RU2774887C1 - Method for obtaining a functional product enriched with fucoxanthinol and methylxanthine - Google Patents

Abstract

FIELD: functional food ingredients.

SUBSTANCE: invention relates to a method for producing a functional product enriched with fucoxanthinol and methylxanthine. A method for obtaining a functional product enriched with fucoxanthinol and methylxanthine includes grinding raw materials from the mussel M. galloprovincialis and isolation of fat-soluble carotenoids from it, separation of carotenoids from a solvent in which to isolate fat-soluble carotenoids, eggs and crushed soft tissues of the mussel M. galloprovincialis are mixed with distilled water, hexane and 96% ethanol in a ratio of 1:1:2:1, the resulting mixture is mixed and settled for 24 hours in the dark at a temperature of 20±2°C until a colored hexane layer containing a carotenoid complex is formed, followed by solvent separation, after which a carotenoid complex containing fucoxanthinol and methylxanthine is dissolved in 96% ethanol in a ratio of 1:5 and the resulting alcohol extract, fish oil and distilled water are combined in a ratio of 1:1:1, and then the mixture is held in the dark for 24 hours at a temperature of 20±2°C until an oil layer enriched with fucoxanthinol and methylxanthine is formed.

EFFECT: above method expands the range of functional products based on natural raw materials of marine origin, provides a stable product with increased physiological efficiency of fucoxanthinol and methylxanthine in combination with fish oil.

1 cl, 1 ex

Description

The invention relates to the field of food and pharmaceutical industry and is intended to obtain a functional product from eggs and soft tissues of the mussel M. galloprovincialis.

Known Dietary biologically active additive (options) (Pat. 2225210, RF, IPC A61K 35/00, 2002), which in one of the options consists of edible fish oil and biologically active substances in the form of a carotenoid concentrate obtained from the gonads of Cucumaria frondosa ( sea cucumber) at a ratio of components: fish oil - 99.30-99.50%, carotenoid concentrate from cucumaria - 0.50-0.70%. A fat-soluble carotenoid concentrate (CC) is isolated from the gonads of the cucumaria Cucumaria frondosa, caught in the Barents Sea in the spring, and then mixed with fish oil. The total content of carotenoids in the product is 243 mg/100 g, among which are identified: lutein + zeoxanthin - 104 mg, cryptoxanthin - 6 mg, astaxanthin - 133 mg. The disadvantages of the method for obtaining a food product are that the authors do not consider the enhancement of the protective properties of carotenoids due to the addition of fish oil [Maeda 2005; Maeda, 2007; Sugawara, 2002]. The disadvantages also lie in the methods of obtaining a concentrate of carotenoids according to US Pat. RF 2181976, 2112527. So, in accordance with patent 2181976, the primary extraction is carried out with acetone at a ratio of raw materials : solvent 1:5. The mixture is homogenized for 5 minutes and then subjected to microwave treatment. It can be argued that microwave exposure is unnecessary, since when lipids are extracted, the membranes of hydrobiont tissue cells, unlike plant cells, are easily destroyed by mechanical action in the presence of a solvent [Kapranova et al., 2019]. Acetone is a precursor. And the use of technical acetone is at least dangerous to health. Next, extraction is carried out for 8-12 hours, then the mixture is centrifuged. The acetone-water fraction is concentrated to 1/3 of the volume, the solution is dehydrated by filtration through anhydrous sodium sulfate, and subsequently concentrated to 1/2 of the original volume. Petroleum ether is added to the resulting solution in a ratio of 1:1, one part of water is added, infused for 3-4 hours, separated in a separating funnel, the petroleum fraction is concentrated to a dense residue. This sequence of chemical manipulations is reduced to the separation of lipid and non-lipid components. In this case, the technique of dehydration of acetone and back extraction with petroleum ether are used. In the known patent, the techniques for separating lipid and non-lipid components are too complicated. Further, acetone cooled to -20°C is added to the residue in the ratio of residue: acetone 1:10. In this case, the dense residue is separated into a precipitate - one of the target products, and an extract of carotenoids, which are isolated in the form of a target product after concentration. In the method, with the help of acetone, they again try to separate the components of a lipid and non-lipid nature. Unfortunately, without specifying the brand of solvent: pure for analysis, chemically pure, technical or freshly distilled. Working with precursors requires special permission and, as a rule, their number is strictly limited in use. The yield of saponins is 85% of their original content in raw materials, and carotenoids - 91%.

