Omega-3 Beverage

Abstract

The present invention provides a beverage in the form of an oil-in-water emulsion comprising a marine oil, black seed oil and fruit juice, wherein the beverage comprises at least 0.4 μg of a mixture of thymoquinone and thymohydroquinone per ml of beverage.

Claims

1. A beverage in the form of an oil-in-water emulsion comprising a marine oil, black seed oil and fruit juice, wherein the beverage comprises at least 0.4 μg of a mixture of thymoquinone and thymohydroquinone per ml of beverage.

2. A beverage according to claim 1, wherein the beverage comprises at least 0.4 μg of thymohydroquinone per ml of beverage.

3. A beverage according to claim 1 or 2, wherein the beverage comprises thymoquinone and thymohydroquinone in a ratio of 1:1 to 1:100.

4. A beverage according to claim 1 or 2, wherein the beverage has been pasteurised at a temperature of at least 60° C.

5. A beverage according to claim 1 or 2, wherein the fruit juice is obtained by mixing water and a fruit concentrate.

6. A beverage according to claim 1, wherein the ratio of black seed oil to marine oil is from 2:1 to 1:20.

7. A beverage according to claim 1 or 2, comprising 0.5-15% by weight of the marine oil based on the total weight of the beverage.

8. A beverage according to claim 1 or 2, comprising 0.05-7% by weight of the black seed oil based on the total weight of the beverage.

9. A beverage according to claim 1, comprising 60-90% by weight of water based on the total weight of the beverage, preferably 65-85%.

10. A beverage according to claim 1 wherein the marine oil is selected from the group of fish oil, such as cod liver oil, salmon and trout oil, and krill oil.

11. A beverage according to claim 1, wherein the content of omega-3 fatty acids in the marine oil is at least 10% by weight, preferably at least 20%, most preferably at least 30%.

12. A beverage according to claim 1, having an absolute viscosity of below 150 kg/(m*s) at room temperature.

13. A beverage according to claim 1, comprising an emulsifier.

14. A process for the manufacture of a beverage according to claim 1, the process comprises the steps of: emulsifying a mixture comprising a marine oil, black seed oil and fruit juice; pasteurising the emulsified mixture at a temperature of at least 60° C.; and filling the emulsified mixture into a suitable air-tight beverage packaging in a substantially oxygen-free environment.

15. A method of obtaining a beverage according to claim 1, wherein the beverage comprises thymoquinone and thymohydroquinone in a ratio of 1:1 to 1:50, comprising the step of: emulsifying a mixture comprising a marine oil, black seed oil and fruit juice.

16. A method of obtaining a beverage comprising black seed oil, wherein the beverage comprises thymohydroquinone only obtained from the black seed oil and the amount of thymohydroquinone in the beverage is larger than the amount of thymoquinone originally present in the black seed oil, the method comprising the step of: emulsifying a mixture comprising black seed oil and fruit juice.

17. A method of obtaining a beverage comprising black seed oil, wherein the beverage comprises thymohydroquinone obtained from reduction of thymoquinone present in the black seed oil, the method comprising the step of: emulsifying a mixture comprising black seed oil and fruit juice.

18. A method of reducing thymoquinone in black seed oil to thymohydroquinone, the method comprising the step of: emulsifying a mixture comprising black seed oil and fruit juice.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0071] It has surprisingly been found that the addition of black seed oil to a nutritional beverage based on a marine oil in water emulsion significantly delays or prevents the generation of oxidation products that are associated with “fishy” smell and unpalatable flavours, even upon prolonged exposure to air (oxygen).

[0072] The superior effect is particularly unexpected in view of the known instability of thymoquinone in aqueous solutions, since thymoquinone is believed to be the major compound responsible for the anti-oxidative properties of black seed oil.

[0073] Surprisingly, thymoquinone appears to be stable when present in a beverage comprising an oil in water emulsion, although a major part of the thymoquinone is reduced to thymohydroquinone, see below. Even more unexpected is the fact that both thymoquinone and thymohydroquinone are stable under heat treatment of the beverage before packaging.

