Consumable Product Comprising Malted Dehulled Oats
20210212349 · 2021-07-15
Assignee
Inventors
Cpc classification
A61K36/899
HUMAN NECESSITIES
A23L7/25
HUMAN NECESSITIES
A23L7/143
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
A23L33/00
HUMAN NECESSITIES
International classification
A23L7/25
HUMAN NECESSITIES
A23L29/00
HUMAN NECESSITIES
A23L7/143
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
Abstract
The disclosure relates to a consumable product comprising malted dehulled oats and/or a leachate of malted dehulled oat, wherein said consumable product induces endogenous production of antisecretory factor (AF) protein and/or fragments thereof in a subject after consumption. The malted dehulled oats comprised in the consumable product disclosed herein is produced by a novel malting process.
The malted dehulled oats and/or a leachate of malted dehulled oats comprised in the consumable product comprises (i) avenanthramide D, wherein the concentration of (i) is higher as compared to the corresponding non-malted dehulled oats, and optionally one or more of the compounds selected from the group consisting of (ii) avenanthramide A, (iii) avenathramide C, (iv) avenanthramide C methyl ester, (v) (Z)-N-feruloyl 5-hydroxyanthranilic acid, (vi) avenanthramide G, and (vii) a compound selected from the group consisting of guaiacol or a derivative thereof, L-tryptophan , DL-phenylalanine, and any combination thereof, wherein the concentration of one or more of (ii-vii) is higher as compared to in the corresponding non-malted dehulled oats.
The disclosure further provides use of the consumable product as food or feed for humans and/or animals, as well as for medical use.
Claims
1. A consumable product comprising malted dehulled oats and/or a leachate of said malted dehulled oats, wherein said malted dehulled oats are produced by a malting process characterized by comprising the steps of: a. dehulling oat kernels, b. wet steeping of the dehulled oat kernels at a temperature from 5 C. to 20 C., c. germinating of said dehulled oat kernels at a temperature from 5 C. to 20 C., d. optionally repeating any one of steps b-c, and subsequent e. drying of said dehulled oat kernels at no more than 80 C. air temperature, wherein the malted dehulled oats comprise avenanthramide D at a higher concentration as compared to the corresponding non-malted dehulled oats and wherein the consumable product induces endogenous production of antisecretory factor (AF) protein and/or fragments thereof in a subject after consumption.
2. A consumable product according to claim 1, wherein the wet steeping of the dehulled oat kernels in step b. is performed at a temperature from 7 C. to 15 C. for 1-5 days.
3. A consumable product according to claim 1, wherein the germinating of said dehulled oat kernels in step c. is performed for 5-9 days at a temperature of 12-15 C.
4. A consumable product according to claim 1, wherein the germinating of said dehulled oat kernels in step c. is performed for 7 days at a temperature not exceeding 15 C.
5. A consumable product according to claim 1, wherein the malted dehulled oats comprise: (i) avenanthramide D, wherein the concentration of (i) is at least 100% higher as compared to non-malted dehulled oats.
6. A consumable product according to claim 1, wherein the malted dehulled oats further comprise one or more of: (ii) avenanthramide A, (iii) avenathramide C, (iv) avenanthramide C methyl ester, (v) (Z)-N-feruloyl 5-hydroxyanthranilic acid, and (vi) avenanthramide G, and wherein the concentration of one or more of (ii), (iii), (iv), (v) and (vi) is higher as compared to non-malted dehulled oats.
7. A consumable product according to claim 1, wherein the malted dehulled oats further comprises: (vii) a compound selected from the group consisting of guaiacol or a derivative thereof, L-tryptophan, DL-phenylalanine, and any combination thereof, wherein the concentration of one or more of (vii) is higher as compared to in the corresponding non-malted dehulled oats.
8. A consumable product according to claim 7, wherein the guaiacol derivative is ferulic acid, sinapic acid, or p-coumaric acid.
9. A consumable product according to claim 1, wherein said consumable product comprises malted dehulled oats and/or a leachate of said malted dehulled oats in an amount sufficient to increase the amount of antisecretory protein and/or fragments thereof in the subject's blood to at least about 1 unit/mL, and/or to increase the amount of ASP Units in the subject's blood to at least about 1 Unit/ml.
