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Title:
KIMCHI BASED ON A RAPE SPECIES
Document Type and Number:
WIPO Patent Application WO/2018/050739
Kind Code:
A1
Abstract:
The present invention relates to a fermented composition comprising at least one plant material selected from the group consisting of rape species; cruciferous vegetables; mustard species, and/or seaweed, preferably rape species and/or seaweed; and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-5 hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis.

Inventors:
LEGARTH JENS HØFFNER (DK)
MADSEN SVEND ROESEN (DK)
HALKIER BARBARA ANN (DK)
Application Number:
PCT/EP2017/073124
Publication Date:
March 22, 2018
Filing Date:
September 14, 2017
Export Citation:
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Assignee:
FERMENTATIONEXPERTS AS (DK)
UNIV COPENHAGEN (DK)
International Classes:
A23C9/127; A23K10/12; A23C9/13; A23C9/133; A23K10/18; A23L33/105; A23L33/135; A61K31/19; A61K36/02; A61K36/31; A61P29/00; A61P31/00
Domestic Patent References:
WO2014131422A22014-09-04
WO2002074300A12002-09-26
WO2013029632A12013-03-07
Foreign References:
KR20110101930A2011-09-16
US20160128357A12016-05-12
Other References:
SHOKO TESAKI ET AL: "4-Hydroxy-3-nitrophenylacetic and Sinapic Acids as Antibacterial Compounds from Mustard Seeds", BIOSCIENCE BIOTECHNOLOGY BIOCHEMISTRY., vol. 62, no. 5, 1998, TOKYO, JAPAN, pages 998 - 1000, XP055418478, ISSN: 0916-8451, DOI: 10.1271/bbb.62.998
EUNA OH ET AL: "Synergistic anti-Campylobacter jejuni activity of fluoroquinolone and macrolide antibiotics with phenolic compounds", FRONIERS IN MICROBIOLOGY, vol. 6, no. 1129, 13 October 2015 (2015-10-13), pages 1 - 9, XP055418529, DOI: 10.3389/fmicb.2015.01129
MICHELA MAIFRENI ET AL: "Lactic acid fermentation of Brassica rapa : chemical and microbial evaluation of a typical Italian product ( brovada )", EUROPEAN FOOD RESEARCH AND TECHNOLOGY, vol. 218, no. 5, April 2004 (2004-04-01), Berlin/Heidelberg, pages 469 - 473, XP055328504, ISSN: 1438-2377, DOI: 10.1007/s00217-004-0877-6
JEONG SOOK NOH ET AL: "Beneficial effects of the active principle component of Korean cabbage kimchi via increasing nitric oxide production and suppressing inflammation in the aorta of apoE knockout mice", BRITISH JOURNAL OF NUTRITION, vol. 109, no. 01, 13 April 2012 (2012-04-13), UK, pages 17 - 24, XP055418432, ISSN: 0007-1145, DOI: 10.1017/S0007114512000633
ANONYMOUS: "Cruciferous vegetables - Wikipedia", 22 August 2016 (2016-08-22), internet, XP055418427, Retrieved from the Internet [retrieved on 20171024]
MO-EUN LEE ET AL: "Starter Cultures for Kimchi Fermentation", JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY., vol. 25, no. 5, 28 May 2015 (2015-05-28), KR, pages 559 - 568, XP055418362, ISSN: 1017-7825, DOI: 10.4014/jmb.1501.01019
BONG YEON-JU ET AL: "Fermentation Properties and Increased Health Functionality of Kimchi by Kimchi Lactic Acid Bacteria Starters", HAN'GUG SIGPUM YEONG'YANGGWAHAG HOEJI - JOURNAL OF THE KOREAN SOCIETY OF FOOD SCIENCE AND NUTRITION, HAN'GUG YEONG'YANG SIGRYANG HAGHOE, PUSAN, KR, vol. 42, no. 11, 30 November 2013 (2013-11-30), pages 1717 - 1726, XP053031871, ISSN: 1226-3311, DOI: 10.3746/JKFN.2013.42.11.1717
JIN-WOO JEONG ET AL., JOURNAL OF MEDICINAL FOOD, vol. 18, no. 6, 2015, pages 677 - 684
HYUN JU KIM ET AL., J. AGRIC. FOOD CHEM., vol. 55, 2007, pages 10486 - 10492
JEONG SOOK NOH ET AL., BRITISH JOURNAL OF NUTRITION, vol. 109, 2013, pages 17 - 24
YE-RANG YUN ET AL., JOURNAL OF MEDICINAL FOOD, vol. 17, no. 8, 2014, pages 886 - 893
Attorney, Agent or Firm:
LARSEN & BIRKEHOLM A/S (DK)
Download PDF:
Claims:
Claims

1. A fermented composition comprising at least one plant material selected from the group consisting of rape species; cruciferous vegetables; mustard species, and/or seaweed,

5 preferably rape species and/or seaweed; and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at 10 least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g.

2. The composition according to claim 1, wherein the one or more lactic acid bacteria 15 present in the composition is selected from the group consisting of Pediococcus

pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum; Lactobacillus rhamnosus; Enterococcus faecium and/or a combination thereof.

3. A composition according to anyone of claims 1-2, wherein the composition comprising at 20 least one plant material and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 25 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g and wherein one or more lactic acid bacteria present in the composition is selected from the group consisting of Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum;

Lactobacillus rhamnosus; Enterococcus faecium and/or a combination thereof.

30

4. The composition according to claim 7, wherein the at least one plant material is selected from the group consisting of rape species; cruciferous vegetables; and/or mustard species, preferably rape species.

35 5. The composition according to anyone of the preceding claims, wherein the composition comprises one or more lactic acid bacterial strains, e.g. two or more lactic acid bacterial strains, such as three or more bacterial strains, e.g. four or more bacterial strains, such as 7 or more bacterial strains, e.g. 10 or more bacterial strains, such as 15 or more bacterial strains, e.g. 20 or more bacterial strains, such as 25 or more bacterial strains, e.g. 30 or more bacterial strains, such as 35 or more bacterial strains, e.g. 40 or more bacterial strains.

5 6. The composition according to anyone of the preceding claims, wherein the one or more lactic acid bacteria stain(s) is selected from the group consisting of one or more of Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Pediococcus pentosaceus HTS (LMG P-22549), Pendiococcus acidilactici NCIMB 30086 and/or

Lactobacillus plantarum LSI (NCIMB 30083).

10

7. The composition according to anyone of the preceding claims, wherein the composition comprises one or more lactic acid bacterial strains in a total amount in the range of 105- 1012 CFU per gram.

15 8. The composition according to anyone of the preceding claims, wherein the plant

material is Brassica rapa.

9. The composition according to anyone of the preceding claims, wherein the plant material, such as rape species, is provided in the form of seeds, press cake, meal or other

20 residues of industrial utilization of said plant material.

10. The composition according to anyone of the preceding claims, wherein the content of the plant material in the composition is at least 50% plant material, such as at least 55% plant material, e.g. at least 60% plant material, such as at least 65% plant material, e.g.

