RU2802223C1 - Method of obtaining biological products from rapana venosa val. - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is intended for obtaining natural products for therapy and prophylaxis. In vivo obtained rapana egg shells are extracted with an alcohol-chloroform mixture in a ratio of 1:3 (egg shells: alcohol-chloroform mixture) in the darkness at a temperature of 20±5°C within 72 hours. Then, by adding distilled water in a ratio of 1:1.5 (spiro-chloroform extract:distilled water), the water-immiscible chloroform layer containing PUFA is separated, which is filtered and distilled off using a rotary evaporator. The resulting residue is combined with a 10% oil solution of vitamin E in a ratio of 1:10.

EFFECT: invention makes it possible to obtain functional products with a high content of polyunsaturated fatty acids from rapana's ovaries.

4 cl, 1 tbl, 1 ex

Description

SUBSTANCE: invention relates to the field of biotechnology and is intended for intravital production of natural therapeutic and prophylactic products enriched with polyunsaturated fatty acids (PUFAs) from the gastropod mollusk rapana.

The Black Sea rapana is an edible predatory gastropod mollusk that feeds mainly on mussels [1]. Rapana reproduction is well studied both in natural and laboratory conditions [2, 3]. During spawning in July, females lay fertilized eggs in egg sacs that have a lipid membrane on the outside and are filled with protein inside, which the developing larvae feed on [4–6]. During the season, one female can lay eggs 3-4 times with an interval of 1-3 days without intermediate mating [1]. Each clutch contains about 100 ovaries, and each ovary contains from 984 to 1221 eggs; In this case, the total weight of the ovules can be greater than the weight of the soft tissues of the female. Rapana egg shells serve as a source of irreplaceable PUFAs [12] and other equally valuable biologically active substances (BAS).

Known Method for obtaining biologically active additives (Pat. 2183414 C1, RU, IPC A23L 1/30 (2000.01), 2000). The invention relates to the food industry, namely the production of biologically active food supplements from rapana meat containing omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) in amounts of at least 45% of the total fatty acids; lipids not less than 0.8%. The method is carried out by means of acid hydrolysis of rapana meat. The disadvantage of this method, firstly, is the use of acid hydrolysis. To isolate fatty acids from rapana meat, there are simpler methods, such as extraction. Secondly, the use of exclusively rapana meat as a feedstock. Sources of PUFAs include not only meat, but also eggs, which the rapana is capable of laying in their lifetime in quantities sufficient to isolate PUFAs.

A known method for producing a therapeutic and prophylactic composition (Pat. 2541463 C1 RU, IPC A61K 35/56, 2014), which provides for the production of a composition of therapeutic and prophylactic action "Cerebromide" from marine mollusks, which can be used in geriatric practice as a prevention of the initial forms of atherosclerosis in elderly patients. The method of obtaining the composition consists in obtaining a mixture of acidic hydrolyzate of rapana meat and alkaline - mussel meat. As part of the composition "Cerebromide" 38 fatty acids were identified, of which 21 are saturated and 17 are unsaturated, including those that are part of the omega-3 and omega-6 fatty acids. The ratio of saturated and unsaturated fatty acids is 1.1:1. The ratio of omega-6 to omega-3 is 1:1.6. The composition includes more than 30 micro and macro elements (Ca, K, Fe, Cu, Zn, Mn, Mg, Co, J, Ni, etc.), which also contributes to the normalization of metabolism and the restoration of impaired body functions. The disadvantages of the method, firstly, is the unjustified grinding of frozen rapana meat in an electric meat grinder. In this method, it is indicated that the minced minced meat is transferred to the reactor, 15 l of a 6% hydrochloric acid solution are added and heated to 80°C. At this temperature and constant stirring, hydrolysis is carried out for 20 hours. It should be noted that the severe hydrolysis conditions indicated in the prototype do not require thorough grinding of the raw material. Secondly, in the separation of the hydrolyzate obtained in the first stage from the thick non-hydrolyzed residue, which is then re-hydrolyzed in 8% hydrochloric acid at the boiling point of the mixture for 24 hours. It is more convenient to carry out the hydrolysis in one stage at a constant temperature of 8% hydrochloric acid for 48 hours, and then separate the hydrolyzate. Thirdly, it is not rational to mix alkaline hydrolyzate from mussels and acid hydrolyzate from rapana meat, and then combine them in a ratio of 10:1.

Known Dietary Supplement "Rapolan-S" (Pat. 151963 U1 RU, IPC A33L1/333, 2015), which is a hydrolyzate obtained from rapana meat by enzymatic hydrolysis. The dietary supplement additionally includes marine collagen, vitamin "C" and edible salt. The technological process for the production of the dietary supplement "Rapolan-S" consists of obtaining and mixing the hydrolyzate of rapana and natural collagen with the further addition of vitamin "C" and food salt, homogenization of the ingredients with further pasteurization for 30 minutes in a two-body boiler with constant stirring and a temperature of up to 60 -70°C. After that, the finished product is packaged in a clean, dried container at a temperature of 50-55°C, sealed, labeled and sent for storage. Among the disadvantages of this method, one can indicate the lack of a description of the method for obtaining a hydrolyzate of rapana (the patent contains a link to the TU of Ukraine (TU U 10.8-25133498-029-2013) and a method for obtaining natural collagen from the skin of a Black Sea bull.

