particles obtained in the first step are mixed with a second medium, in a ratio from 1 : 3 to 1 : 80 by weight, at a temperature from 10 to 120°C for 20 min to 20 hours to obtain a mixture, and the mixture is subjected to separation whereby a plant extract is separated from a plant residue. The second medium is selected from water, ethanol, ethyl acetate, acetone and combinations thereof. When food grade products are produced water or ethanol is used. Preferably water is used, where the second step can be called as hydrothermal extraction.

In an embodiment in the second step the ratio of the particles (or suspension) obtained from the first step carried out in the presence of the first medium to the second medium is from 1 : 3 to 1 : 80 by weight, preferably from 1 : 5 to 1 : 50, respectively. In a preferable embodiment, in the second step the particles are mixed with a second medium in a ratio from 1 : 10 to 1 : 30 by weight. In an embodiment, in the second step, the ratio of the particles (or suspension) obtained from the first step carried out in the presence of the first medium to the second medium is from 1 : 5 to 1 :40, respectively.

In an embodiment, the particles or the suspension obtained from the first step is mixed with the aqueous medium, typically using mixing speed 100-1500rpm. Any suitable mixing devices may be used.

In an embodiment, the second step is carried out in the presence of an acid. The acid is selected from ascorbic acid, oxalic acid, citric acid, acetic acid, malic acid, benzoic acid and HCI, preferably ascorbic acid is used. The concentration of the acid in the second medium is from 0.01 to 0.2 % by weight, preferably from 0.05 to 0.1 % by weight. Preferably the second medium is water and the acid is ascorbic acid. The mild acidic conditions have stabilizing effect on anthocyanins. In an embodiment in the second step the mixing temperature may range from 20 to 95°C, preferably from 25 to 90°C. In an embodiment, in the second step the mixing may be carried out in at least two mixing steps, where the temperature in the first mixing step is 10-50°C, preferably 30-47°C, and the temperature in the second mixing step is 50-120°C, preferably 60-95°C.

In an embodiment, the mixing is carried out under a pressure from 1 to 10 bar, preferably from 1 to 2 bar.

In an embodiment, where the mixing is carried out in at least two mixing steps, the mixing time in the first mixing step is 30 min-12 h, preferably, 1-4 h, and the mixing time in the second mixing step is 30 min 12 h, preferably 30 min - 7 h.

Optionally, enzymes may be used in the in the second step of the process.

In an embodiment, in the second step, at least one enzyme may be added to the second medium. Preferably the second medium is water. The enzyme is suitably selected from carbohydrate hydrolyzing enzymes, suitably from cellulase, pectinase, xylanase and combinations thereof. The enzyme is dosed based on their main activity (e.g. 100-200 nkat/g or 0.01-1 % by weight). The enzyme is diluted in water before mixing with the second medium. Depending on the enzyme the treatment is carried out either at a pH optimal for each enzyme or at the intrinsic pH of the used material. When an enzyme is used, the second step is carried at a temperature of 40-47°C for 1-4 hours, and then from 30 min to 7 hours at 50-90°C.

In the second step the separation of the plant extract from the plant residue (solid materials), is carried out using suitable methods, such as centrifuging, filtration, decanting and the like.

In an embodiment, the plant extract, obtained in the second step may be used as such in the manufacture of products, or it may be concentrated, frozen or dried. The concentration may be carried out as vacuum evaporation, pressure filtration, or using a decanter centrifuge.

In an embodiment, the plant extract, obtained in the second step is dried. The drying may be carried out by freeze-drying, spray-drying, flash-drying drying at elevated temperature, in drying cabinets, and the like. Suitably the drying is carried out at a temperature from 40 to 150°C, suitably spray-drying at 80- 95°C. The second medium obtained in the drying step may be recycled to the first or second step of the process. Pretreatment of plant material

In an embodiment of the invention, the plant material is pretreated prior to subjecting it to the process. The pretreatment may be carried by subjecting the plant material to methods selected from heat treatment, fermentation, enzymatic treatment, extraction with ethanol, pressing, squeezing, crushing and combinations thereof.