25 at least 70% plant material, such as at least 75% plant material, e.g. at least 80% plant material, such as at least 85% plant material, e.g. at least 90% plant material, such as at least 95% plant material.

11. The composition according to anyone of the preceding claims, wherein the composition 30 further comprises one or more ingredient selected from the group consisting of cereals,

(e.g. wheat, barley, rye, rice, maize (cob maize silage (CCM) or ripe), triticale, oat);

vegetables (e.g. potatoes, beans, peas, maize, soy); and carbohydrate ingredient (e.g. whey, curd, skim milk and the like).

35 12. The composition according to anyone of the preceding claims, wherein the one or more lactic acid bacterial strain are viable cells.

13. The composition according to anyone of the preceding claims, wherein the composition has a pH value in the range of pH 3-5, such as in the range of pH 3.5-4.5, e.g. in the range of pH 3.8-4.4, 3.85-4.1, 3.9-4.0, such as in the range of pH 4.0-4.3, e.g. about pH 4.2.

14. The composition according to anyone of the preceding claims, wherein the plant material is not a cabbage species.

15. The composition according to anyone of the preceding claims, wherein the amount of HDMPPA is increased relative to a fermented composition comprising a cabbage species, such as Korean cabbage, or fermented mainly using Lactobacillus kimchi.

16. The composition according to anyone of the preceding claims, wherein the composition does not comprise Lactobacillus kimchi.

17. The composition according to anyone of the preceding claims, wherein HDMPPA is present in at least 2 times the concentration/amount found in non-fermented plant material, such as at least 5 times, e.g. at least 10 times, such as at least 15 times.

18. The composition according to anyone of the preceding claims, wherein HDMPPA is absent in a non-fermented plant material.

19. A substance for use as a medicament comprising a fermented composition according to anyone of claims 1-18.

20. A substance for use in the treatment, alleviation or prophylaxis of inflammatory conditions in a mammal (such as a human or an animal), said substance comprises a fermented composition comprising according to claims 1-18.

21. A combination product comprising a fermented composition, a substance obtained from the fermented composition, HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent.

22. A combination product comprising a fermented composition, a substance obtained from the fermented composition, HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent, for use in the treatment and/or alleviation of a microbial infection with reduced incidence of anti-microbial resistance of the targeted microorganism by the antimicrobial agent.

23. A combination product according to claim 22, wherein the incidence of antimicrobial resistance of the targeted microorganism by the antimicrobial agent is reduced by at least 10%, such as by at least 20%, e.g. by at least 30%, such as by at least 40%, e.g. by at least 50%, such as by at least 60%, e.g. by at least 70%, such as by at least 80%.

Description:
KIMCHI BASED ON A RAPE SPECIES

Technical field of the invention

The present invention relates to a fermented plant material comprising a high content of lactic acid bacterial strains and a high content of active ingredients. In particular the present invention relates to a new method for producing a novel fermented composition, based on a rape species, comprising a high content of lactic acid bacterial strains and a high content of HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid), and said fermented composition show nutritional and/or pharmacological effects.

Background of the invention

Kimchi is a traditional fermented Korean side dish made of vegetables, such as cabbage, red pepper, garlic, ginger and onion, where cabbage is the main component. During preparation the ingredients are mixed with Lactobacillus kimchii, being a bacteriocin- producing lactic acid bacterium of the genus Lactobacillus.

Kimchi has gain increased attention due to the nutritional and health benefits obtained from the product.

The reason kimchi is considered to have the various benefits is that kimchi contains lots of dietary fiber and is a low-calorie product. One serving provides up to 80% of the daily recommended requirement of vitamin C and carotene. Kimchi is generally rich in vitamins A, thiamin (Bl ), riboflavin (B2), calcium and iron, as well as lactic acid bacteria

(Lactobacillus kimchii) . The paper "Health" placed kimchi on its list of the world's five healthiest ingredients because of its richness in vitamins, promote digestion and a possible reduction in the growth of cancer. The compound found in kimchi which is considered to provide a significant part in the various advantages of kimchi is HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) . HDMPPA is a compound originally found in kimchi as one of the major molecules. HDMPPA is believed by researchers to be an active immune defence in the central nervous system, CNS (see Jin-Woo Jeong et al . 2015), to have anti-oxidative activity and prevent and/or treating atherosclerosis (Hyun Ju Kim, el al . 2007), to be lipid lowering, to have anti-tumor and anti-atherogenic effects, to have anti-viral effects against acute respiratory syndrome (Ye-Rang Yun et al. 2014).

The present invention provides a method for proving higher amounts of HDMPPA and a composition having increased amount of HDMPPA which is ready to eat, and easy to produce.

Summary of the invention

The inventors of the present invention surprisingly found a fermented plant material comprising high content of lactic acid bacterial strains and a high content of HDMPPA having nutritional and/or pharmacological effects. The inventors of the present invention found a method for producing a novel fermented composition comprising a high content of lactic acid bacterial strains and a high content of HDMPPA and at the same time having nutritional and/or pharmacological effects.

Thus, one aspect of the invention relates to a fermented composition comprising at least one plant material selected from the group consisting of rape species; cruciferous vegetables; mustard species, and/or seaweed, preferably rape species and/or seaweed; and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g. A further aspect of the present invention relates to a fermented composition comprising at least one plant material and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g and wherein one or more lactic acid bacteria present in the composition is selected from the group consisting of Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum;

Lactobacillus rhamnosus; Enterococcus faecium and/or a combination thereof.

Another aspect of the present invention relates to the use of a fermented composition according to the present invention as an ingredient.

A further aspect of the present invention relates to the use of a fermented composition according to the present invention in a food product; in a food supplement product; in an herbal medicine product; in a natural medicine product; in a medicinal product and/or as an adjuvant product accompanied by one or more other drugs.

An even further aspect of the present invention relates to a substance for use as a medicament comprising a fermented composition according to the present invention. Another aspect of the present invention relates to a substance for use in the treatment, alleviation, stabilisation or prophylaxis of inflammatory condition in a mammal (such as a human or an animal), said substance comprises a fermented composition according to the present invention. Still another aspect of the present invention is to provide a process for preparing a fermented composition according to the present invention, the method comprises the steps of:

(i) providing a plant material;

(ii) providing one or more lactic acid bacteria strain(s);

(iii) subjecting the plant material to fermentation by combining the plant material provided in step a) with the one or more lactic acid bacterial strain(s) provided in step b),

(iv) continuing the fermentation until the fermented composition is provided having a pH value below 5.0. In a further aspect, the present invention relates to a preferred embodiment of the present invention relates to a combination product comprising a fermented composition, a substance obtained from the fermented composition, HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent. An even further aspect of the present invention relates to a combination product comprising a fermented composition, a substance obtained from the fermented

composition, HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent, for use in the treatment and/or alleviation of a microbial infection with reduced incidence of antimicrobial resistance of the targeted microorganism by the antimicrobial agent.