In the method according to patent 2112527 for the extraction of carotenoids, tissues are ground using a meat grinder, and 600 ml of ethanol and 600 ml of chloroform are added to 300 g of minced meat, i.e. a mixture of Folch with a ratio of components 1:1. The mixture is stirred for two hours, squeezed, 600 ml of distilled water is added to the obtained extract, the resulting mixture is infused for 8-12 hours. Re-extraction is carried out with ethanol at room temperature and at boiling temperature at a ratio of sediment: ethanol (1:3). The resulting extracts are combined. The dense precipitate obtained in this way is dried at a temperature of from 60°C to 80°C, crushed to the state of flour. The resulting product is a protein feed additive containing up to 80% protein, while the yield of the protein additive from the mass of the feedstock is 78%. The combined extracts are filtered, evaporated in a rotary evaporator, the resulting residue is extracted 3 to 5 times with acetone at a ratio of residue: acetone 1:5. The result is a precipitate containing saponins and an acetone extract containing carotenoids. Selected is not the most optimal ratio of residue : acetone 1:5 extraction in five repetitions. A large excess of solvent complicates further sample preparation. To isolate the complex of carotenoids from the acetone extract, it is filtered, evaporated to dryness and the resulting residue is dissolved in ethanol at a ratio of residue : ethanol 1:3, cooled for 8-10 hours at a temperature of -20°C, filtered and the resulting filtrate is evaporated to a dense residue , which contains a concentrate of carotenoids with a content of target products up to 90% (dry residue content from 85% to 93%). This technology is also very complex and is aimed at biochemical research.

Known Means based on biologically active compounds of marine hydrobionts, which has a cancer-preventive effect and increases the therapeutic activity of antitumor antibiotics (Pat. 2659682 C1, RF, IPC A61K 35/616, 2018), for which components are obtained: initial carotenoids from freshly caught, frozen or dried brown algae species Laminaria japonica, Fucus evanescens, F. Vesiculosus; carotenoids from the sea star Patiria pectinifera or from the extract of the sea urchin Scaphechinus mirabilis. Each resulting ingredient is transferred into a soluble state. Lecithin is used as a solvent. Lecithin emulsions, taken in equal mass fractions, are combined and mixed in a closed container at room temperature until a homogeneous preparation is obtained, which contains: from 1.0% to 1.5% fucoxanthin; 1.0% to 1.5% astaxanthin; 0.3% to 0.4% lutein; 0.2% to 0.4% zeaxanthin; from 0.7% to 1.0% echinochrome. The finished product is placed in gelatin capsules, based on the recommended daily dose of 4-6 mg of active ingredients per dose. The composition of the product is confirmed by HPLC. The disadvantage of this method is very complex chemical manipulations at the stage of carotenoid isolation, expensive physicochemical methods for studying the target product, as well as the use of gelatin encapsulation, which leads to a significant increase in the cost of the target product, despite the rather high yield of carotenoids.

The closest technical solution is the Method for obtaining an oil composition enriched with polyunsaturated fatty acids and carotenoids from the mussel M. galloprovincialis (Pat. 2743019, RF, IPC A23D 9/00, 2020). The method involves obtaining an alcoholic extract from marine hydrobionts, combining the oil with the alcoholic extract and water, and separating the oil fraction. As a raw material for obtaining an alcohol extract, sexual products collected during mass spawning are used - eggs and spermatozoa, or gonads of the mussel M. galloprovincialis, as an oil composition - a 10% oil solution of vitamin E.

A 10% oil solution of vitamin E, an alcoholic extract from marine hydrobionts and distilled water are combined in a ratio of 1:1:1, then the mixture is stirred for 5 minutes, settled for 30-40 minutes and the oil fraction that does not contain alcohol is separated. The disadvantage of the invention is that in order to obtain the target product enriched in carotenoids and polyunsaturated fatty acids, an alcoholic extract is first prepared, and then re-extraction is carried out, saturating the oil solution with biologically active components.