[0074] Consequently, the use of black seed oil in the inventive nutritional beverage is highly advantageous, both in that the marine oils are protected from oxidation and in that the therapeutically beneficial compounds thymoquinone and its reduced form thymohydroquinone are stabilized by the marine oil in water emulsion, thus allowing thymoquinone and thymohydroquinone to be ingested in an easy manner through a beverage.

[0075] Thus, it has been found an efficient way to stabilize and possibly reverse the oxidation process to reduce the oxidation levels of marine oils, ensuring the beneficial effect of low oxidized marine oils, while at the same time providing a nutritional beverage comprising thymoquinone and thymohydroquinone in a stable form.

[0076] The present invention has made possible a beverage beneficial for achievement and maintenance of good health combining the beneficial effects of low oxidized marine oil and black seed oil. The marine oil and the black seed oil may even work synergistically.

[0077] The inventive beverage may advantageously comprise additional beneficial ingredients or compounds.

[0078] Carnitine (β-hydroxy-γ-N-trimethyl-aminobutyric acid, 3-hydroxy-4-N,N,N-trimethylaminobutyrate) is a particularly preferred additional ingredient/compound. Carnitine is a quaternary ammonium compound involved in metabolism in most mammals, plants and some bacteria. Carnitine is the generic expression for a number of compounds that include L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine. It is most accumulated in cardiac and skeletal muscles as it accounts for 0.1% of its dry matter. Carnitine is involved in transporting fatty acids across the mitochondrial membrane, by forming a long chain acetyl carnitine ester and being transported by carnitine palmitoyl transferase I and carnitine palmitoyl transferase II. Carnitine also plays a role in stabilizing acetyl-CoA and coenzyme A. During the past 30 years, dietary supplementation with carnitine has been widely studied in a number of conditions including exercise performance, cardiovascular health, epilepsy, autism, diabetes/metabolic syndrome, overweight, fertility etc. The results from the different trials are conflicting and one of the main arguments against carnitine supplementation has been lack of sufficient uptake in the target tissue, e.g. muscle.

[0079] The manner in which carnitine is formulated or supplemented seems to be highly relevant for the bioavailability and utilization of this compound. The incorporation of carnitine in the beverage according to the invention is believed to provide a superior bioavailability of carnitine.

[0080] The beverage according to the invention may also comprise other additives such as proteins, including hydrolysed proteins and peptides, both of animal and plant origin, amino acids or amino acid derivatives such as leucine, beta alanine, creatine, BCAA (branched chain amino acids), GABA (gamma-aminobutyric acid), L-glutamine, L-theanine, citrulline, natural or modified polysaccharides such as xanthan gums, alginates, celluloses, starches, hyaluronic acid, choline, chitosan etc., stilbenoides such as pterostilbene, isothiocyanates such as sulforaphane, herbs such as; valerian root, lemon balm, ashwagandha, ginseng, maca, rhodiola, holy basil, hormones such as melatonin, vitamin D3, antioxidants such as curcuminoids, resveratrol, pycnogenol, Q10, lipoic acid, astaxanthin, lycopene, lutein, quercetin, glutathione, polyphenols and flavonoids, cannabinoids, minerals such as magnesium, selenium, potassium, iron, calcium, zinc, vitamins such as vitamin E, D, B, K and C and/or preservatives, or any combinations thereof.

[0081] The beverage according to the invention may be prepared according to procedures well-known in the art, for example as disclosed in WO 2007/064222 A1. However, it should be noted that due to its excellent stability towards marine oil oxidation, the inventive beverage may be manufactured by standard commercial processes commonly used for beverages not comprising easily oxidized marine oils.

[0082] Thus, a major advantage of the beverage according to the invention is that its production is not dependent on any specialized processes to avoid oxidation of the marine oil. Further, as discussed below, the beverage has an excellent shelf life even at room temperature storage.