Brief description of the figure

Figure 1 shows a chromatogram of the amount of HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) in a fermented rapeseed/seaweed (1); fermented rapeseed (2); and non-fermented rapeseed (3). The amount is determined by Ultra High Pressure Liquid Chromatography-Quadruple Time of Flight Mass Spectroscopy (UPLC- QTOF-MS) by analysing an 80% methanol extract. From this chromatogram it is shown that fermented rapeseed/seaweed (1) has significant more HDMPPA relative to fermented rapeseed (2), which has significant more HDMPPA relative to non-fermented rapeseed (2).

Detailed description of the invention

Over the years, the interest in probiotics and prebiotics has kept increasing and the numbers of applications for which they are used have continuously enlarged and the nutritional and/or pharmacological effects are expanding. In this respect the inventors of the present invention surprisingly found that a combined fermented composition comprising probiotics and prebiotics show unexpected and improved nutritional and pharmacological effects.

Prebiotics are well known to the skilled person and refer to chemicals that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that, when consumed by a human or an animal, contribute to the well-being of the host. The most common example of using prebiotics is in the gastrointestinal tract, where prebiotics can alter the composition of micro-organisms in the gut microflora. Probiotics are also well known to the skilled person and are live microorganisms that, when administered to a human or an animal, are believed to provide health benefits. Thus, one aspect of the present invention relates to a process for preparing a fermented composition according to the present invention, in particular a fermented composition comprising a prebiotic and a probiotic, the method comprises the steps of: (i) providing a plant material;

(ii) providing one or more lactic acid bacteria strain(s);

(iii) subjecting the plant material to fermentation by combining the plant material provided in step a) with the one or more lactic acid bacterial strain(s) provided in step b),

(iv) continuing the fermentation until the fermented composition is provided having a pH value below 5.0.

In a preferred embodiment of the present invention the fermented composition comprises one or more probiotic bacterial strain(s). Preferably, different strain(s) of probiotic bacteria may be used according to the present invention. In an embodiment of the present invention one or more probiotic bacterial may be lactic acid-producing bacteria, Lactic acid bacteria. Because of the activity of lactic acid bacteria they are often linked with food fermentations as acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins, toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s), are produced by several lactic acid bacteria strains and provide an additional hurdle for spoilage and pathogenic

microorganisms.

Furthermore, lactic acid bacteria may produce lactic acid and other metabolic products which contribute to the organoleptic, textural, nutritional and pharmacological profile of the fermented composition.

The industrial importance of the lactic acid bacteria may be evidenced by their generally regarded as safe (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the lactic acid bacteria, and which may be used in the present invention, are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella; these genera belong to the order Lactobacillales. In the present invention, the one or more lactic acid bacterial strain(s) provided in step (iii) and used for fermentation may be selected from lactic acid bacteria selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and

Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella. Preferably, the one or more lactic acid bacterial strain(s) provided in step (iii) and used for fermentation may be selected from lactic acid bacteria selected from the group consisting of lactic acid bacteria of the genus Enterococcus, Lactobacillus, Pediococcus or Lactococcus, or combinations thereof. In an embodiment of the present invention, the one or more lactic acid bacterial strain(s) provided in step (iii) and used for fermentation may be selected from the group consisting of one or more Enterococcus spp., Lactobacillus spp., Lactococcus spp., Pediococcus spp., and a combination hereof. Preferably, the one or more lactic acid bacterial strain provided in step (iii) and used for fermentation may be selected from the group consisting of one or more one Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, Pediococcus acidililactili, Pediococcus pentosaceus, Lactococcus Lactis, Lactococcus Cremoris, Lactococcus Diacetylactis, Leuconostoc Cremoris and a combination hereof.

In a further embodiment, the one or more lactic acid bacterial strain(s) provided in step (iii) and used for fermentation comprise Lactobacillus plantarum, Enterococcus faecium and/or Lactobacillus rhamnosus.

In still another embodiment, the one or more lactic acid bacterial strain(s) provided in step (iii) and used for fermentation comprise one or more of Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Pediococcus pentosaceus HTS (LMG P-

22549), Pendiococcus acidilactici NCIMB 30086 and/or Lactobacillus plantarum LSI (NCIMB 30083) or a combination hereof.

In order to increase productivity and effectivity two or more lactic acid bacterial strains may be provided, such as three or more lactic acid bacterial strains, e.g. four or more lactic acid bacterial strains, such as 7 or more lactic acid bacterial strains, e.g. 10 or more lactic acid bacterial strains, such as 15 or more lactic acid bacterial strains, e.g. 20 or more lactic acid bacterial strains, such as 25 or more lactic acid bacterial strains, e.g. 30 or more lactic acid bacterial strains, such as 35 or more lactic acid bacterial strains, e.g. 40 or more lactic acid bacterial strains.

In an embodiment of the present invention a starter culture or an inoculum may be provided comprising one or more lactic acid bacterial strain(s) as defined herein. The term "inoculum" relates to a source material, such as the one or more lactic acid bacterial strain(s), used for the inoculation of a new culture. The inoculum may be employed to prime a process of interest. "Inoculation" refers to the placement of a microorganism(s) (e.g. one or more lactic acid bacterial strain(s)) that will grow when implanted in a culture medium such as a fermentation tank comprising media to be fermented, e.g. a plant material.

A primary inoculum may be provided and refers to the generation of an initial inoculum in a series of repeated similar of essentially identical inoculation process, for example one or more repetitions of a fermentation process. An aliquot of the product of the formation process may be used to inoculate a new process of fermentation. Thus, the inoculation may be a fermented feed product which comprises viable lactic acid producing bacteria in sufficient amount to prime a lactic acid fermentation process of a another feed product, or similar feed, to be fermented, e.g. a plant material.

The inoculum according to the present invention may be a in a liquid form, dry form, or essentially dry form. The moisture content of the inoculum may be adjusted in order to optimize the fermentation process. In one embodiment the inoculum may be provided as essentially pure viable bacteria (such as bacteria in freeze dried form) or bacteria suspended in a suitable media prior to the application (such as a water, buffer or a growth media) .

The proportion of the inoculums added to the plant material may vary. In case it is considered that the load of undesirable microbes are significant in the plant material or the fermentation system, the proportion of the inoculum in the fermentation mixture

(inoculum + plant material + additional water) may be increased to insure that the fermentation is directed by the microbes (e.g. lactic acid bacteria) of the inoculums. Thus, the inoculum may be provided with a concentration of lactic acid bacteria in the inoculum sufficient to outgrow other (non lactic acid bacteria, yeast or moulds present in the plant material.