The objective of the method for obtaining a functional product enriched with fucoxanthinol and methyloxanthin is to expand the range of functional products based on natural raw materials of marine origin; proposal of a new technology for the isolation of carotenoids, incl. fucoxanthinol and mityloxanthin, from eggs and soft tissues of the mussel M. galloprovincialis; strengthening the protective properties of fucoxanthinol and mityloxanthin through fish oil.

The technical result from the solution of the task is manifested: in the increase in the physiological effectiveness of fucoxanthinol and methyloxanthin in synergy with fish oil; obtaining a stable food composition in which oxidative processes are minimized.

The claimed technical result is achieved due to the fact that in the claimed method, including the grinding of raw materials from the mussel M. galloprovincialis and the isolation of fat-soluble carotenoids from it, as well as the separation of carotenoids from the solvent, a number of changes are provided in comparison with the prototype. So, to isolate fat-soluble carotenoids, the eggs and crushed soft tissues of the mussel M. galloprovincialis are mixed with distilled water, hexane and 96% ethanol in a ratio of 1:1:2:1. The mixture is stirred and left for 24 hours in the dark at a temperature of 20±2°C until a colored hexane layer containing a carotenoid complex is formed, followed by separation of the solvent. The carotenoid complex containing fucoxanthinol and methyloxanthin is dissolved in 96% ethanol in a ratio of 1:5 and the resulting alcohol extract, fish oil and distilled water are combined in a ratio of 1:1:1. The resulting mixture is left in the dark for 24 hours at a temperature of 20±2°C until an oil layer is formed, enriched with fucoxanthinol and methyloxanthin.

Common with the prototypes is the extraction of carotenoids from marine invertebrates. New in the proposed method is:

to obtain the target product, only 96% ethanol and hexane are used as organic solvents;

- saturation of fish oil with fucoxanthinol and methyloxanthin is carried out by mixing equal parts of an alcohol extract containing fucoxanthinol and methyloxanthin, fish oil and water in order to enhance the properties of pigments in the resulting functional product;

- experimentally established by the authors, the ratio of eggs and crushed soft tissues of the mussel M. galloprovincialis, distilled water, hexane and 96% ethanol - 1:1:2:1;

- defending the mixture for 24 hours at a temperature of 20±2°C in the dark;

- for the preparation of an alcoholic extract, the dry residue colored in red-brown color, formed after the distillation of hexane, and 96% ethanol are mixed in a ratio of 1:5;

- to enrich fish oil, it is mixed with an alcohol extract and distilled water in a ratio of 1:1:1; defend the mixture in the dark for 24 hours at a temperature of 20±2°C to carry out the process of transition of mityloxanthin and fucoxanthinol and other fat-soluble carotenoids from the alcohol extract to fish oil; separation of the oil layer.

These distinctive features allow us to conclude that there is novelty in the proposed technical solution. Conducted patent research, as well as the study of available scientific publications related to the subject of the invention, did not find solutions that have features similar to the proposed invention. The temperature regimes and the claimed sequence of operations of the method are selected by the authors experimentally within the boundaries of the optimal and effective achievement of the claimed technical result. The method is designed for use both in laboratory conditions and in industrial production. With the industrial application of the method, the conditions for its implementation will not change.

Scientists from the FRC InBYuM (RF) and Japan [Ivanov et al., 2009; Maoka et al., 2011] proved that the soft tissues and eggs of the Black Sea mussels M. galloprovincialis contain physiologically active carotenoids, for example, fucoxanthinol and mytiloxanthin in sufficiently high concentrations. Get fucoxanthinol chemical methods is very difficult [US Pat. 2548103, RF, IPC C12R 23/00, C09 B 61/00, 2015)]. It is much easier to isolate it with other carotenoids, such as methyloxanthin. With the help of fish oil, you can enhance the effectiveness of the effects of fucoxanthinol and methyloxanthin on the body.

Chemical properties, in particular, solubility in 96% ethanol and the process of transition from dilute ethanol (70% and below) to hexane, are similar for fucoxanthinol and methyloxanthin at low concentrations [Fox, 1976].