EXEMPLARY EMBODIMENTS

[0083] Exemplary beverages according to the invention are described in the following:

Example 1—Beverage Comprising Marine Oil and Black Seed Oil Added Plant Proteins

[0084]

TABLE-US-00001 Pr 100 g Marine oil 2.0 g Black seed oil 300 mg Plant proteins (from Sunflower seeds, Chia seeds 3.6 g and Lupin seeds), Pectin 0.4 g Blueberry concentrate 4.0 g Goji berry concentrate 6.0 g Elderberry concentrate 1.5 g Strawberry concentrate 3.5 g Water 78.7 g

Example 2—Beverage Comprising Marine Oil and Black Seed Oil Added Whey Proteins and Hydrolysed Collagen

[0085]

TABLE-US-00002 Pr 100 g Marine oil 4.0 g Black seed oil 850 mg Plant proteins (from Sunflower seeds, Chia seeds 1.5 g and Lupin seeds), Whey protein concentrate 4.0 g Natural Peach aroma 0.2 mg Collagen hydrolysed 15 g Pectin 0.3 g Blueberry concentrate 3.0 g Goji berry concentrate 7.0 g Apple concentrate 1.5 g Strawberry concentrate 5.0 g Water. 71.15 g

Example 3—Beverage Comprising Marine Oil, Black Seed Oil and

[0086]

TABLE-US-00003 Pr 100 g Marine oil 2.5 g Black seed oil 0.9 g Plant proteins (from Sunflower and Chia seeds), 3.6 g L-Carnitine 0.2 g Pectin 0.4 g Goji berry concentrate 6.0 g Elderberry concentrate 1.5 g Strawberry concentrate 3.5 g Blueberry concentrate 4.0 g Water 77.4 g 

Example 4—Beverage Comprising Marine Oil, Black Seed Oil and Carnitine

[0087]

TABLE-US-00004 Pr 100 g Marine oil 3.0 g Black seed oil 0.85 g L-Carnitine 0.25 g Lecitin 3.0 g Cinnamon aldehhyde 10 mg Pectin 0.35 g Black currant concentrate 4.0 g Pomegranate concentrate 2.0 g Apple concentrate 6.0 g Natural Raspberry aroma 0.2 mg Water 80.34 g

Example 5—Beverage Comprising Marine Oil, Black Seed Oil and Carnitine, Added Nicotinamide, Curcumin and Vitamin D3

[0088]

TABLE-US-00005 Pr 100 g Marine oil 4.0 g Black seed oil 2.0 g Acetyl -L-Carnitine 0.4 g Whey proteins 2.5 g Pectin 0.4 g Bilberry concentrate 3.0 g Goji berry concentrate 2.0 g Elderberry concentrate 1.0 g Apple concentrate 5.0 g Cinnamon aldehyde 2 mg Sweet apple aroma 0.3 mg Curcumin 0.3 g Vitamin D3 5 μg Nicotinamide 0.3 g Water 79.5 g

Example 6—Beverage Comprising Marine Oil, Black Seed Oil and Carnitine

[0089]

TABLE-US-00006 Pr 100 g Marine oil 2.4 g Black seed oil 0.31 g  L-Carnitine 0.2 g Whey proteins 6.0 g Pectin 0.38 g  Collagen 3.3 g Black currant concentrate 2.5 g Apple concentrate 6.5 g Pear concentrate 5.5 g Water 73.0 g 

Example 7—Beverage Comprising Marine Oil, Black Seed Oil and Carnitine

[0090]

TABLE-US-00007 Pr 100 g Water 74.57 g Marine oil 2.55 g Black cumin oil 0.6 g Pectin 0.3 g Hydrolysed Collagen 4.00 g Whey protein concentrate 2.00 g Guardian rosemary extract 201 0.02 g Guardian toco 50 IP, antioxidant 0.01 g Nicotinamide 0.10 g Carnitine 0.15 g Pear juice concentrate 7.50 g Black current concentrate 4.00 g Apple juice concentrate, sweet, normal acid 4.00 g Natural Peach aroma Frey & Lau 0.2 g

Example 8—Beverage Comprising Marine Oil, Black Seed Oil and Carnitine

[0091]