In an embodiment of the invention, the plant material is heat treated for the removal of harmful microbes. Suitably the heat treatment is carried out at 75990°C for 5-10 min.

In an embodiment of the invention, the plant material is fermented using lactic acid bacteria or yeasts to modify the phenolic compounds and the carbohydrate components of the seed. Preferably the starter culture is selected from the genera Lactococcus, Lactobacillus, Pediococcus, Oenococcus and native yeasts originating from the plant material used in these fermentations. In the fermentation, typically frozen plant material and water, suitably ultra-pure water, are mixed together (1 : 1) and heated, suitably at 80°C for 5 min. The mixture is cooled, suitably in an ice bath and if needed berry or fruit material is crushed. The pH of the mixture is adjusted to approx. pH 5.0, suitably with 5 N sodium hydroxide. The microbes are pre-grown in food-grade media. The fermentation is carried out in a bioreactor (a vessel) etc., for example for 3 days at 30°C under constant mixing. Lactic acid bacteria fermentations are purged with sterile nitrogen gas to create anaerobic conditions.

In an embodiment of the invention, the plant material is pretreated with carbohydrate hydrolyzing enzymes. The plant material is pressed or squeezed after the enzyme incubation. Suitably the enzyme is selected from cellulase, pectinase, xylanase and combinations thereof.

In the enzyme treatment, the enzymes are dosed based on their main activity (e.g. 100- 200 nkat/g or 0.01-1 % by weight). Thawed, mashed and heated (40-45 °C) plant material is incubated at 40-45 °C for 2-4 hours. Enzyme is diluted in water before mixing with the mashed and heated plant materials. The treatments are carried out at the intrinsic pH of the used material (about pH 3). After enzyme incubations, juice is extracted by a juice pressing device. Enzyme treatment typically increases the yields of the plant extract and compounds therein. In an embodiment of the invention, the plant material is dried prior to introducing to the process, for removing excess water, until it has water content of not more than 15 wt%, preferably 0.1-10 wt%. The drying may be carried out as convective drying, such as hot- air drying, vacuum drying or steam drying, flash drying, microwave drying with or without vacuum drying, or freeze-drying. The drying may be carried out using a fluid-bed drier at a temperature from 35 to 70°C preferably 35-45°C. Suitably the freeze drying is carried out at a temperature from -40 to 0°C, and convective drying at a temperature from 40 to 70°C, preferably from 40 to 50°C. Any conventional drying devices suitable for the drying can be used. In an embodiment, the plant material is crushed prior to the processing, suitably using compression crushing to break lumps of skin, leaves, peels, pulp etc from the seeds and to cause minimum damage to the seeds. The crusher may be selected from roll crusher, a ball crusher, manual type crusher, a kneader grinder or a combination thereof. A kneader grinder may be used as the crusher, by which the seeds of the berry are detached from the dry pulp and skin portion without breaking the seeds. A suitable grinder is a falling number mill containing a rotating rotor and a stationary stator. This mill provides a kneading and slightly cutting and striking effect, wherein the grinding energy is, however, not sufficient to break the seeds, but they are detached from the matrix. A disc mill or an impact mill with a guided impact is preferably used, whereby a gentler grinding process is achieved.

The plant extracts (extracts contained in the second medium or dry extracts), obtained with process of the invention are rich in phenolic compounds, such as ellagic acid, ellagitannins and their derivatives and other bioactive compounds, and they also may contain C18 fatty acids.

Said plant extracts can be used as natural preservatives in cosmetic products and skin care products including masks and peeling creams, hygiene products, household products, machine parts such as membranes and filters, nutraceuticals, food products, food supplements, animal feeds, petfood, packages and in pharmaceutical products.

The plant extracts with antimicrobial activity, obtained with process of the invention have particularly good effect against Staphylococcus aureus, as can be seen from the examples, they have also effect against Pseudomonas aeruginosa and Escherichia coli. In skin care and hygiene products, the plant extracts may be utilized to stabilize, balance and protect healthy skin microbiome, as these fractions inhibit the growth of many pathogenic microbes without effecting the growth of beneficial microbes.