Accordingly, in one embodiment of the invention, the proportion of the inoculums in the combination of the plant material and the one or more lactic acid material as defined in step (iii) is in the range of 0.1 to 99.9 vol-%; such as 1 to 99 vol-%; e.g. 5 to 70 vol-%; such as 10 to 50 vol-%; e.g. 25 to 35 vol-%; such as 0.1 to 10 vol-%; e.g. 0.5 to 5 vol- %; such as 1 to 2.5 vol-%; or around 1 to 2 vol-%. The fermentation process according to the present invention, may preferably be essentially a homofermentative process. "Essentially homofermentative" means, that the predominant bacterial flora driving the fermentation is homofermentative. In the present context the term "essentially homofermentative" relates to a fermentation process where, 60% or more of the bacteria are homofermentative, such as 70% or more of the bacteria are homofermentative, e.g. 80% or more of the bacteria are homofermentative, such as 85% or more of the bacteria are homofermentative, e.g. 90% or more of the bacteria are homofermentative, such as 95% or more of the bacteria are homofermentative, e.g. 99% or more of the bacteria are homofermentative.

In an embodiment of the present invention the fermentation is essentially a

homofermentation, such as a homolactic fermentation.

The term "homolactic fermentation" when used according to the present invention indicates that the major fermentation product is lactic acid, and the levels of acetic acid and ethanol are either below taste threshold, around taste threshold or slightly above taste threshold. Preferably, "essentially homofermentative" indicates a ratio of lactic acid to acetic acid or lactic acid to ethanol in (mM/mM) of more than 1 : 1, such as 2 : 1 or more, e.g. 10: 1 or more, such as 20: 1 or more, e.g. 50 : 1 or more, or such as 100: 1 or more.

In another embodiment of the present invention the method according to the present invention is essentially a heterofermentative fermentation.

In the present context the term "essentially heterofermentative" means, that the predominant bacterial flora driving the fermentation is heterofermentative. In the present context the term "essentially heterofermentative" relates to a fermentation process where, 60% or more of the bacteria are heterofermentative, such as 70% or more of the bacteria are heterofermentative, e.g. 80% or more of the bacteria are heterofermentative, such as 85% or more of the bacteria are heterofermentative, e.g. 90% or more of the bacteria are heterofermentative, such as 95% or more of the bacteria are heterofermentative, e.g. 99% or more of the bacteria are heterofermentative.

In an embodiment of the present invention the heterofermentation according to the present invention results in a major part of the fermentation products are acetic acid and ethanol, and the ratio of acetic acid and ethanol to lactic acid in (mM/mM) is more than 1 : 1, such as 2: 1 or more, e.g. 10: 1 or more, such as 20: 1 or more, e.g. 50: 1 or more, or such as 100: 1 or more. In a further embodiment of the present invention, the fermented composition provided in step iv) has an acetic acid and/or ethanol concentration of at least 50 mM, such as at least 100 mM, such as 100-1000 mM, such as 100-500 mM, such as 100-300 mM, such as 100- 200 mM, such as 150-500 mM, such as 200-500 mM or such as 300-500, mM lactic acid.

5

The fermentation according to the present invention may be continued for 10 days or less, such as 9 days or less, e.g. 8 days or less, such as 7 days or less, e.g. 6 days or less, such as 5 days or less, e.g. 4 days or less, such as 3 days or less, e.g. 2 days or less, such as 36 hours or less, e.g. 24 hours or less.

10

Even the fermentation should be continued as quickly as possible, the inventors also found that a certain time of fermentation may be required to provide the desired nutritional and/or pharmacological effects of the fermented composition. Hence, in an embodiment of the present invention the fermentation should be continued for more than 8 hours, such as

15 more than 10 hours, e.g. more than 12 hours, such as more than 15 hours, e.g. more than 17 hours, such as more than 20 hours, e.g. more than 24 hours, such as in the range of 24 hours to 10 days, e.g. in the range of 20 hours to 8 days, such as in the range of 15 hours to 6 days, e.g. in the range of 12 hours to 4 days, such as in the range of 10 hours to 2 days, e.g. in the range of 8 hours to 36 hours, such as in the range of 8 hours to 24

20 hours.

Controlling the temperature may be one of the best ways to improve the quality and the effects of the fermented composition. In an embodiment of the present invention, the fermentation is performed at a temperature below 50°C, such as below 47°C, e.g. below 25 45°C, such as below 43°C, e.g. in the range 15-45°C, such as 18-43°C, such as 25-40°C, such as 30-40°C, such as 15-20°C or such as 40-45°C.

The fermentation process may preferably involve a temperature gradient, said temperature gradient involves 3 stages; a starting temperature, a temperature increases and a steady 30 state fermentation temperature.

In an embodiment of the present invention the starting temperature may be in the range of 18-30°C, such as in the range of 20-28°C, e.g. in the range of 22-26°C, such as in the range of 24-25°C.

35

The temperature increase of the fermentation process of the present invention may preferably be a slow temperature increase from the starting temperature to the steady state fermentation temperature. In an embodiment of the present invention the temperature increase is provided without addition of heat. In the present context the term "without addition of heat" relates to a fermentation temperature increase wherein the heat provided is produced by the fermentation itself without the use of electrical, mechanical or fuel based heat. In an embodiment of the present invention, the steady state fermentation temperature may be below 50°C, such as below 47°C, e.g. below 45°C, such as below 43°C, e.g. in the range 20-45°C, such as in the range 22-43°C, such as in the range 25-40°C, such as in the range 30-40°C, such as in the range 40-45°C. In yet an embodiment of the present invention, the fermentation may be a one-step fermentation of the plant material.

In the present context, the term "one-step fermentation" relates to a fermentation process wherein the same type of plant material is subjected to the same fermentation conditions, or substantially the same fermentation conditions. Hence, the term "one-step

fermentation", exclude the option of taking out a part of the plant material during fermentation, leaving the remaining plant material to be further fermented and followed by mixing the part which was taken out with the further fermented material. Preferably, the term "one-step fermentation" also includes a temperature increase provided without addition of heat.

In a further embodiment of the present invention, the fermented composition does not involve subsequent supplementation of plant material and/or one or more lactic acid bacterial strain(s) to the fermented composition.

The moisture content of the plant material to be fermented may be another relevant parameter to control in order to control the fermentation process and the resulting fermented composition. Thus, in an embodiment of the present invention the moisture content during the fermentation may be in the range of 20-60% (w/w), such as in the range of 30-50% (w/w), e.g. preferably in the range of 35-45% (w/w).

In a preferred embodiment of the present invention, the fermentation may be continued until the fermented composition has a pH below pH 6.5, such as below pH 6.0, e.g. below pH 5.5, such as below pH 5.0, e.g. in the range of pH 3.0-6.5, such as in the range of pH 3.0-6.0, e.g. in the range of pH 3.1-5.5, such as in the range of pH 5.0-3.2, such as in the range 3.3- 4-2, such as 3.4-4.0, such as 3.5-3.8, such as 3.7-4.2, such as 3.7-4.0, or such as 3.8-4.2. In yet another embodiment, the fermented composition provided in step iv) has a lactic acid concentration of at least 50 mM, such as at least 100 mM, such as 100-1000 mM, such as 100-500 mM, such as 100-300 mM, such as 100-200 mM, such as 150-500 mM, such as 200-500 mM or such as 300-500, mM lactic acid.