10. A consumable product according to claim 1, which is a food, feed, a food supplement and/or a nutraceutical.
11. A consumable product according to claim 1, which is in the form of a liquid, a solid or a combination thereof.
12. A consumable product according to claim 1, which has antisecretory properties, anti-diarrhoeal properties and/or anti-inflammatory properties.
13. A method for treatment, amelioration and/or prevention of a condition responsive to increase of levels of antisecretory factor protein and/or antisecretory protein fragments in the blood of a patient comprising administering to a subject/patient in need thereof a sufficient amount of a consumable product according to claim 1.
14. A method for treatment, amelioration and/or prevention of a condition according to claim 13, wherein said condition is selected from the group consisting of diarrhoea, inflammatory disease, oedema, autoimmune disease, cancer, tumour, leukaemia, diabetes, diabetes mellitus, glioblastoma, traumatic brain injury, intraocular hypertension, glaucoma, compartment syndrome, Alzheimer's disease, Parkinson's disease, encephalitis, and Meniere's disease.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0125] Oats (Oats) is a well-known food or food ingredient. It is generally consumed as dehulled precooked (steamed) flakes or as oats flour. Oats is an important source for a number of valuable nutrients, among them -glucans. -glucans form very viscous water solutions, making worth filtering difficult. Oats also contains high levels of phytic acid, making essential mineral absorption in the gut less efficient.
[0126] The oats kernel is surrounded by a hard hull, considered as inedible. Consequently, a number of processes have been developed to dehull the oats kernel. Dehulling oats includes the risk of also removing the germ together with the hull. Hence oats intended for malting for e.g. beer brewing are not dehulled. Thus, as a rule, oats are malted with hull.
[0127] Seed development has produced an oats variety with an undeveloped hull, hull-less oat, also called naked oat. Naked oats is used, mainly as a food ingredient. Still, the lack of hull is necessarily compensated by the development of a strong pericarp.
[0128] The malting of oats has been investigated extensively, mainly with the purpose of improving worth yield and reducing of phytic acid content. During the malting process a vast number of dormant enzymes are activated, such as hydrolases, amylases, proteases, lipases and phytases.
[0129] Industrial malting consists of cleaning of the grain, steeping, germination, drying and sprout removal. The processes are performed batch-wise in grain beds. Moisture content of the grain in steeping is determined by contact time in water. Germination time is determined by the intended use of the finished malt, moisture content and temperature during germination. The generated metabolic heat is controlled by cooling with air. During germination, the grains are stirred by mechanical devices. Drying with warm or hot air induces the formation of taste and aroma substances.
[0130] Malting of seeds implies that the seeds are steeped in water for different length of time and temperatures. After steeping, the seeds are germinated for different lengths of time and temperature. As seeds are not sterile, malting also implies the growth of fungi and bacteria during steeping and germination. If the malted product is intended for beer production, the worth cooking functions also acts as a pasteurization. Hence, the growth of microorganisms can be controlled to a large extent. The heat evolved during germination is normally cooled by cold air blown through the grains.
[0131] When utilizing malted oats for other intended uses than in beer-production, though, the oats hulls make the product less palatable. What is more, malting of oats with hulls without pasteurisation can give a final product with unhealthy or less advantageous levels of microorganisms. Also, when cooking the worth, the hulls form a porous filter cake when the worth is filtered prior to fermentation.
[0132] Dehulling oats prior to malting would consequently reduce the problems listed above. But, dehulling dramatically increases the risk of removing the germ, making germination impossible. Further, moistened dehulled oats form impermeable beds due to the high level of hydrocolloids on the kernel surface.
[0133] To solve the problems referred to above a novel malting process is herein disclosed wherein a malted dehulled oats product is produced which is suitable for food, feed and/or medical food purposes. The malting process is described in detail in example 1.
[0134] The novel malting process described herein is a low-temperature malting process that allows malting of dehulled oats in a process that is easily scalable to industrial use.
[0135] In the process, the oats lot is refined by sieving and by using gravity tables so that the final 1000 grain weight exceeds 30 grams/1000 kernels. Such as that the final 1000 grain weight exceeds 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 grams/1000 kernels.