The plant extracts, obtained with process of the invention are suitably incorporated in food products, in cosmetic products, in hygiene products, in household products, in pharmaceutical products, in machine parts, nutraceuticals, food products, food supplements in animal feeds, in petfood, in packaging materials, particularly in packaging materials of products, such as food which is easily spoiled. Examples of such products are topical products like creams, ointments, skin care products, diapers, female hygiene pads, tampons, incontinence pads and diapers and the like.

Examples of said easily spoiled food products are poultry products, marinades, milk based products, such as yoghurts, drinks, sour cream products, fermented milk based products; jams, beverages, berry soups, conserves, pastes, purees, babyfood; nutritional food products, particularly for special use, such as hospital use and hose administration; grain products, such as bread, cereals, snack products, muesli, precooked porridge, fermented grain based products and gluten-free products.

The plant residues contain typically high amounts of ellagic acid (approx. 80 g/kg), C18 fatty acids, fibers and lignin, and thus they provide an excellent additive particularly for nutraceuticals, food products, petfood, food supplements and feeds.

The present invention provides several advantages. The seeds, leaves, by-products, side streams and waste materials originating from industry processing plant materials, such as berries, fruit and oil plants can be effectively utilized in the simple and economic process of the invention, for obtaining plant extracts with antimicrobial activity. The leaves may be harvested separately, or simultaneously with berries or fruit.

Said plant extracts with antimicrobial activity may be used as effective antioxidants, antimicrobial agents and preservatives.

Dumping of the waste materials to the landfills can be avoided or at least significantly reduced. This is also a clear environmental and ecological benefit. Nutritionally rich and valuable waste materials and by-products from the plant processing industry can be utilized in a simple and efficient way in various products, such as food products and animal feeds, as well as in packages, cosmetics and in pharmaceutical products. The antimicrobial effect of the plant extract is dose dependent.

The invention provides improved storage time and microbiological safety to the products, as these plant extracts with antimicrobial activity act as preservatives. It is possible to decrease the amount of synthetic preservatives in the products and replace them by these natural compounds. In addition, in cosmetic products these natural compounds also balance skin microbiota, as they effectively inhibit the growth of skin pathogens, such as Staphylococcus.

The invention provides improved microbiological preservation, improved inhibition of oxidation reactions and increased antioxidant status to the products where they are incorporated. In general, this means improved stability and microbiological safety. The plant extracts are typically readily soluble in water, which is an important technological benefit. The plant extracts contain no or only very limited amounts of free sugars, which improves stability of the products. Thus, the plant extracts are not sticky and easily stored in frozen form. The whole procedure to prepare plant extracts may be carried out asa food grade process, no toxic or harmful reagents or process steps can be avoided. The extracts are particularly suitable also for food purposes.

EXAMPLES

The following examples are illustrative of embodiments of the present invention, as described above, and they are not meant to limit the invention in any way.

EXAMPLE 1

Wet grinding of berry press cake, hydrothermal extraction and antimicrobial activity

Frozen berry pomace from berry processing industry obtained from bilberry (100%), bilberry (50%) and lingonberry (50%), cloudberry (100%) and raspberry (100%) was subjected to wet grinding in a Masuko Supermasscolloider stone mill. Trial settings are presented in Table 1. The pomace was blended with water to obtain a slurry having a dry matter content of 25-40% by weight depending on the initial raw material. Water was used to improve throughput in the grinder. The cloudberry pomace was first ground without dilution to obtain a smooth paste. The grinding was performed at 1500 rpm and gap width varied from 0.3 to 1.5 mm. The grinding stone was a stone type MKE10-46 made of silicon carbide and resins with a diameter of 10". Also, stones made of aluminum oxide could be used. The quality of the material was controlled by moving the lower stone to set the clearance between the grinding stones. The pomace was subjected to compression and shear forces between the stones, which determined the particle size of the output material. Average particle size distributions of the obtained suspensions were measured after one pass, two passes and three passes. After one pass the product had about D50 = 270 μιη, but after three passes it was much finer, about D50 = 29 μιη.