Since it is believed that the probiotic effect of the one or more lactic acid bacterial strain(s) peaks after 6 hours to 10 days, such as 12 hours to 9 days, e.g. 18 hours to 8 days, such as 24 hours to 7 days, e.g. 2-6 days, such as after 3-5 days, e.g. after 4-5 days, such as 6 hours to 4 days, such as 6 hours to 3 days, e.g. 6 hours to 2 days, such as 6 hours to 24 hours, e.g. 6 hours to 18 hours, such as 6 hours to 12 hours the fermentation process may be stopped within this time period. After the fermentation is stopped the fermented composition may be dried in order to prolong the viability (CFU) of the one or more lactic acid bacterial strain(s). The fermented composition according the present invention may be a liquid, a slurry, or a dry powder.

The process according to the present invention may further comprises a step of reducing the moisture content from the moisture content obtained from the fermentation process to a fermented composition moisture content. In an embodiment of the present invention the composition may be subjected to drying.

Any moisture reducing methods may be used which are sensitive to the pre- and probiotic components of the fermented composition in order to maintain the activity of the components in the fermented composition and that ensure high viability of the lactic acid bacteria present in the fermented composition. The method to reduce to moisture content as described in WO 2013/029632 may be preferred (WO 2013/029632 is hereby incorporated by reference). In order to improve the fermentation process, the plant material, according to the present invention, may be pre-treated before combining the plant material with the one or more lactic acid bacterial strain(s) as described in step (iii).

Such pre-treatment may involve grinding, cutting, chopping, slicing, and/or fractionizing the plant material.

In a preferred embodiment of the present invention, the plant material provided in step (i) has, optionally after a pre-treatment, an average diameter of 5 mm or less, such as an average diameter of 4 mm or less, such as an average diameter of 3 mm or less, such as an average diameter of 2 mm or less, such as an average diameter of 1 mm or less, such as an average diameter in the range 25 μηη to 5 mm, such as 0.1 mm to 4 mm, such as an average diameter in the range of 0.5 mm to 2.5 mm, such as an average diameter in the range 0.5 mm to 2 mm.

Determination of the average diameter of the plant material may e.g. be performed by microscopic inspection, laser diffraction or sieving.

In an embodiment of the present invention, the plant material may preferably be selected from one or more of rape species; cruciferous vegetables; and/or mustard species.

Preferably, the plant material may preferably be selected from one or more of rape species. Preferably, the rape species is a rapeseed product, such as rapeseed meal, or rapeseed cake, preferably rapeseed cake. In another embodiment of the present invention, the plant material may be Brassica rapa.

The plant material may be provided in the form of seeds, press cake, meal or other residues of industrial utilization of said plant material. Preferably, the plant material may be provided in the form of cake or meal, even more preferably, the plant material may be provided in the form of cake.

In an embodiment of the present invention the combination of the plant material provided in step a) and the one or more lactic acid bacterial strain(s) provided in step b) may be further supplemented with one or more further ingredient selected from the group consisting of cereals, (e.g. wheat, barley, rye, rice, maize (cob maize silage (CCM) or ripe), triticale, oat); vegetables (e.g. potatoes, beans, peas, maize, soy); and/or carbohydrate ingredient (such as whey, curd, skim milk and the like).

When the combination of the plant material provided in step a) and the one or more lactic acid bacterial strain(s) provided in step b) may be further supplemented with cereals, preferably wheat, the cereal constitutes 1-15% (w/w) of the composition, such as 5-10% (w/w) of the composition.

When the combination of the plant material provided in step a) and the one or more lactic acid bacterial strain(s) provided in step b) may be further supplemented with vegetables, preferably starch containing vegetables such as potatoes, the vegetables constitutes 0.1- 5% (w/w) of the composition, such as 0.5-2% (w/w) of the composition When the combination of the plant material provided in step a) and the one or more lactic acid bacterial strain(s) provided in step b) may be further supplemented with carbohydrate ingredient, such as whey, the carbohydrate ingredient constitutes 0.1-5% (w/w) of the composition, such as 0.5-2% (w/w) of the composition.

In an embodiment of the present invention, the fermentation process may preferably be an anaerobic fermentation process or a substantially anaerobic fermentation process.

In the context of the present invention the term "substantially anaerobic fermentation" relates to fermentation where no oxygen or other respiratory metabolism or oxidative phosphorylation is being established. Hence, the term "substantially anaerobic

fermentation" means that oxygen (or other electron acceptor compounds) is not systematically added to the fermentation. The oxygen naturally present and included when setting up the fermentation process may be accepted.

The process according to the present invention was shown to be suitable for producing a novel fermented plant material comprising a high content of lactic acid bacterial strains and a high content of HDMPPA and showing nutritional and/or pharmacological effects. In a preferred embodiment of the present invention the fermented composition comprises at least one plant material selected from the group consisting of rape species; cruciferous vegetables; mustard species, and/or seaweed, preferably rape species or the combination of rape species and seaweed; and one or more lactic acid bacteria, wherein the

composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g.

HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) is a compound originally found in kimchi as one of the major molecules. Kimchi is a fermented Korean vegetable side dish comprising cabbage red pepper, garlic ginger and lactic acid bacteria. HDMPPA is believed by researchers to be an active immune defence in the central nervous system, CNS (see Jin-Woo Jeong et al. 2015), to have anti-oxidative activity and prevent and/or treating atherosclerosis (Hyun Ju Kim, el al. 2007), to be lipid lowering, to have anti-tumor and anti-atherogenic effects, to have anti-viral effects against acute respiratory syndrome (Ye-Rang Yun et al. 2014). The amount of HDMPPA in kimchi is approximately 1 mg/lOOg kimchi (Jeong Sook Noh et al., (2013)).

The inventors of the present invention surprisingly found that the fermented plant material 5 according to the present invention comprises significant more HDMPPA than the fermented product according to the present invention, relative to the amount of HDMPPA found in non-fermented plant material or in kimchi products described in the prior art.

In an embodiment of the present invention, the fermented composition comprises at least 10 2 mg HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g. at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 15 70 mg/100 g, such as at least 80 mg/100 g.

The significant increase in HDMPPA provided in the fermented composition according to the present invention may be provided by a unique combination of lactic acid bacteria used for the fermentation. Hence, the one or more lactic acid bacteria present in the composition 20 may be selected from the group consisting of Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum; Lactobacillus rhamnosus; Enterococcus faecium and/or a combination thereof. Preferably, the lactic acid bacteria present in the

composition may be Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum; Lactobacillus rhamnosus; and Enterococcus faecium.

25

In a preferred embodiment of the present invention, the fermented composition comprising at least one plant material and one or more lactic acid bacteria, wherein the composition comprises at least 2 mg HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) per 100 g fermented composition, on a dry matter basis, such as at least 3 mg/100 g, e.g.