TABLE-US-00008 Pr 100 g Water 71.16 g  Marine oil 2.70 g Black cumin oil 0.31 g Pectin (Genu Pectin YM-115-L) 0.38 g Whey protein concentrate 6.50 g Sunflower protein powder 0.10 g Hydrolysed collagen 2.80 g Guardian rosemary extract 201 0.02 g Guardian toco 50 IP, antioxidant 0.01 g Carnitine 0.20 g Pear juice concentrate 5.70 g Blackcurrant juice concentrate 3.50 g Apple juice concentrate, sweet, normal acid 6.40 g Natural Raspberry aroma IFF 0.22 g

Example 9—Beverage Comprising Marine Oil and Black Seed Oil

[0092]

TABLE-US-00009 Pr 100 g Water 70.66 g Marine oil 2.7 g Black cumin oil 0.31 g Pectin 0.38 g Whey protein concentrate 9.50 g Sunflower protein powder 0.3 g Guardian rosemary extract 201 0.02 g Guardian toco 50 IP, antioxidant 0.01 g Vitamin D3 # Pear juice concentrate 5.70 g Blackcurrant juice concentrate 3.80 g Apple juice concentrate, sweet, normal acid 6.40 g Natural Raspberry aroma IFF 0.22 g

Example 10—Beverage Comprising Marine Oil and Black Seed Oil Added Oat Beta Glucans and Proteins, Choline Bitartrate and Vitamin D3

[0093]

TABLE-US-00010 Pr 100 g Marine oil 4.0 g Black seed oil 850 mg Oat beta glucans 3.0 g Whey protein concentrate 1.0 g Natural Peach aroma 0.2 mg Choline bitartrate 0.2 g Pectin 0.15 g Black current concentrate 3.0 g Pear concentrate 5.0 g Apple concentrate 4.5 g Vitamin D3 5 micrograms Water. 78 g

[0094] The exemplary beverages were prepared according to well-known procedures as described above, subjected to heat treatment and filled in closed (air tight) aseptic containers, i.e. single dose containers of 250 ml (Tetrapak carton).

[0095] Marine Oil

[0096] Commercial Fish Oils: [0097] CodMarine 70/100 TG—Batch no: PF9470118) produced by PharmaMarine, Ålesund, Norway. The oil was analyzed to have an omega-3 content of 25%, of which DHA amounted to 11% and EPA to 9%, and a totox value of 6 (PV: 0 and ANv: 6). [0098] (Omega FF100-1—Batch no: 833018-77) produced by Vesteraalens as, Havnegata 17, 8400 Sortland. The oil was analyzed to have an omega-3 content of 25.3%, of which DHA amounted to 11.3% and EPA to 8.3%, and a totox value 1.6 (PV: 0.3 and AV: 1)

[0099] Black Seed Oil

[0100] Cold pressed ‘Black Cumin’ oil from Egypt, supplied by All Organic Treasures, Germany. (Lot no: 1804A0751.) The oil was analysed by the producer and comprises 0.35 mg/ml thymoquinone (TQ) and thymohydroquinone (THQ) in a ratio of 60:1.

[0101] Remaining Ingredients were Supplied by:

[0102] Strawberry and Elderberry juice concentrate delivered by BerryPharma gmba.

[0103] Bilberry, Blueberry and Goji berry concentrate delivered by Storms as.

[0104] Pectin delivered by Alimenta

[0105] Protein powders; Sunflower, Chia, Lupin and Coconut, delivered by All Organic

[0106] Treasures AOL

[0107] Cinnamon aldehyde delivered by Storms as

[0108] Nicotinamide and L-Carnitine delivered by Lonza

[0109] Vitamin D3 delivered by GoJohnsen

[0110] Hydrolyzed Collagen delivered by Junca Gelatine

[0111] Whey protein concentrates delivered by Arla Foods

[0112] Peach Aroma delivered by Einar Willumsen

[0113] Apple Aroma delivered by Storms as

[0114] Raspberry Aroma delivered by IFF

[0115] Curcumin delivered by Naturex

[0116] VitaCholin from Balchem

[0117] PromOat from Lantmannen

[0118] Sensory Analysis of Mixtures of Fish Oil and Black Seed Oil Upon Storage in the Presence of Air

[0119] As discussed above, the potential of black seed oil in preventing lipid oxidation of fish oil has previously been described and examined (Lutterodt et al., LWT—Food Science and Technology 43, 2010, 1409-1413). The amount of lipid oxidation was determined indirectly by measuring the electron spin resonance (ESR) intensity of a 1:1 mixture of fish oil and black seed oil, i.e. by measuring the presence of free radicals. However, no mention is made of whether the anti-oxidative effect of the black seed oil was sufficient to prevent the formation of unpleasant taste and/or smell.