Of course, in the proposed method, fucoxanthinol and mytiloxanthin will be isolated along with a number of other carotenoids and protein impurities that will not affect the properties of fucoxanthinol and methyloxanthin in any way.

Fucoxanthin metabolites, fucoxanthinol and mytiloxanthin, are widely distributed in marine bivalves. Hydrobionts accumulate fucoxanthin consumed with food and convert it into a more active antioxidant mytiloxanthin (Maoka et al., 2011). Fucoxanthinol, the main metabolite of fucoxanthin, formed under the action of enzymes in the body, has a 1.5-2 times higher biological activity than the original carotenoid [Konishi, 2006; Sachindra, 2007; Sugawara, 2006].

Mityloxanthin showed excellent antioxidant activity by inhibiting lipid peroxidation [Maoka et al., 2016]. The high biological activity of carotenoids is an experimentally confirmed fact (Apryshko, 2005). Antitumor and anticarcinogenic properties of methyloxanthine are known [Borodina, 2008].

The source of fucoxanthinol and mytiloxanthin are the eggs and tissues of the mussel M. galloprovincialis, which contain these pigments in amounts sufficient for their isolation [Ivanov, 2009; Maoka et al., 2011].

The carotenoid composition and percentage of carotenoids in the body of M. galloprovincialis have already been studied; the content of fucoxanthin is 3.1%, fucoxanthinol - 7.2%, and methyloxanthin - 11.5% of the mass of all carotenoids [Maoka et al., 2011]. Mityloxanthin is not found in algae and therefore can only be formed through metabolic transformation processes that appear to be specific to molluscs.

Mityloxanthin, being a metabolite of fucoxanthin, has a lower bioavailability than other nutrients of the carotenoid class [Hashimoto, 2012; Maoka et al., 2016]. The bioavailability of fucoxanthin and its metabolites is increased in fish oil. It has been experimentally proven that an oily solution of fish oil, containing polyunsaturated omega-3 fatty acids in its composition, significantly increases the effectiveness of carotenoids [Maeda 2005; Maeda, 2007; Sugawara, 2002].

Fish oil contains a high content of 5, 8, 11, 14, 17-eicosapentaenoic (C20:5ω3) and 4, 7, 10, 13, 16, 19-docosahexaenoic (C22:6ω3) polyunsaturated fatty acids, which have a beneficial effect on health [Simopoulos , 1991]. It is well known that fish oil exhibits a synergistic effect with, for example, sesamin in preventing fatty liver in mice [Yanagita et al., 2005]. Upon contact with air, liquid fish oil gradually oxidizes and loses its beneficial properties. But in fish oil enriched with carotenoids, the oxidation process will slow down, since carotenoids are the strongest antioxidants [Apryshko et al., 2005]. An example of the implementation of the method.

Example 1

Oocytes obtained by thermal stimulation of spawning and deposited at 1500 rpm [US Pat. 2599834], as well as crushed soft tissues taken from 50 spawning female mussels M. galloprovincialis, were poured with distilled water and hexane (1:1:2), and one part of 96% ethanol was added. After thorough mixing, the mixture was settled in a separating funnel for 24 hours in the dark at a temperature of 20±2°C until the layers were separated. A red-brown colored (you entered it into the text above, and you must repeat it in the example!) hexane layer was separated. The solvent was distilled off on a rotary evaporator.

The identification of fucoxanthinol and mityloxanthin in the carotenoid "complex" was carried out spectrophotometrically in the visible region (400-800 nm). Absorption maxima in diethyl ether: mityloxanthin - 470 nm, fucoxanthinol - 445 and 470 nm.

The resulting carotenoid "complex" was dissolved in 96% ethanol in a ratio of 1:5, the alcohol extract was mixed with fish oil and distilled water in a ratio of 1:1:1. Defended in a separating funnel for 24 hours in the dark at a temperature of 20±2°C until the formation of an oil layer enriched with fucoxanthinol and mityloxanthin.