30 at least 4 mg/100 g, such as at least 5 mg/100 g, e.g. at least 7 mg/100 g, such as at least 10 mg/100 g, e.g. at least 15 mg/100 g, such as at least 20 mg/100 g, e.g. at least 30 mg/100 g, such as at least 40 mg/100 g, e.g. at least 50 mg/100 g, such as at least 60 mg/100 g, e.g. at least 70 mg/100 g, such as at least 80 mg/100 g and wherein one or more lactic acid bacteria present in the composition is selected from the group consisting

35 of Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum;

Lactobacillus rhamnosus; Enterococcus faecium and/or a combination thereof.

In an embodiment of the present invention, neither the composition nor the fermented composition comprises Lactobacillus kimchi. In a preferred embodiment of the present invention the at least one plant material (when the composition comprises HDMPPA) is selected from the group consisting of rape species; cruciferous vegetables; mustard species, and/or seaweed, preferably rape species and/or seaweed. Preferably, the plant material is not a cabbage species. Even more preferably, the plant material is (when the composition comprises HDMPPA) a combination of rape species and seaweed.

In an embodiment of the present invention the fermented composition was analysed for HDMPPA by subjecting an 80% methanol extraction of the fermented composition to Ultra High Pressure Liquid Chromatography-Quadruple Time of Flight Mass Spectroscopy

(UHPLC-Q-ToF-MS). Apart of a significant amount of HDPPA in the fermented plant material, an even more significant amount of a molecule was found in a fermented rapeseed/seaweed fraction (1); whereas fermented rapeseed (2); non-fermented seaweed (3); and non-fermented rapeseed (4), showed no or insignificant amounts of the molecule.

Furthermore, the amount of HDMPPA may be increased relative to kimchi, such as a fermented composition comprising a cabbage species, such as Korean cabbage, or a fermented plant material mainly using Lactobacillus kimchi.

In the present context the term "mainly using" relates to a concentration of more than 50% of the total content of lactic acid bacteria in the composition or in the fermented composition being Lactobacillus kimchi. The inventors surprisingly found that HDMPPA may be present in at least 2 times the concentration/amount found in non-fermented plant material, such as at least 5 times, e.g. at least 10 times, such as at least 15 times. It was surprisingly found that HDMPPA may be present in at least 2 times the concentration/amount found in kimchi, such as at least 5 times, e.g. at least 10 times, such as at least 15 times.

The fermented composition according to the present invention may comprise the same one or more lactic acid bacterial strain(s) as provided in step (ii).

In an embodiment of the present invention at least one of the one or more lactic acid bacterial strain(s) as provided in step (ii) are not present in the fermented composition, such as at least two, e.g. at least 3, such as at least 4, e.g. at least 5, such as at least 10, e.g. at least 15. In another embodiment of the present invention the fermented composition comprises one or more lactic acid bacterial strains, e.g. two or more lactic acid bacterial strains, such as three or more bacterial strains, e.g. four or more bacterial strains, such as 7 or more bacterial strains, e.g. 10 or more bacterial strains, such as 15 or more bacterial strains, e.g. 20 or more bacterial strains, such as 25 or more bacterial strains, e.g. 30 or more bacterial strains, such as 35 or more bacterial strains, e.g. 40 or more bacterial strains.

In a further embodiment of the present invention the one or more lactic acid bacterial strain(s) present in the fermented composition may be selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella. Preferably, the one or more lactic acid bacterial strain(s) present in the fermented composition are preferably lactic acid bacteria of the genus Enterococcus, Lactobacillus, Pediococcus, Lactococcus, or a combination thereof.

In an even further embodiment of the present invention the one or more lactic acid bacteria stain(s) may be selected from the group consisting of one or more Enterococcus spp. , Lactobacillus spp. , Lactococcus spp. , Pediococcus spp. , and a combination hereof. Preferably, the one or more lactic acid bacterial strain(s) is/are selected from the group consisting of one or more of Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, Pediococcus acidililactili , Pediococcus pentosaceus, Lactococcus Lactis, Lactococcus Cremoris, Lactococcus Diacety lactis, Leuconostoc Cremoris and a combination hereof. In yet a further embodiment of the present invention, the main lactic acid bacterial strain(s) present in the composition may be Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum; Lactobacillus rhamnosus; or Enterococcus faecium. Preferably, the main lactic acid bacteria present in the composition may be Lactobacillus plantarum.

In another embodiment of the present invention the one or more lactic acid bacteria stain(s) may be selected from the group consisting of one or more of Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Pediococcus pentosaceus HTS (LMG P-22549), Pendiococcus acidilactici NCIMB 30086 and/or Lactobacillus plantarum LSI (NCIMB 30083).

In order to provide the desired effects, the fermented composition should have a high content of viable lactic acid bacteria. In an embodiment of the present invention the fermented composition comprises one or more lactic acid bacterial strain(s) in a total amount in the range of 10 5 -10 12 CFU per gram, such as in the range of 10 5 -10 12 CFU per gram, e.g. in the range of 10 7 -10 n CFU per gram, such as in the range of 10 8 -10 n CFU per gram, e.g. in the range of 10 9 -10 10 CFU per gram. In an embodiment of the present invention the content of the plant material in the fermented composition is at least 50% plant material, such as at least 55% plant material, e.g. at least 60% plant material, such as at least 65% plant material, e.g. at least 70% plant material, such as at least 75% plant material, e.g. at least 80% plant material, such as at least 85% plant material, e.g. at least 90% plant material, such as at least 95% plant material.

The fermented composition may be provided in many forms. Preferably, the fermented composition may be a liquid, a slurry, or a dry powder. In an embodiment of the present invention the fermented composition may be mixed with one or more other fermented compositions comprising one or more other lactic acid bacterial strain(s) and/or one or more other plant materials. Such mixed fermented compositions may provide additional effects and/or synergistic effects. The fermented composition according to the present invention may be used as an ingredient.

The fermented composition obtainable by the processes of the invention may form part of (or be) a food ingredient. Thus, an aspect of the invention relates to a food/feed ingredient comprising the fermented composition according to the invention. In the present context, the terms "food" and "feed" may be used interchangeable, however, the term "food" refers specifically to eatable material suitable for human consumption, whereas the term "feed" refers specifically to eatable material suitable for animal consumption. In an embodiment of the present invention the term "animal(s)" may include pigs, piglets, cattle, and horses, poultry such as chickens, turkeys, hens, geese, dogs, cats and ducks, and fish such as salmon and trout.

The food/feed ingredient may also form part of a food/feed product. Thus, in a further aspect the invention relates to a food/feed product comprising the food/feed ingredient according to the invention.

An additional aspect relates to the use of the fermented composition according to the invention as a food/feed ingredient. Yet an embodiment of the present invention relates to a food/feed product comprising the fermented composition, preferably as a dry feed/food ingredient according to the invention. Preferably, the food/feed product comprises in the range 5-50% by weight of the dry feed/food ingredient, such as in the range 5-30%, such as 10-30% or such as 10- 20%.