Table 1. Grinding settings of berry pomace.

Suspensions were either freeze-dried or used as such for hydrothermal extraction.

For hydrothermal extraction water was added to 50 g of the berry suspension (freeze- dried or as such) to get the final dry weight content lg dry material / 20 ml water. Extraction was carried out for 1 hour at 80°C (with 500 rpm mixing). The mixture was filtered using Miracloth ^® filter material with 22-25 μιη pore size to obtain a filtrate and a residue. The filtrate was freeze dried.

The antimicrobial activity of the freeze-dried filtrates (berry materials) and their combinations was tested against selected microbes including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Antimicrobial activities were measured in liquid cultures. Freeze-dried materials of 2.5 mg ml ¹ were suspended into microbial cultures. Microbial culture without berry material was used as positive control, and culture with antibiotic (chloramphenicol) was used for negative growth control. The microbial cultures were incubated in their optimal growth conditions, and growth by cell counts was followed by plate count during 24 h of cultivation. The results show clear bactericidal activity (about 2 logarithmic units) of the hydrothermal extracts of wet ground berry fractions against Staphylococcus aureus compared to positive control culture with no antimicrobial agents. Raspberry was the best inhibitor. Moderate growth inhibition was detected with Escherichia coli too. Cloudberry was the best inhibitor. Slight inhibition was detected against Pseudomonas aeruginosa. Raspberry was the best inhibitor. Better antimicrobial activities were associated to those samples which were not freeze-dried before hydrothermal extraction.

Figure 1 shows the antimicrobial effect of hydrothermal extracts of wet ground press cake against Staphylococcus aureus in liquid culture during cultivation for 24 hours. Culture with antibiotic chloramphenicol (50 μg/ml) was used as negative control. Effect of extract was tested in one concentration in the culture: 2.5 mg/ml. Effects of same extracts against Escherichia coli are shown in Figure 2.

EXAMPLE 2

Wet grinding of berry press cake from berry liquer producing industry, hydrothermal extraction and antimicrobial activity

Cloudberry pomace from liquor production was subjected to wet grinding in a Masuko Supermasscolloider stone mill (model MKZA10-15J). The pomace was obtained from two production batches after short (4 days) and long (4 months) ethanol extraction periods. The first batch was 400 g and the second was 1 kg. First the pomace was ground without dilution to obtain a smooth paste. Based on the moisture analyzer the dry matter content was between 30-34%. Gap width between the grinding stones was 0.8 mm. For the second pass the once ground pomace was blended with water to obtain a slurry having 10% solids content. In the second pass the gap width was 0.3 mm. The grinding was performed at 1500 rpm. The grinding stone was a stone type MKGA10-46 made of aluminum oxide and resins with a diameter of 10". The quality of the material was controlled by moving the lower stone to set the clearance between the grinding stones. The pomace was subjected to compression and shear forces between the stones, which determined the particle size and homogeneity of the output material, the suspension. Suspensions were either freeze- dried or used as such for hydrothermal extraction.

For hydrothermal extraction water was added to 50 g of the berry suspension (freeze- dried or as such) to get the final dry weight content lg dry material / 20 ml water. Extraction was carried out for 1 hour at 80°C (with 500 rpm mixing). The mixture was filtered using Miracloth ^® filter material with 22-25 μιη pore size. The filtrate was freeze dried.

The antimicrobial activity of the freeze-dried filtrate (berry fraction) was tested against selected microbes including Staphylococcus aureus and Pseudomonas aeruginosa as described in Example 1, with the exception that the tested concentration was 5 mg/ml.

The results showed strong bactericidal activity of the hydrothermal extracts of wet milled berry fractions against Staphylococcus aureus compared to positive control culture with no antimicrobial agents. The pomace exposed to short ethanol extraction was better inhibitor compared to pomace exposed to long extraction. Strong inhibition was also detected against Pseudomanas aeruginosa. In this case, better antimicrobial activities were associated to the sample which was exposed to longer ethanol extraction before the wet-milling process. Figure 3 shows the antimicrobial effect of hydrothermal extracts of wet-milled press cakes against Staphylococcus aureus in liquid culture during cultivation for 24 hours. Culture with antibiotic chloramphenicol (50 μg/ml) was used as negative control. Effect of extract was tested in one concentration in the culture: 5 mg/ml. Effects of same extracts against Pseudomonas aeruginosa are shown in Figure 4.