[0136] The selected oats lot is dehulled by a dehuller. In the disclosed process, the dehuller is preferably a rotating disc with radial groves, but the person skilled in the art will understand that any commercially available dehuller can be used, as long as it leaves dehulled oats with the specified minimum germinability. A commercially available dehuller can be selected from the non-limiting group of Bailer BSSA Stratopact HKE5OHP Ex and Streckel &Schrader. The feed and disc speed are typically selected so that 30 -70% of the kernels are dehulled at each passage.
[0137] The germinability of the dehulled oats is tested to exceed 95%, such as no less than 80.
[0138] 81, 82, 83, 84, 85, 85, 87, 88, 89, 90, 91, 92, 93, 94 or 95% in petri-dish, or at least 82%, such as at least 77, 76, 78, 79, 80, 81 or 82% in H.sub.2O.sub.2.
[0139] The selected dehulled oats kernels are steeped with cold water (w), optionally alternatingly in dried conditions (d) at temperatures between 5-15 C., or 7 C.-15 C., such as at temperatures not exceeding 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 C., such as at a temperature between 5-12 C., 5-15, 12 C., 7-12 C., 12-15 C., 10-15 C. or 7-10 C., fora total of 1-3 days, such as for 20-26 hours, such as for 20, 21, 22, 23, 24, 25 or 26 hours, such as for no less than 1, 2 or 3 days. Kernel moisture content is herein kept between 30-50%, such as between 30-35%, 30-40%, 30-45%, 35-40%, 35-45%, 35-50%, 40-45%, 40-50% or 45-50%. The kernel moisture should in this process step not exceed 30, 35, 40, 45 or 50%.
[0140] In the present context, the malting comprises wet steeping in which the oats is partly or entirely soaked with water. Additionally, or alternatively, the wet steeping may involve spraying with water.
[0141] After steeping, the dehulled oats is germinated for 7-9 days at 5-20 C., preferably at 7-12 C., at 7-15 C., or at 12-15 C., such as for at least 7, 8 or 9 days at a temperature not exceeding 12, 13, 14, 15 or 20 C., such as at a temperature of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 20 C.
[0142] The heat evolved is cooled by cold air. Due to the impermeable beds that can be formed, only shallow beds are used, with no more than 0.5 m bed height, such as with max 0.1, 0.2, 0.3, 0.4 or 0.5 m bed height. Any movement of the grains is performed at slow speed.
[0143] The germinated grain is initially dried at low air temperature not exceeding 35 C., such as at a temperature between 15-35, 20-35, 25-35 or 30-35 C. In the later stages of drying, when moisture content is below 20%, drying air temperature is raised to a maximum temperature of 65 C., max 65-70 C. or max 65-80 C. The drying air temperature should not exceed 80 C. at any time.
[0144] By this novel malting method, a healthy malted dehulled oats product with a high level of enzymatic activity is produced as disclosed in the present disclosure and as analysed in example 2 herein.
[0145] It has been found that the process for malting the oats impacts the properties of the consumable product into which it is incorporated. Importantly, the malting should take place at a low temperature such as from about 5 C. to about 20 C. and subsequent drying should take place at an air temperature of 80 C. or less. It will be appreciated that in this document the expression a temperature of 80 C. or less means a temperature equal to or less than 80 C.
[0146] Thus, there is provided a consumable product as described herein, wherein the malted dehulled oats is obtained from a process comprising the steps of: [0147] a. malting dehulled oats at a temperature from about 5 C. to about 20 C., and [0148] b. drying said dehulled oats at no more than 80 C.
[0149] In a further example, there is provided a consumable product as described herein, wherein the malted dehulled oats is obtained from a process comprising the steps of: [0150] a. wet steeping of dehulled oats at a temperature from about 5 C. to about 20 C., [0151] b. germinating/growing at a temperature from about 5 C. to about 20 C., [0152] c. optionally repeating any one of steps a-b, and subsequent [0153] d. drying of said dehulled oats at no more than 80 C.
[0154] Steps a. and/or b. described herein may independently take place at a temperature of about 8 C. or from about 13 C. to about 15 C.