The raw weight of eggs obtained from 50 mussels was 3 g. The content of carotenoids in the soft tissues of 50 mussels M. galloprovincialis was 36 mg per 1000 g of fresh weight. The content of fucoxanthinol in 3 g of eggs is 0.01 mg; in 1000 g of soft tissues - 3 mg, and methyloxanthin - 0.01 mg and 4 mg, respectively. The resulting product was a yellow-orange oily solution with a characteristic fish oil odor and contained at least 3 mg of fucoxanthinol and 4 mg of methyloxanthin.

The resulting product in the form of an oily extract of marine carotenoids is promising for long-term storage, since it is not subject to oxidative deterioration and is also suitable for use as a biologically active component. Considering the daily dose of 2.4-8 mg of hard-to-find and expensive fucoxanthinol and methyloxanthin in pure form, as well as the low probability of eating 50 mussels per day, the minimum amount of fish oil enriched with fucoxanthinol and methyloxanthin allows not only to increase the effectiveness of fucoxanthinol and methyloxanthin, but also allows dosed intake of these valuable natural compounds for the treatment and prevention of various diseases.

Literature sources taken into account:

1. Apryshko G.N. Antitumor drugs from marine algae of the Mediterranean basin / G.N. Apryshko, M.V. Not good, V.N. Ivanovna. Milchakova // Algology. - 2005. - V. 15, No. 4. - S. 485-490.

2. Borodina A.V. Anticancer drugs in some marine food products / A.V. Borodina, M.V. Nekhoroshev // Russian Biotherapeutic Journal. - 2008. - V.7, No. 1. - S. 36.

3. Ivanov V.N. The content of lipids and carotenoids in the swept sexual products of the Black Sea Mytilus galloprovincialis Lam. / V.N. Ivanov, N.V. Karavantseva, M.V. Nekhoroshev // Marine Ecological Journal. - 2009. - V. 4, No. 1. - S. 84.

4. Pat. 2599834, Russian Federation. IPC A23L/10, A23L 17/50. A method for obtaining a biologically active substance from the Mediterranean mussel Mytilus galloprovincialis Lam / L.L. Nikonova (RU), M.V. Nekhoroshev (RU); applicant and patent holder Institute of Marine Biological Research (Russia) - No. 2014138314/13; dec. 09/22/2014, publ. 10/20/2016. Bull. No. 29.

5. Pat. 2548103, Russian Federation. IPC C12R 23/00, C09B 61/00. Method for obtaining trans-fucoxanthinol / N.V. Pospelova (RU), M.V. Nekhoroshev (RU), Vladimir Vaiser (IL); applicant and patentee Institute of Biology of the Southern Seas. A.O. Kovalevsky (RU) - No. 101365 (UA); dec. 03.10.2014, publ. 04/10/2015. Bull. No. 10.

6 Fox D.L. Animal biochromes and structural colors physical, chemical, distributional & physiological features of coulored bodies in the animal world / D.L. fox. California: Berkeley [u.a.] Univ. of California Pr. 1976. - 432 p.

7. Hashimoto T. Pharmacokinetics of fucoxanthinol in human plasma after the oral administration of kombu extract / T. Hashimoto, Y. Ozaki, M. Mizuno, M. Yoshida, Y. Nishitani, T. Azuma, A. Komoto, T. Maoka , Y. Tanino, K. Kanazawa // British Journal of Nutrition. - 2012. - Vol.107, iss. 11:1566-9. https://doi.org/10.1017/S0007114511004879.

8. Kapranova L.L. Fatty acid composition of gonads and gametes in the Black Sea bivalve mollusk Mytilus galloprovincialis Lam. at different stages of sexual maturation / L.L. Kapranova, M.V. Nekhoroshev, L.V. Malakhova, V.I. Ryabushko, S.V. Kapranov, T.V. Kuznetsova // Journal of Evolutionary Biochemistry and Physiology. - 2019. - Vol. 55, iss. 6. - P. 448-455. https://doi.org/10.1134/S0022093019060024.

9. Konishi I. Halocynthiaxanthin and fucoxanthinol isolated from Halocynthia roretzi induce apoptosis in human leukemia, breast and colon cancer cells / I. Konishi, M. Hosokawa, T. Sashima, H. Kobayashi, K. Miyashita // Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology. - 2006. - Vol. 142, iss. 1-2. - P. 53-59. https://doi.org/10.1016/j.cbpc.2005.10.005.