In a further aspect according to the present invention the fermented composition or the ingredient according to the present invention may be used in a food product; in a food supplement product; in an herbal medicine product; in a natural medicine product; in a medicinal product and/or as an adjuvant product accompanied by one or more other drugs. Preferably, the fermented composition or the ingredient according to the present invention may be used as an adjuvant product. In the present context the term "adjuvant" means contributing to or enhancing an existing medical regimen. A preferred embodiment of the present invention relates to a substance for use as a medicament comprising a fermented composition according to the present invention.

In the present context, the term "substance" relates to a drug, a complex material or a combination hereof that a may improve the well-being of a mammal. The complex material may preferably be the fermented composition according to the present invention.

In an embodiment of the present invention, the term "mammal" according to the present invention may be a human or an animal. Preferably, the substance according to the present invention may be used in the treatment, alleviation or prophylaxis of an inflammatory condition in a mammal (such as a human or an animal), said substance comprises a fermented composition according to the present invention. In the present context the term "inflammatory condition" relates to a substance showing antioxidative activity, active in the defence in the central nervous system (CNS), antitumor activity, lipid lowering, anti-atherogenic activity, e.g. atherosclerosis, and/or antiviral effects against acute respiratory syndrome. In the context of the present invention the term "treatment" relates to the use of an agent, such as the substance or the fermented composition according to the present invention, in an attempt to cure or mitigate a disease, a condition, or an injury in a mammal . The term "alleviation" used in the present invention relates to the action of an agent, such as the substance or the fermented composition according to the present invention, to make a disease, a condition or an injury less intense and/or reduce symptoms in a mammal.

In the context of the present invention the term "prophylaxis" relates to the use of an agent, such as the substance or the fermented composition according to the present invention, in an attempt to prevent a disease, a condition or an injury in a mammal and/or for the protective treatment of a mammal.

In an embodiment of the present invention one dose or a single dose relates to 50 gram fermented product according to the present invention or less, such as 40 g or less, e.g. 30 gram or less, such as 25 gram or less, e.g. 20 gram or less, e.g. 15 gram or less, such as 10 gram or less.

In another embodiment of the present invention the substance and/or the fermented composition may be used as an adjuvant product accompanied by one or more other drugs. A preferred embodiment of the present invention relates to a combination product comprising a fermented composition, a substance obtained from the fermented

composition, HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent. An even further preferred embodiment of the present invention relates to a combination product comprising a fermented composition, a substance obtained from the fermented composition, HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) according to the present invention, or sinapic acid in combination with an antimicrobial agent, for use in the treatment and/or alleviation of a microbial infection with reduced incidence of anti- microbial resistance of the targeted microorganism by the antimicrobial agent.

Modern life is closely connected to the use of antimicrobial agents, such as antibiotic agents. Antibiotic agents cure diseases, such as pneumonia and bacterial abdominal infections, and are indispensable in connection with major surgery. Not only humans use increasing amounts of antibiotics, but also modern livestock farming has become more and more dependent on antibiotics as animals may get ill exactly like humans. Animals, that previously would die or be killed, are now cured and able to reproduce and be delivered at slaughterhouses. The biggest problem of using all these antimicrobial agents, and in particular using excessive amounts of antimicrobial agents is that the targeted microorganisms develop resistance against the antimicrobial agents which becomes ineffective. Every year more than 25,000 European citizens die from infections caused by bacteria that have developed resistance towards antimicrobial agents. The result is that treatments that used to work efficiently have lost their effect.

In the context of the present invention, the term "antimicrobial resistance" relates to a microorganism's resistance to an antimicrobial agent that was once able to treat an infection by that microorganism. A person cannot become resistant to an antimicrobial agent, resistance is a property of the microbe, not a person or another organism infected by a microbe.

Resistance against an antimicrobial agent may arises through one of three ways: natural resistance in certain types of bacteria; genetic mutation; or by one species acquiring resistance from another. Resistance can appear spontaneously due to random mutations; or more commonly following gradual buildup over time, and because of misuse of antibiotic agents or antimicrobial agents. Resistant microbes are increasingly difficult to treat, requiring alternative medications or higher doses - which may be more costly or more toxic. Hence, if antibiotics lose their potency, we weaken or even lose a fundamental basis of modern society.

In an embodiment of the present invention the antimicrobial agent is an antibiotic agent. In the present context the term "antibiotic agent" relates to any type of antimicrobial drug used in the treatment and prevention of bacterial infections.

The SOS pathway or the SOS response plays a critical role in the acquisition of mutations that lead to the emergence of antibiotic resistant microorganisms, and may be used to evaluate the extent of resistance of an antimicrobial agent. SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and

mutagenesis are induced.

In an embodiment of the present invention the incidence of antimicrobial resistance of the targeted microorganism by the antimicrobial agent is reduced by at least 10%, such as by at least 20%, e.g. by at least 30%, such as by at least 40%, e.g. by at least 50%, such as by at least 60%, e.g. by at least 70%, such as by at least 80%.

The inventors of the present invention surprisingly found that the fermented composition, the substance obtained from the fermented composition, the HDMPPA (3-(4'-hydroxyl- 3',5'-dimethoxyphenyl)propionic acid) of the present invention or the sinapic acid may inhibit the SOS response induced by an antimicrobial agent.

In an embodiment of the present invention the fermented composition, the substance obtained from the fermented composition or the HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) may be obtained from lactic acid fermentation of a plant material. Preferably, the fermented composition, the substance obtained from the fermented composition or the HDMPPA (3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid) may comprise at least one plant material and one or more lactic acid bacteria.

In yet an embodiment of the present invention the fermented composition, the substance obtained from the fermented composition or the HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) comprises one or more lactic acid bacteria and one or more plant materials.

In another embodiment of the present invention the HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) or the sinapic acid may be chemically produced.

In the present context, the term "plant material" provided for the combination product of the present invention, relates to organisms (such as plants, seaweed or algae) capable of performing photosynthesis.

In an embodiment of the present invention, the plant material, provided for the combination product of the present invention, may be selected from at least one proteinaceous plant material. The plant material, provided for the combination product of the present invention, and the proteinaceous plant material, may be selected from at least one of Brassica spp.; seaweed; algae; sun flower; palm; soya, field beans, Lupins; or a combination hereof. Preferably, the plant material, and the proteinaceous plant material, may be selected from Brassica spp.; seaweed/ algae; or a combination hereof. The Brassica spp. may preferably be selected from one or more of rape species; cruciferous vegetables; cabbage species; and/or mustard species. Preferably, the Brassica spp. may preferably be selected from one or more of rape species. Preferably, the rape species is a rapeseed product, such as rapeseed meal, or rapeseed cake, preferably rapeseed cake. In another embodiment of the present invention, the Brassica spp., when provided for the combination product of the present invention, may be selected from one or more species such as Brassica napus; Brassica oleracea; Brassica campestris; and/or Brassica rapa. The seaweed and/or algae may be selected from one or more of brown algae, red algae, green algae, such as kelps, Laminaria saccharina, Laminaria digitata, and/or Laminaria hyperborean. In an even further embodiment of the present invention, the plant material, provided for the combination product of the present invention, comprises a combination of Brassica spp., in particular Brassica napus; or Brassica campestris, and seaweed/algae.