[0155] The present disclosure is based on the unexpected and surprising finding that a consumable product comprising malted dehulled oats, produced with a malting process according to the present invention, comprises a combination of (i) avenanthramide A, (ii) avenanthramide C methyl ester, (iii) avenanthramide D and (iv) certain compounds as described herein to such an increased amount that it induces endogenous production of antisecretory factor (AF) protein and/or fragments thereof in a subject after consumption.
[0156] Surprisingly, it was found that that the combination of the compounds (i)-(iv) in the concentrations described herein increases the Antisecretory Factor (AF) activity, and/or improves the endogenous formation of AF in a subject after consumption.
[0157] Thus, there is provided a consumable product comprising malted dehulled oats and/or a leachate of said malted dehulled oats comprising in particular (i) avenanthramide D, wherein the concentration of (i) is higher as compared to the corresponding non-malted dehulled oats, and wherein the consumable product induces endogenous production of antisecretory factor (AF) protein and/or fragments thereof in a subject after consumption.
[0158] The malted dehulled oats and/or a leachate of said malted dehulled oats comprised in the consumable product may further comprise one or more of:
[0159] (ii) avenanthramide A,
[0160] (iii) avenanthramide C,
[0161] (iv) avenanthramide C methyl ester,
[0162] (v) (Z)-N-feruloyl 5-hydroxyanthranilic acid, and optionally
[0163] (vi) avenanthramide G;
[0164] wherein the concentration of one or more of (ii), (iii), (iv) (v) and (vi) is higher as compared to the corresponding non-malted dehulled oats.
[0165] The malted dehulled oats and/or a leachate of said malted dehulled oats comprised in the consumable product may further comprise:
[0166] (vii) a compound selected from the group consisting of guaiacol or a derivative thereof, L-tryptophan, DL-phenylalanine, and any combination thereof;
[0167] wherein the concentration of one or more of (vii) is higher as compared to the corresponding non-malted dehulled oats.
[0168] The guaiacol derivative described herein may be ferulic acid, sinapic acid and/or p-coumaric acid.
[0169] The consumable product described herein may comprise malted dehulled oats and/or a leachate thereof in an amount sufficient to induce endogenous production of antisecretory factor (AF) protein and/or fragments thereof in a subject after consumption. The specific amount of the consumable product may be adjusted depending on the condition to be treated. For instance, the consumable product may comprise malted dehulled oats and/or a leachate thereof in an amount sufficient to increase the amount of antisecretory protein and/or fragments thereof in the subject's blood to more than 0.5 Units/ml blood, such as to at least 0.6, 0.7, 0.8, 0.9 or at least 1 Units/ml blood. The skilled person may determine the amount using methods known in the art such as the RTT method and/or the Antisecretory Factor Complex Assay described herein.
[0170] The consumable product described herein may be food, feed, a food supplement, and/or a nutraceutical. The food or feed may be for human and/or animal consumption. Generally, food is intended for human consumption while feed is intended for animal consumption. The consumable product described herein may be a liquid, a solid and/or a combination thereof. For instance, the liquid may be a beverage. In a further example, the consumable product may be an infusion. When the food or feed is a solid it may be dry or semi-dry.
[0171] The food described herein may be a medical food. Additionally, or alternatively, the food described herein may be a FSMP, i.e. a food for special medical purposes. It will be appreciated that a FSMP may be food for individuals who suffer from certain diseases, disorders and/or medical conditions, and/or for people whose nutritional requirements cannot be met by normal foods. In a further example, the food described herein may be a nutraceutical. As used herein, a nutraceutical is a food or feed providing an extra health benefit in addition to basic nutritional value in food or feed. The food and/or food supplement for human consumption may be in the form of a liquid, a solid or a combination thereof. In an example, the food for human consumption may be in the form of a liquid, i.e. a liquid food for humans
[0172] The feed described herein may be given to animals such as poultry or livestock animals. The feed for animals may be in the form of a liquid, a solid or a combination thereof. In an example, the feed for animals may be in the form of a liquid, i.e. a liquid feed for animals. Examples of poultry include chickens, hens, ducks, geese, pigeons, quails, turkeys, pheasants and ostriches. Examples of livestock animals include cattle such as cows, horses, donkeys, goats, pigs and sheep. In a further example, animals that can be treated with the consumable product described herein include camels, deer, elks, yaks, lamas, alpacas and water buffalos. In still a further example of animals that can be treated with the consumable product described herein include pets such as dogs, cats, rabbits, guinea pigs and hamsters. In a particular example, the feed described herein is horse feed. In a further example, the feed described herein is pig feed. In still a further example, the feed described herein is dog or and/or cat feed. In still a further example, the feed described herein is fish feed.