[0120] The characteristic smell and taste of oxidised omega-3 fatty acids from marine oils can be detected in much lower concentrations than in other fats, such as vegetable oils (Frankel, Lipid Oxidation 2.sup.nd edition, Elsevier February 2005, Olsen et al. Journal of Agric. Food Chem. January 2005). The measurement of such low concentrations with classical chemical analytical methods can be challenging, and sensory analysis has in many cases proven superior to these methods for the early detection of fish oil oxidation (Frankel, Lipid Oxidation 2.sup.nd edition, Elsevier February 2005,).

[0121] Volatile secondary oxidation products from omega-3 fatty acids have strong flavours even at very low concentrations (Frankel, Lipid Oxidation 2.sup.nd edition, Elsevier February 2005), and such flavours can render marine oil products less marketable and lead to poor product reputation and loss of sales (Olsen et al., Journal of Agric. Food Chem. January 2005)

[0122] Fatty acids can be auto-oxidized, photo-oxidized, or “enzymatically” oxidized. Auto-oxidation happens when polyunsaturated fatty acids are exposed to air (oxygen), photo-oxidation upon exposure to UV light, and “enzymatic oxidation” through enzymatic (lipase) hydrolysis of triglycerides yielding free fatty acids that are particularly sensitive to oxidation in contact with air (oxygen) (Frankel, Lipid Oxidation 2.sup.nd edition, Elsevier February 2005).

[0123] To investigate whether black seed oil is in fact capable of sufficiently preventing lipid oxidation of fish oil, such that no unpleasant taste and/or smell is formed, a preliminary study was performed.

[0124] In the preliminary study, samples of pure fish oil and pure black seed oil was compared to samples containing mixtures of fish oil and black seed oil. The samples were subjected to various temperatures (4 and 20° C.) and stirring to maximize their interaction with oxygen. Blinded sensory evaluation of the samples, i.e. smell and taste, was performed by three judges.

[0125] The preliminary study revealed that the addition of black seed oil to fish oil significantly delays or prevents the generation of oxidation products that are associated with unwanted smell and flavour, even upon prolonged exposure to air (oxygen). Surprisingly, even low amounts of black seed oil (about 10 weight % relative to the fish oil) was effective in preventing oxidation.

[0126] Although black seed oil was shown to prevent oxidation of fish oil in an oil in oil mixture, the applicant was reluctant to use black seed oil in a water-based beverage since thymoquinone, the major compound believed to be responsible for the anti-oxidative effect, as well as various therapeutic and prophylactic properties, of black seed oil, is known to be highly unstable in water (Salmani et al. Molecules 2014, 19, 5925-5939).

[0127] Stability Test of an Exemplary Beverage

[0128] To evaluate whether black seed oil was suitable for oxidative protection of a marine oil in a water-based beverage, the exemplary beverage 6 was prepared. The beverage was pasteurized and packaged in 250 ml air tight containers suitable for consumers.

[0129] Containers with beverage from two different batches were stress tested by being stored at a temperature of 40° C. for 4 weeks.

[0130] The beverages were analysed for M0233 Volatile Compounds (JAOCS, Vol. 82, no. 2, 2005, 97-203) before and after the stress test. The analysis revealed that no oxidation products were formed during the stress test.

[0131] Containers with exemplary beverages 7 and 9 were also opened to evaluate the stability of the beverage towards oxygen/air over time. The opened containers were stored at 23° C. (i.e. room temperature) for one week. The sensory properties of the beverages were then evaluated by a panel of three persons. No unpleasant taste and/or smell was detected.