EXAMPLE 3

Dry milling of raspberry leaves, hydrothermal extraction and antimicrobial activity Fresh wild raspberry leaves were collected from the nature after berry harvesting (September) and frozen. Frozen leaves were freeze-dried and milled using ball mill (Retsch) 2 x lmin, 29 Hz.

For (hydrothermal) extraction 200 ml of water was added to 10 g of the leaf powder. Extraction was carried out for 1 hour at 80°C (with 500 rpm mixing). The mixture was filtered using Miracloth ^® filter material with 22-25 μιη pore size. The filtrate was freeze- dried.

Phenolic profiles were determined from 100 mg of the freeze-dried samples homogenised and extracted with 1 ml methanol. The samples were run by using Waters Alliance HPLC- DAD (High performance liquid chromatography- photodiode array detection) with a Hypersil BDS-C18 column (4.6 x 150 mm, 5 μιη, 25 °C). The solvents were 5% formic acid (A) and acetonitrile (B) and the gradient was from 5% B to 90% B in 35 minutes at a flow rate of 0.700 ml/min. The injection volume was 20 μΙ and the data were collected in a range of 190-600 nm. Ellagic acid and ellagitannins were detected according to their specific spectra.

HPLC chromatograms of the hydrothermal extract of the raspberry leaf material were rich in ellagic acid and ellagitannins. The HPLC chromatogram of the hydrothermal extract of raspberry leaf is shown in Figure 5 and UV absorption spectra of the main compounds in Figure 6. Peaks 1 and 2 are typical for ellagitannins and peak 3 is typical for ellagic acid.

The antimicrobial activity of the freeze-dried leaf material was tested against selected microbes including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Antimicrobial activities were measured in liquid cultures. Freeze-dried materials of 1 ; 2.5 and 5 mg ml ¹ were suspended into microbial cultures. Microbial culture without berry material was used as positive control, and culture with antibiotic (chloramphenicol) was used for negative growth control. The microbial cultures were incubated in their optimal growth conditions, and growth by cell counts was followed by plate count during 24 h of cultivation.

The results show very strong bactericidal activity of the hydrothermal extracts of milled raspberry leaf fraction against Staphylococcus aureus compared to positive control culture with no antimicrobial agents. Inhibition was clearly dependent on the concentration of the extract. The highest concentration, 5 mg/ml, totally eliminated the bacteria within 7 hours of cultivation. Slight growth inhibition was detected against Escherichia coli and Pseudomanas aeruginosa.

Figure 7 shows the antimicrobial effect of hydrothermal extracts of milled raspberry leaves against Staphylococcus aureus in liquid culture during cultivation for 24 hours. Culture with antibiotic chloramphenicol (50 μg/ml) was used as negative control. Effect of extract was tested in three concentrations in the culture: 1, 2.5 and 5 mg/ml Example 4

Pretreatment of berry material: Fermentation of cloudberries, pressing and sieving

Fermentation

Frozen, ripe cloudberries (Rubus chamaemorus) were used as the berry material. The berry material was first heat treated and then inoculated with approximately 10 ⁶ cfu g ¹ of washed LAB cells. Pedicoccus pentosaceus VTT E-072742 from VTT Culture Collection was used as a starter culture in the fermentation of cloudberries (http://culturecollection.vtt.fi/). Prior to fermentations, the strain was refreshed in de Man Rogosa Sharpe broth for 1 day in a 100% carbon dioxide atmosphere which was created using anaerobic jars and Anaerocult C strips. The cells were collected from refreshed cultures by centrifugation and washed once in Ringer's solution. The fermentations were performed in 6 kg scale in a 15-1 capacity bioreactor for 3 days at 30 °C under constant mixing (130 rpm). The bioreactor was purged with sterile-filtered nitrogen gas in order to create anaerobic conditions. The viable counts of lactic acid bacteria and yeasts were determined before and after the fermentations using plate count technique. The results were expressed as colony-forming units (CFU) per gram of wet weight. The fermented berry mash was stored frozen. Pressing and drying