[0173] Moreover, it will be appreciated that the consumable product described herein may be feed for ruminants such as cows, sheep and/or camels. The feed for ruminants may be in the form of a liquid, a solid or a combination thereof. In an example, the feed for ruminants may be in the form of a liquid, i.e. a liquid feed for ruminants.
[0174] In the present context, the term feed is used to describe materials of nutritional value fed to animals. Each species has a normal diet composed of feeds or feedstuffs which are appropriate to its kind of alimentary tract and which are economically sensible as well as being nutritious and palatable. Animals such as agricultural animals at pasture often have a diet which is very variable and subject to naturally occurring nutritional deficiencies. The feed disclosed herein may help to remedy or at least alleviate such deficiencies as well as disease, condition and/or symptom brought on by a stressful situation and or environment.
[0175] The presently disclosed feed can further comprise forage feed, such as hay, ensilage, green chop. i.e. any feed with a high cellulose content relative to other nutrients. The presently disclosed feed can further comprise feed grain such as cereal and other grains and pulses used as animal feed. The aforementioned feed grain may include wheat, barley, oats, rye, maize, peas, raps, rape seed, rape seed meal, soybean meal, and sorghum.
[0176] In a further example, the feed described herein may be provided in pelleted form.
[0177] The presently disclosed feed can further comprise feed supplements, i.e. nutritive materials which are feedstuffs in their own right, and which are added to a basic diet such as pasture and/or forage to supplement its deficiencies, such as minerals and aromatics. Feed supplements typically include trace elements and macrofeeds, feed additives or supplements, such as protein supplements and/or minor feed ingredients, such as essential amino acids and vitamins.
[0178] The consumable product can be a feed supplement in itself.
[0179] Albeit the present disclosure mainly is directed to a consumable product in the form of food or feed, it is also envisaged that the consumable product may be administrated to a subject in other ways than oral intake. For instance, the consumable product may be provided in a form making it suitable for topical, ocular, subcutaneous and/or systemic administration.
[0180] The food described herein may form part of a functional food. For instance, the functional food may be muesli, bread, biscuits, gruel, oatmeal, grains, flakes, pasta, omelette and/or pancake. In an example, the functional food is a beverage, or a food intended to drink. Alternatively, the functional food is not a beverage, or a food intended to drink but a solid or semi-solid foodstuff
[0181] Due to the presence of the malted dehulled oats and/or leachate of malted dehulled oats as described herein, the consumable product such as the food and/or feed possesses properties associated with induction of antisecretory factor (AF) protein and/or fragments thereof such as anti-diarrhoeal properties and/or anti-inflammatory properties.
[0182] Consequently, the consumable product may be used in treatment, prevention and/or prophylaxis of abnormal physiological conditions caused by pathologically high levels of body fluid discharge. Additionally, or alternatively, the consumable product may be used in the treatment, prevention and/or prophylaxis of a condition which is responsive to increase of antisecretory factor protein and/or antisecretory protein fragments in the blood of a patient. The condition(s) described herein may be selected from the group consisting of diarrhoea, inflammatory diseases, oedemas, autoimmune diseases, cancer, tumours, leukaemia, diabetes, diabetes mellitus, glioblastoma, traumatic brain injury, intraocular hypertension, glaucoma, lipid raft dysfunction, compartment syndrome, Alzheimer's disease, Parkinson's disease, encephalitis, and Meniere's disease.
[0183] The consumable product described herein may be provided in the form of a medicament. Thus, there is provided a consumable product as described herein such as a functional food product and/or a pharmaceutical product for use as a medicament.
[0184] The present disclosure will be further explained hereinafter by means of non-limiting examples and with reference to the appended drawings.