In the event the plant material, provided for the combination product of the present invention, comprises a combination of Brassica spp., in particular Brassica napus; or

Brassica campestris, and seaweed/algae. The seaweed/algae may preferably be subjected to the pre-treatment to an extent that result in an average diameter which is at most 75% of the average diameter of the Brassica spp., such as at most 50% of the average diameter.

In yet an embodiment of the present invention, the ratio between Brassica spp. and seaweed/algae is at least 1 : 1, such as at least 1 :2, e.g. at least 1 :3, such as at least 1 :4, e.g. at least 1 : 5, such as at least 1 :6, e.g. at least 1 :7, such as at least 1 :8, e.g. at least 1 :9, such as at least 1 : 10.

In an embodiment of the present invention, the dose of HDMPPA (3-(4'-hydroxyl-3',5'- dimethoxyphenyl)propionic acid) or sinapic acid may be added in an amount of 0.1-10 μg/ml, such as 0.5-8 μg/ml, 1-6 μg/ml, 2-5 μg/ml, such as about 4 μg/ml. In yet an embodiment of the present invention, the dose of fermented composition or the substance obtained from the fermented composition, may be added in an amount of 1- 1000 μg/ml, such as 5-600 μg/ml, 10-200 μg/ml, 15-100 μg/ml, 20-75 μg/ml, 22-50 μg/ml, such as about 25 μg/ml. In the present invention the incidence of anti-microbial resistance of the microorganism towards the antimicrobial agent may also be expressed in terms of expression of sulA; the ratio of cell mass/DNA content; phage transduction mediated by recA; and/or the mutagenesis and expression of DinB. In a further embodiment of the present invention the combination product inhibited the expression of su I A by antimicrobial agents by at least 25%, such as at least 40%, e.g. at least 50%, such as at least 60%, e.g. at least 70%, such as at least 80%, e.g. at least 85% The combination product of the present invention prevents an increase in the ratio of cell mass/DNA content, induced by an antimicrobial agent, of at least 10%, such as by at least 20%, e.g. by at least 30%, such as by at least 35%. The combination product according to the present invention also significantly reduced the phage transduction mediated by recA, as well as the mutagenesis and expression of DinB induced by an antimicrobial agent by at least 10%, such as at least 20%, e.g. at least 30%, such as at least 40%, e.g. at least 50%. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1

Preparation of a fermented composition comprising rapeseed according to the present invention.

The present invention was exemplified by mixing about 80% rapeseed meal (having an average diameter of about 2 mm), about 14% wheat 1% potato flakes (vegetable source), and 5% Perlac (carbohydrate source) in a fermentation tank. The moisture content was adjusted to about 40% humidity and a combination of the lactic acid-producing bacteria Pediococcus pentosaceus, Pediococcus acidilactici and Lactobacillus plantarum was added. The air intake to the fermentation tank was taken directly from the air in the room wherein the fermentation tank was situated. The ingredients were mixed at room temperature and the temperature was slowly increased from the heat generated from the fermentation to a steady state temperature of about 42°C.

After 20 hours of fermentation the fermentation broth had a pH of 4.2 and a lactic acid concentration of about 140 mM. The fermented composition had a bacterial count of 3.6x l0 5 CFU/gram and analyzing the fermented composition showed very small amounts of acetic acid and ethanol were produced, hence the mixture was considered being 95-

100% homofermentative. The content of HDMPPA in the plant material before fermentation and the content of HDMPPA in the fermented plant material (after fermentation) is illustrated in figure 1. The resulting fermented composition was subjected to spin flash drying in order to protect the lactic acid bacteria and keep a high number of viable cells. Example 2

In order to identify the amount of HDMPPA in the fermented and unfermented plant materials, extracts of the plant materials were provided comprising 80% methanol. The extracts were subjected to untargeted metabolomics analysis by Ultra High Pressure Liquid Chromatography coupled to a Q-ToF mass spectrometer (UHPLC-Q-ToF-MS).

Chromatography was performed on a Dionex UltiMate® 3000 Quaternary Rapid Separation UHPLC+ focused system (Thermo Fisher Scientific, Germering, Germany). Separation was achieved on a Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 μηη, 100 A, Phenomenex, Torrance, CA, USA). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B, respectively.

Gradient conditions were as follows: 0.0-0.5 min, 2% B; 0.5-14.0 min 2-20% B; 14.0-20.0 min 20-45% B, 20.0-24.5 min 45-100% B, 24.5-26.5 min 100%, 26.5-26.55 min 100-2% B and 26.55-30.0 min 2% B. The mobile phase flow rate was 300 μΙ min-1. The column temperature was maintained at 25°C. Four wavelengths (205 nm, 220 nm, 250 nm and 390 nm) were monitored by a UV-VIS detector. The liquid chromatography was coupled to a Compact micrOTOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive or negative ionization mode. The ion spray voltage was maintained at -3900 V in negative mode. Dry temperature was set to 250°C and dry gas flow was set to 8 L min-1. Nebulizing gas was set to 2.5 bar and collision energy to 15 eV. Nitrogen was used as dry gas, nebulizing gas and collision gas.

The m/z range was set to 50-1400. AutoMSMS mode was used to obtain MS and MS/MS spectra of the three most abundant ions present at each time point with smart exclusion to also include less abundant ions. All files were calibrated based on compound spectra collected from Na+-formiate clusters at the beginning of each run.

Methanol extract samples (80% methanol) of rape species (rape seed) according to the present invention, and non-fermented rape species (rape seed) were run in autoMSMS mode to acquire MS and MS/MS spectra of molecules present in the different extracts. The m/z range was set to 50-1400. Na + -formiate clusters were used as calibrant and injected at the beginning of each sample run. All files were automatically calibrated based on the compound spectra collected from the Na + -formiate clusters by post processing.

Results

A dominating peak was found having a "mass to charge ratio" (m/z) of (in negative mode) :

MSI mass =225.0772

Retention time: 11.86 min (under the set conditions)

From comparative studies with pure samples of HDMPPA this dominating peak showed to be HDMPPA. As it appears from the chromatogram in figure 2, a significant amount of HDMPPA (3-(4'- hydroxyl-3',5'-dimethoxyphenyl)propionic acid) may be found in fermented rapeseed (1) relative to non-fermented rapeseed (2). From this chromatogram it is shown that fermented rapeseed (1) has significant more HDMPPA relative to non-fermented rapeseed (2) .

References

WO 2013/029632

Jin-Woo Jeong et al., Journal of Medicinal Food, 18 (6) 2015, 677-684 Hyun Ju Kim et al., J. Agric. Food Chem., 2007, 55, 10486-10492 Jeong sook Noh et al., British Journal of Nutrition; (2013), 109, 17-24 Ye-Rang Yun et al., Journal of Medicinal Food, 17 (8) 2014, 886-893