After fermentation, the berry mash was treated with a hydraulically operated high- pressure tincture press using 5 litres filling material to separate juice and insoluble press cake. The press cake from juice pressing was dried in a fluid bed dryer using +45°C air flow, until the water content of the berry press cake was below 15 % by weight. After that, the dried berry press cake was dry sieved using different sieve sizes or using a suction apparatus. A skin fraction having average particle less than 1250 μιη was separated and seed fraction having average particle size of more than 750 μιη was collected.

The mass yields of cloudberry press cake (fermented and non-fermented samples) were 8-10 %. About 5 % of the press cake consisted of peels and pulp, and remaining 95% were seeds. The press cake was used as such in the process or frozen for later use. EXAMPLE 5

Composition and sensory properties of hydrothermal extraction residue of wet ground berry press cake

Berry side-streams, i.e. berry pomace from berry processing industry, or berry leaves, as described in Example 3 were subjected to wet or dry grinding and hydrothermal extraction. Composition of the residue was analysed for 1) fibre, protein and dry weight; 2) phenolic compounds, fatty acids and other lipid-soluble compounds and 3) sensory properties.

Fibre, protein and dry weight

The dry matter content of the hydrothermal extraction residues was analysed using an oven method and the protein content with the Kjeldahl method. Total, soluble, and insoluble dietary fibres were analysed using an enzymatic-gravimetric method (AOAC Official method 991.43). In the residues from the wet-ground side-streams dry matter content was approximately 45 %, but in the residue of dry-ground raspberry leaves it was approx. 25 % (Table 2). Almost or over 70 % of the dry matter in the berry residues was insoluble fibre, with wet- ground cloudberry having also high soluble fibre content (8 % of dry matter) (Table 3). Protein content in berry materials was 6-7 % (d.m.), and in the residue from raspberry leaves it was over 20 %.

Table 2. Dry matter, fibre and protein content of the extraction residues, presented as % in moist sample.

Sample (moist sample) \ dry i insoluble soluble fibre, protein matter \ fibre (%) fibre total (%) (%) (%) (%)

Wet-ground cloudberry seed 43.5 i 29.4 0.4 29.8 3.2 pomace, hydrothermal residue ;

Wet-ground raspberry seed i 46.1 i 32.3 0.0 32.3 3.1 pomace, hydrothermal residue

Dry-ground raspberry leaves, 26.5 i 13.8 1.1 14.9 5.5 hydrothermal residue Table 3. Dry matter, fibre and protein content of the extraction residues, presented as % in moist sample.

Phenolic compounds, fatty acids and other lipid-soluble compounds

Hydrothermal extraction residues were dried prior to analyses.

Phenolic profiles were determined from 100 mg of air-dried samples homogenised and extracted with 1 ml methanol. The samples were run by using Waters Alliance HPLC-DAD (High performance liquid chromatography- photodiode array detection) with a Hypersil BDS-C18 column (4.6 x 150 mm, 5 μιη, 25 °C). The solvents were 5% formic acid (A) and acetonitrile (B) and the gradient was from 5% B to 90% B in 35 minutes at a flow rate of 0.700 ml/min. The injection volume was 20 μΙ and the data were collected in a range of 190-600 nm. Ellagic acid and quercetin were quantified at 360 nm and anthocyanins at 520 nm by using ellagic acid, kaempherol and cyanidin-3-glucoside as external standards for calibration curves.