REFERENCES
[0185] 1. Lange S. and Lnnroth I., International Review of Cytology, Vol 210 (2001), 39-75 [0186] 2. WO 97/08202; [0187] 3. WO 05/030246; [0188] 4. WO 2007/126364; [0189] 5. WO 2018/015379 [0190] 6. WO 98/21978 [0191] 7. WO 07/126363 [0192] 8. SE 9000028-2 (publication No. 466,331) [0193] 9. A study of avenanthramides in oats for future applications by Elne Karlberg, Uppsala University School of Engineering, published in June 2010 [0194] 10. WO 2010/108277 [0195] 11. WO 2015/179676 [0196] 12. WO 2007/52153 [0197] 13. US 4,581,847 [0198] 14. WO 2007/117815 [0199] 15. WO 2017/09004 [0200] 16. WO 00/38535 [0201] 17. WO 201 5/1 81 324 [0202] 18. Food Chemistry 253 (2018) 93-100, section 2.5 page 95 [0203] 19. Chambers, M. et al. (2012) A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol., 30, 918-920. [0204] 20. Smith, C. A. et al. (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem., 78, 779-787. [0205] 21. Stanstrup, J. et al. (2013) Metabolite profiling and beyond: approaches for the rapid processing and annotation of human blood serum mass spectrometry data. Anal. Bioanal. Chem., 405, 5037-5048. [0206] 22. Zhu, Z.-J. et al. (2013) Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database. Nat. Protoc., 8, 451-460. [0207] 23. Ganna, A. et al. (2016) Large-scale non-targeted metabolomic profiling in three human population-based studies. Metabolomics, 12, 4. [0208] 24. Lin Shi, Johan A Westerhuis, Johan Rosn, Rikard Landberg, C. and Brunius (2018) Variable selection and validation in multivariate modelling. Bioinformatics. [0209] 25. Shi, L. et al. (2018) Plasma metabolites associated with type 2 diabetes in a Swedish population: a casecontrol study nested in a prospective cohort. 849-861. [0210] 26. Brunius, C. et al. (2016) Large-scale untargeted LC-MS metabolomics data correction using between-batch feature alignment and cluster-based within-batch signal intensity drift correction. Metabolomics, 12, 173. [0211] 27. Stekhoven, D. J. and Bhlmann, P. (2012) Missforest-Non-parametric missing value imputation for mixed-type data. Bioinformatics, 28, 112-118. [0212] 28. De Bruijn, W. J. C. et al. (2016) Mass Spectrometric Characterization of Benzoxazinoid Glycosides from Rhizopus-Elicited Wheat (Triticum aestivum) Seedlings. J. Agric. Food Chem., 64, 6267-6276 [0213] 29. Hanhineva, K. et al. (2011) Qualitative characterization of benzoxazinoid derivatives in whole grain rye and wheat by LC-MS metabolite profiling. J. Agric. Food Chem., 59, 921-927 [0214] 30. Koistinen, V. M. et al. (2018) Metabolic profiling of sourdough fermented wheat and rye bread. Sci. Rep., 8, 1-11. [0215] 31. Sumner, L. W. et al. (2007) Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211-221.
EXAMPLES
Example 1
The Novel Oat Malting Process The aim of this experiment was to find a new low-temperature malting process that would allow malting of dehulled oats in a scale-able process.
[0216] The oats lot is refined by sieving and by using gravity tables so that the final 1000 grain weight exceeds 30 grams/1000 kernels.
[0217] The germinability is tested to exceed 95% in petri-dish, or at least 82% in H.sub.2O.sub.2.
[0218] The selected oats lot is dehulled by a dehuller (Bhler BSSA Stratopact HKE50HP Ex). The feed and disc speed are selected so that 30 -70% of the kernels are dehulled at each passage.
[0219] Dehulled kernels with germs are sorted out by gravity tables. The germinability of the dehulled kernels is tested to exceed 95%, or at least 82% in H.sub.2O.sub.2.
[0220] The selected dehulled oats kernels are steeped with cold water (w) at temperatures between 7 C. and 15 C. and in dry conditions (d), for a total of 1-3 days (20-26hours) (2w+10d+2w+10d+2w=26/20h). Kernel moisture content is between 30-50%.