10. Maeda H. Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabetic KK-Ay mice / H. Maeda, M. Hosokawa, T. Sashima, K. Miyashita // Journal of Agricultural and Food Chemistry. - 2007. - Vol. 55, iss. 19. - P. 7701-7706. https://doi.org/10.1021/jf071569n.

11. Maeda H. Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues/ H. Maeda, M. Hosokawa, T. Sashima, K. Funayama, K. Miyashita //Biochemical and biophysical research communications . - 2005. - Vol. 332, iss. 2. - P. 392-397. https://doi.org/10.1016/j.bbrc.2005.05.002.

12. Maoka T. A Series of 190-Hexanoyloxyfucoxanthin Derivatives from the SeaMussel, Mytilus galloprovincialis, Grown in the Black Sea, Ukraine / T. Maoka, T. Etoh, A. V. Borodina, A. A. Soldatov // Journal of Agricultural and Food Chemistry. - 2011. - Vol. 59. - P. 13059-13064. https://doi.org/10.1021/jf2035115.

13. Maoka T. Anti-oxidative activity of mytiloxanthin, a metabolite of fucoxanthin in shellfish and tunicates / T. Maoka, A. Nishino, H. Yasui, Y. Yamano, A. Wada // Marine Drugs. - 2016. - Vol. 14, iss. 5: 93. https://doi.org/10.3390/mdl4050093.

14.SachindraN. M. Radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites / N. M. Sachindra, E. Sato, H. Maeda, M. Hosokawa, Y. Niwano, M. Kohno, K. Miyashita // Journal of Agricultural and Food Chemistry. - 2007. - Vol. 55, iss. 21. - P. 8516-8522. https://doi.org/10.1021/jf071848a.

15. Simopoulos A. P. Omega-3 fatty acids in health and disease and in growth and development / A. P. Simopoulos // American Journal of Clinical Nutrition. - 1991. - Vol. 54.-P 438-63. https://doi.org/10.1093/ajcn/54.3.438.

16. Sugawara T. Brown algae fucoxanthin is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells and mice/ T. Sugawara, V. Baskaran, W. Tsuzuki, A. Nagao // Journal of Nutrition. - 2002. - Vol. 132, iss. 5. - P. 946-51. https://doi.org/10.1093/jn/132.5.946.

17. Sugawara T. Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, flicoxanthinol / T. Sugawara, K. Matsubara, R. Akagi, M. Mori, T. Hirata // Journal of Agricultural and Food Chemistry. - 2006. - Vol. 54, iss. 26. - P. 9805-9810. https://doi.org/10.1021/jf062204q

18. Yanagita T. Conjugated linoleic acid-induced fatty liver can be attenuated by combination with docosahexaenoic acid in C57BL/6N mice / T. Yanagita, Y. M. Wang, K. Nagao, Y. Ujino, N. Inoue// Journal of Agricultural and food chemistry. - 2005. - Vol. 53. - P. 9629-9633. https://doi.org/10.1021/jf052203i.

Claims (1)

A method for obtaining a functional product enriched with fucoxanthinol and mityloxanthin, including grinding raw materials from the mussel M. galloprovincialis and isolating fat-soluble carotenoids from it, separating carotenoids from the solvent, characterized in that to isolate fat-soluble carotenoids, the eggs and crushed soft tissues of the mussel M. galloprovincialis are mixed with distilled water, hexane and 96% ethanol in a ratio of 1:1:2:1, the resulting mixture is stirred and left for 24 hours in the dark at a temperature of 20 ± 2°C until a colored hexane layer containing a carotenoid complex is formed, followed by separation of the solvent, after which the carotenoid complex containing fucoxanthinol and methyloxanthin is dissolved in 96% ethanol in a ratio of 1:5 and the resulting alcohol extract, fish oil and distilled water are combined in a ratio of 1:1:1, and then the mixture is left to stand in the dark for 24 hours at temperature 20±2°C until the formation of an oil layer enriched fucoxanthinol and methyloxanthin.