HPLC chromatograms of the extraction residues at the wave length 360 nm are presented in Figure 8. Press cake residues contained some phenolic compounds which varied in different press cakes. Main phenolic compounds in bilberry press cake extraction residue were anthocyanins. Their contents were 2.75 g / kg of the dried extraction residue. Main compound in bilberry-lingonberry press cake extraction residue was quercetin. Its content was 130 mg / kg of the dried extraction residue. Main compound in cloudberry and raspberry press cake residues was ellagic acid. Its content in cloudberry was 248 mg/ kg of the dried extraction residue and in raspberry 100 mg / kg of the dried extraction residue. As a conclusion, press cakes contained health promoting phenolic compounds, and thus they are potent food ingredients. Especially in bilberry press cake the anthocyanin content was very high, at the sa me order of magnitude as in the berries.

In Figure 8 the HPLC chromatograms of A: bilberry press cake residue, B : lingonberry- bilberry press cake residue; C: cloudberry press cake residue and D : raspberry press ca ke residue, are presented . Ellagic acid, quercetin and anthocyanins are ma rked in the figure with arrows.

Fatty acids were determined on an Agilent GC-MS by using an FFAP silica capillary column. The air-dried samples (20 mg) were spiked with C17 triacylglycerol and free fatty acid . The samples were mixed with petroleum ether (bp 40-60 °C), transesterified with sodium methoxide and acidified with sodium hydrogen sulphate. Esterified and free fatty acids were identified by using commercial mixtures of relevant reference substances. For the determination of sterols, tocopherols, squalene and amyrins the transesterified samples were trimethylsilylated with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) containing 1% trimethylchlorosilane (TMCS) and run by GC-MS on a DB-5MS column (30 m) . Identification was based on the retention times and mass spectra of relevant reference substances and comparison with the Palisade Complete 600K Mass spectral library (Palisade Mass Spectrometry, USA) .

Fatty acid compositions of the extraction residues are shown in Tables 4 and 5. All extraction residues were rich in fatty acids. Highest amounts were detected in raspberry press ca ke residue. Most of the fatty acids were bound ones (over 90 %) . Highest amounts of free fatty acids were found in bilberry press cake residue (about 10 %) . Main fatty acids in all extraction residues were oleic acid, linoleic acid and a-linolenic acid, which are also the main fatty acids in the corresponding berry seeds. The fatty acid composition of the extraction residues was found to be very beneficial for human health, as they were rich in essential fatty acids. Other lipid-soluble compounds are presented in Table 5. Amounts of other lipid-soluble compounds analysed by GC-MS a re presented in Table 6. Amyrins, plant sterols and squalene were found especially in lingonberry and bilberry extraction residues, and they are associated with cardiovascular health. Raspberry extraction residue was most rich in tocopherols. Thus all these extraction residues would very promising as food ingredients. Table 4. Fatty acid composition of the extraction residues of the press cakes

Fatty acid (ug/mg) Wet-milled bilberry pomace Wet-milled lingonberry-bilberry pomace Wet-milled cloudberry seed pomace Wet-milled raspberry seed pomace dry weight hydrothermal residue hydrothermal residue hydrothermal residue hydrothermal residue

C14:0 myristic 0,03 0,02 0,03 0,02

C16:0 palmitic 2,57 1,60 1,43 1,89

C16:l palmitoleic 0,10 0,08 0,10 0,08

C18:0 stearic 0,63 0,27 0,83 0,73

C18:l oleic 8,53 5,14 7,74 7,52

C18:2 linoleic 11,99 10,33 14,91 21,85

C18:3 a-linolenic 10,74 11,58 10,81 12,82

C20:0 arachidic 0,09 0,08 0,52 0,27

C20:l eicosenoic 0,08 0,10 0,47 0,14

C20:2 eicosadienoic 0,04 0,04 0,14 0,04

C22:0 behenic 0,08 0,14 0,20 0,12

FFA C1&0 palmitic 0,71 0,26 0,10 0,10

FFA C18:0 stearic 0,13 0,03 0,04 0,05

FFA C18:1 oleic 0,53 0,17 0,34 0,48

FFA C18:2 linoleic 1,63 0,69 0,75 0,60

FFA C18:3 a-linolenic 0,81 0,42 0,47 0,19

FFA C20:0 arachidic 0,06 0,02 0,01 0,00

FFA C22:0 behenic 0,02 0,04 0,00 0,00

Bound fatty acids 34,88 29,40 37,19 45,47

Free fatty acids 3,90 1,64 1,72 1,42

Total fatty acids 38,78 31,04 38,90 46,88

Table 5. Fatty acid composition of the extraction residues of the press cakes, % of dry weight.