[0221] After steeping, the dehulled oats is germinated for 7-9 days at 12-15 C. The heat evolved is cooled by cold air. Due to the impermeable beds that can be formed, only shallow beds are used, with max 0.5 m bed height. Any movement of the grains is performed at slow speed.
[0222] The germinated grain is initially dried at low air temperature, max 35 C. In the later stages of drying, when moisture content is below 20%, drying air temperature is raised to max. 65 C.
[0223] By this novel malting method, a healthy malted dehulled oats product with a high level of enzymatic activity is produced.
TABLE-US-00001 TABLE 1 Micromalting JW 281 MICROMALTING Kaura 6.3.-17.3.2004 Box number 1 2 3 4 5 OATS ANALYSIS Moisture % 12.2 12.2 12.2 12.2 12.2 Protein % Germinat.capacity 82 82 82 82 82 (H.sub.2O.sub.2) % Sorting mm 1.5 1.5 1.5 1.5 1.5 Fraction I, > 2.8 mm % Fraction II, > 2.5 mm % MALTING PROCESS Steeping program 2 w + 10 d + 2 w + 10 d + 2 w = 26/20 h Wet/dry steeping 15/15 temp. C. Moisture after 1. wet 30.5 30.5 30.6 30.5 steep % Moisture after 2. wet steep % Moisture after 42.0 42.9 43.1 43.4 43.2 steeping % Spraying day 1 1 1 1 1 Moisture after 46 46 46 50 46 spraying % Germination program 9/12 7/15 7/15 7/15 7/15 days/ C. Germination time 9 7 7 7 7 days Germination 2 days/% 94/79 70/79 76/80 74/71 80/74 Green malt 32.4 42.2 42.5 46.6 42.6 moisture % Kilning program EM* PM EM EM freeze drying Respiration 5.0 5.3 7.2 9.0 7.7 losses % Rootlet losses % 2.1 5.3 5.2 5.0 11.2 Total losses % 7.1 10.6 12.4 14.0 18.9 *EM (Enzyme malting)
Example 2
[0224] In this example, analysis was performed on 6 oat samples. Sample 51 was un-malted oat, i.e., oats that had not been subjected to malting, with hull. Sample S2 was oats with hull that had been subjected to malting. Sample S3 was dehulled oats that had been subjected to malting. Sample S4 was un-malted dehulled oat. Sample S5 was naked oats that had been subjected to English malting. Sample S6 was dehulled oats that had been subjected to Nordic malting, i.e., a novel malting process as described in this document.
[0225] Oat sample extracts were thawed at room temperature for 30 min and a 100 L aliquot of each sample was transferred into a 1.5 ml microcentrifuge tube. Cold extraction solution (900 L) was mixed with samples using a multi-tube vortexer (VWR International, Inc) for 10 min and incubated at 4 C. for 2 h. The mixtures were centrifuged for 12 min at 13000 rpm at 4 C. The supernatant from each sample was kept in refrigerator at 4 C. until they were injected on the LC-MS instrument. Each oat sample was prepared in triplicates. Quality control samples (QC) were achieved by pooling aliquots of all the study oat samples (i.e., 6 varieties with and without treatments) and were used to monitor the stability and functionality of the system throughout the instrumental analyses.
Analytical Protocol of Untargeted LC-MS Metabolomics
[0226] Oat extract samples were analyzed by LC-qTOF mass spectrometry -MS (Agilent Technologies 6550 iFunnel Q-TOF LC/MS, United States). Sample solution (5 L) was injected for reversed-phase (RP) chromatographic analyses using both positive and negative electrospray ionization modes. Separation was performed using an Acquity UPLC High Strength Silica T3 column (2.1100 mm, 1.8 m; Waters) at 45 C. The mobile phase was delivered at 400 L/min and consisted of eluent A (water, Milli-Q purified; Millipore) and eluent B (methanol, Sigma-Aldrich), both containing 0.04% (vol:vol) of formic acid (Sigma-Aldrich), delivered in a profile: 0-10.5 min 100% B, 10.5-15 min: 5% B. The dual electrospray ionization source (ESI) was operated using the following conditions: Drying gas (nitrogen) temperature of 175 C. and flow of 10 L/min, nebulizer pressure of 45 PSI, capillary voltage of 3500 V, fragment or voltage of 175 V, and a skimmer of 65V. For data acquisition, a 2-GHz extended dynamic range mode was used, and the instrument was set to acquire over the mass range of m/z 50-1700. Data were collected in centroid mode at an acquisition rate of 1.67 spectra/s with an abundance threshold of 200 counts. The automatic data-dependent MS/MS analyses were performed on the QC samples, and the 4 most abundant ions were selected for fragmentation from every precursor scan cycle.