[0132] Analysis of Exemplary Beverages for Content of Thymoquinone (TQ) and Thymohydroquinone (THQ)

[0133] The exemplary beverages 7, 8 and 9 were analyzed for the presence of thymoquinone (TQ) and its reduced form thymohydroquinone (THQ) after storage at room temperature for different time periods. The analysis was performed by SINTEF Industry, Department of Biotechnology and Biomedicine. The compounds of interest were extracted from 10 ml of the respective sports drinks with 1 ml of dichloromethane, by vigorous mixing followed by phase separation by centrifugation. The lower phase (dichloromethane) was dried by addition of excess NaSO.sub.4 and analyzed by GC-MS with electron impact positive ionization, in selected ion monitoring (SIM) mode, following the m/z 164 (TQ) and 166 (THQ) molecular ions. Quantification was performed against a standard curve of TQ and THQ, run in duplicate, in dichloromethane ranging from 0.1 to 100 μg/ml. The final concentrations—corrected for sample concentration upon liquid-liquid extraction—in the sports drinks as found by this approach were:

TABLE-US-00011 Beverage Storage TQ THQ no. (months) (μg/ml) (μg/ml) 7 6 0.078 1.90 8 1 0.035 0.43 9 3 0.032 0.62

[0134] Matrix effects may have had an impact on the consistency of the results, but it is clear that thymoquinone and thymohydroquinone are stabilized in the inventive beverages, even at room temperature over an extended period of time. It is noted in the analysis report that TQ/THQ were highly unstable in methanol, i.e. more than 80% degradation over three days.

[0135] The large amount of thymohydroquinone was surprising but is believed to be the result of thymoquinone being reduced by help of oxidoreductases present in the fruit juices of the beverage (K. Jensen, B. L. Moller (2010) Plant NADPH-cytochrome P450 oxidoreductases, Phytochemistry, 71 (2-3), 132-141). Many of these oxidoreductases, e.g. NADPH-cytochrome P450 oxidoreductase, are known to reduce quinones, including thymoquinone (Armutcu, F., Akyol, S., & Akyol, O. (2018). The interaction of glutathione and thymoquinone and their antioxidant properties. Electronic Journal of General Medicine, 15 (4), em59).

[0136] The results show that the marine oils present in the beverage are protected from oxidation during storage in a substantially oxygen free environment (i.e. in the closed container) and even for a significant time period when in contact with air. Further, this protective effect is present even when the beverage has been subjected to heat treatment (i.e. pasteurizing conditions) before packaging.

[0137] Thymoquinone (TQ) is believed to be the major anti-oxidative compound in pure black seed oil, as well as having various therapeutic/prophylactic properties.

[0138] Thymohydroquinone (THQ) is however known to be a more potent anti-oxidant than thymohydroquinone (K. H. Khalife & G. Lupidi (2007) Non-enzymatic reduction of thymoquinone in physiological conditions, Free Radical Research, 41:2, 153-161; Armutcu, F., Akyol, S., & Akyol, O. (2018). The interaction of glutathione and thymoquinone and their antioxidant properties. Electronic Journal of General Medicine, 15 (4), em59). In addition, THQ is also known for various therapeutic/prophylactic properties. However, the amounts of THQ in pure black seed oil is very low. Consequently, the conversion of TQ to THQ in the inventive beverage is believed to be highly advantageous in that it provides a more potent anti-oxidant. The high amounts of THQ may explain the excellent stability and shelf life of the inventive beverage.

[0139] The above results show that after an initial reduction of an amount of thymoquinone, both thymoquinone and thymohydroquinone are stabilized in the beverage according to the invention and can provide an anti-oxidative effect. Consequently, the results show that the beverage according to the invention can provide thymoquinone and thymohydroquinone in a stable form.

[0140] The ability of black seed oil to prevent deterioration of marine oils in an oil-in-water beverage, even in the presence of oxygen, is a novel and unexpected finding. Even more surprising is the fact that the invention can provide consumers with a beverage comprising thymoquinone/thymohydroquinone in a stable form. The latter entails that the many therapeutic and/or prophylactic effects of thymoquinone and thymohydroquinone may easily be achieved by use of a palatable and nutritious beverage.