Fatty acid Wet-milled bilberry pomace Wet-milled lingonberry-bilberry pomace Wet-milled cloudberry seed pomace Wet-milled raspberry seed pomace

% hydrothermal residue hydrothermal residue hydrothermal residue hydrothermal residue

C14:0 myristic 0,08 0,08 0,08 0,03

C16:0 palmitic 6,63 5,17 3,68 4,03

C16:l palmitoleic 0,25 0,27 0,26 0,17

C18:0 stearic 1,61 0,88 2,13 1,56

C18:l oleic 22,00 16,57 19,88 16,04

C18:2 linoleic 30,93 33,29 38,31 46,60

C18:3 a-linolenic 27,70 37,31 27,78 27,33

C20:0 arachidic 0,23 0,25 1,34 0,58

C20:l eicosenoic 0,20 0,32 1,22 0,29

C20:2 eicosadienoic 0,09 0,14 0,37 0,08

C22:0 behenic 0,22 0,44 0,52 0,27

FFA C16:0 palmitic 1,84 0,84 0,26 0,22

FFA C18:0 stearic 0,34 0,11 0,11 0,10

FFA C18:l oleic 1,38 0,56 0,87 1,02

FFA C18:2 linoleic 4,21 2,21 1,93 1,27

FFA C18:3 a-linolenic 2,09 1,36 1,22 0,41

FFA C20:0 arachidic 0,15 0,06 0,02 0,00

FFA C22:0 behenic 0,05 0,12 0,00 0,00

Bound fatty acids % 89,94 94,73 95,59 96,97

Free fatty acids % 10,06 5,27 4,41 3,03

Total fatty acids % 100,00 100,00 100,00 100,00

Table 6. Amounts of other lipid-soluble compounds analysed by GC-MS.

Wet-milled bilberry pomace Wet-milled lingonberry-bilberry pomace Wet-milled cloudberry seed pomace Wet-milled raspberry seed pomace dry weight hydrothermal residue hydrothermal residue hydrothermal residue hydrothermal residue

Squalene 0,100 0,116 0,040 0,052 gamma-Tocopherol 0,030 0,021 0,074 0,222 alpha-Tocopherol 0,091 0,123 0,080 0,087

Sum of tocopherols 0,120 0,144 0,154 0,309

Campesterol 0,061 0,060 0,062 0,060 beta-Sitosterol 0,579 0,754 0,300 0,316

Sum of sterols 0,640 0,814 0,362 0,376 alpha-Amyrin 0,087 0,119 0 0

beta-Amyrin 0,079 0,276 0 0

Sum of amyrins 0,166 0,395 0 0

Sensory properties of the hydrothermal extraction residues were screened. The hydrothermal residues in the evaluation were originating from wet-milled cloudberry and raspberry seed pomace and from wet-milled bilberry press-cake and wet-milled bilberry- lingonberry mixture press-cake. Free descriptive analysis of appearance, odour, taste and structure was performed with 5-6 panellists. Extraction residues were evaluated as such to have a preliminary estimate of their potentiality as fibre-rich food ingredients. Results are presented as a set of descriptive attributes / sample.

For the evaluated hydrothermal residues, many berry-like attributes were given (Table 7). The taste attributes were mostly either positive or neutral. Therefore, the hydrothermal extraction residues can be seen as promising food ingredients which could enhance the nutritional properties of food products without affecting negatively on their sensory properties.

Table 7. Attributes given by 5-6 panellists to describe the sensory properties of the hydrothermal residues. Samples evaluated as such.

While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described embodiments that fall within the spirit and scope of the invention. It should be understood, that the invention is not limited in its application to the details of construction and arrangements of the components set forth herein. Variations and modifications of the foregoing are within the scope of the present invention.