[0227] Collision energies were 10, 20 and 40 volt (V). Continuous mass axis calibration was performed by monitoring two reference ions, m/z 121.050873 and m/z 922.009798 for positive mode and m/z 112.98558700 and 966.000725 for negative mode, from an infusion solution throughout the runs. All the oat samples were analysed randomly in one batch. Two blank samples and one priming quality control sample provided by the Chalmers Mass Spectrometry Infrastructure were injected before the analytical sequence. Two pooled QCs described as above were injected at the beginning and end and as every 10.sup.th injection throughout the sequence.
Detection and Quantification of Avenanthramides
[0228] The method workup was identical, but the mass spectrometer used for analysis differed. The detection and quantification were performed as described in Food Chemistry 253 (2018) 93-100 section 2.5 page 95. The LC-MS/MS system used was a QTRAP 6500+ LC-MS/MS (SCIEX A/B, Stockholm, Sweden). Avenanthramides were ionized using positive electrospray ionization in multiple reaction monitoring (MRM) mode for each of the avenanthramides, which were as follows: B (2c) m/z 329.9.fwdarw.176.9 (collision energy (CE)-15 V); C (2f): m/z 315.9.fwdarw.162.9 (CE-15 V); A (2p): m/z 299.9.fwdarw.146.9 (CE-25 V); 2fd: m/z 342.fwdarw.172.95 (CE-10 V) and 2pd m/z 326.fwdarw.173 (CE-12 V). Dwell times were 50 ms. For all mass spectrometry analyses, the ion source temperature was set to 500 C., entrance potential 10 V and drying curtain gas flow 30 L/min. Identity of avenanthramides was confirmed using neutral loss scanning for loss of m/z 153, which is characteristic of the main avenanthramides (Xie et al., 2017).
Data Pre-Processing
[0229] Raw data files from RP (ESI+), RP (ESI) were converted to mzML format using ProteoWizard msconvert (Chambers et al., 2012). Data deconvolution was performed with xcms, a freely available software under open-source license, implemented in R (Smith et al., 2006). Specifically, feature detection in each chromatogram was performed using the centWave algorithm implemented in the xcmsSet function and obiwarp was applied for retention time correction. The term feature refers to a mass spectral peak, i.e. a molecular entity with a unique mass-to-charge ratio and retention time as measured by an LC-MS instrument. Parameters were the values suggested by xcms online (https://xcmsonline.scripps.edu/) and from recently relevant publications (Stanstrup et al., 2013; Zhu et al., 2013; Ganna et al., 2016; Shi et al., 2018). Parameters were: peak width=c(10, 60), ppm=15, prefilter intensity (3, 1000), bandwidth (2), mzdiff (0.01). Quality of data acquisition and processing was examined by visualization of the total ion chromatogram and the base peak chromatogram for each sample, extracted-ion chromatograms for multiple features, and assessment of differences between adjusted and raw retention times per sample. Within-batch signal intensity normalization was performed using R package batchcorr (Brunius et al., 2016). Features passing a QC test (CV<0.3) were determined as qualified features and were further subjected to statistical analyses. In total, 3511 and 3809 features were retained after a stringent normalization procedure for RP (ESI+) and RP (ESI), respectively. Missing values were imputed by using random forest algorithm implemented in R package missForest (Stekhoven and Bhlmann, 2012).
Metabolite Identification
[0230] Metabolite identification was accomplished based on accurate mass and MS/MS fragmentation matched against online databases (i.e. Metlin, FooDB and MassBank) or the literature (De Bruijn et al., 2016; Hanhineva et al., 2011; Koistinen et al., 2018). The confidence level of annotation was categorized according to the Metabolomics Standard Initiative (MSI) (Sumner et al., 2007).
Results
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[0242] It will be appreciated that they axis in