The objective of the Method for obtaining biological products from Rapana venosa Val. is the development of a technology for the intravital production of biologically active PUFAs in organic form from rapana ovaries.

The technical result of solving the task is to obtain products containing a high concentration of PUFAs that are essential for the life of the human body, which can be used independently, and also become the basis for the production of cosmetic, pharmaceutical and food preparations.

The claimed technical result is achieved due to the fact that in the Method for obtaining biological preparations from Rapana venosa Val. temperature 20±5°C for 72 hours. Then, by adding distilled water in a ratio of 1:1.5 (spiro-chloroform extract: distilled water), the water-immiscible chloroform layer containing PUFAs is separated, which is filtered and distilled off using a rotary evaporator. The resulting residue is combined with a 10% oil solution of vitamin E in a ratio of 1: 10. Intravital production of rapana egg cells includes the collection of rapana mollusks from their natural habitat during the breeding season, transplanting into aerated plastic cages filled with sea water with a temperature of 20 ± 5 ° C , in which the water is changed once every 24 hours, and rapanas are collected from the walls of the cages. At the same time, for the extraction of polyunsaturated fatty acids, an alcohol-chloroform mixture is used in a ratio of 1:1 (96% ethanol: chloroform). In addition, molluscs of rapana with a shell height of up to 115 mm, aged 2+ years, which are fed with live mussels with a valve size of 40 mm or more, are subject to collection.

Distinctive features of the proposed method from the known are:

- in intravital obtaining of rapan sheep and using them as a source of polyunsaturated fatty acids. The resulting ovotes are easier to process than rapana meat, while the raw weight of the ovotes, which one mollusk is able to lay, exceeds the raw weight of its soft tissues. In egg cells, the content of biologically active substances is maximum in comparison with the soft tissues of the mollusk;

- in the extraction of biologically active PUFAs by extraction, and not by hydrolysis. The proposed extraction regimes were selected experimentally and contribute to a high yield of PUFAs from rapana's ovaries. The isolation of fatty acids by extraction has a number of advantages over hydrolysis: the technology is simpler, the cost of the process is low; during extraction, other biologically active substances, such as carotenoids, pass into solution along with PUFAs.

The claimed method meets the criteria of novelty and inventive step, because for the first time, conditions were proposed for the intravital isolation of PUFAs from rapana ovaries. The method does not require complex and expensive equipment or expensive chemical reagents. The 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 claimed technical solution, which confirms the conclusions about the compliance with the patentability criteria.

As shown in Table 1, the ratio of omega-6 / omega-3 PUFAs in omega-6 / omega-3 PUFAs is low (sob / co3<2), so functional products from oats can be used as a prevention of cardiovascular diseases [8].

In humans, fatty acids of the omega-6 and omega-3 families play an important physiological role in prostaglandin biosynthesis [9]. Arachidonic (С20:4ω6) and docosahexaenoic (С22:6ω3) acids are precursors of prostaglandins, compounds that stop inflammatory reactions and play a major role in the normal functioning of the immune system [10, 11]. Docosahexaenoic (C22:6ω3) acid is necessary for the adaptation of body cells to changes in environmental conditions [12]. Eicosapentaenoic (С20:5ω3) acid is one of several omega-3 PUFAs that protects cell structure from malignant transformation [9].

An example of the implementation of the method

Rapana were collected during the breeding season - in August 2022 in the Sevastopol Bay in the amount of 50 specimens. at a depth of 9 to 15 m under a mussel farm (outer roadstead of the Sevastopol Bay) at a water temperature of 19°C. The sizes of shells of mollusks ranged from 30 to 115 mm, and their age was from 2+ years. Within three days in laboratory conditions received ovoteki. To do this, freshly caught mollusks were transplanted into aerated plastic cages (lined on top to prevent mollusks from crawling out) filled with sea water at a temperature of 20 ± 2°C. Once a day, the water in the cages was changed, and the walls of the container were used to collect egg cells, which are a source of valuable PUFAs. During the experiment, the mollusks were fed with live mussels with a valve size of 40 mm, grown on a mussel farm.

The extraction of total lipids was carried out with an alcohol-chloroform mixture (1:1 - 96% ethanol: chloroform). To do this, 300 g of an alcohol-chloroform mixture was added to 100 g of freshly picked ovaries. Everything was thoroughly mixed and left to be extracted in a dark place. After 72 hours, the mixture was transferred to a separating funnel, 150 ml of distilled water was added, and the chloroform layer, immiscible with alcohol and water, was separated, the volume of which was 150 ml. The chloroform layer was filtered through a pleated filter. Chloroform was evaporated on a rotary evaporator. The content of total lipids was determined gravimetrically. 100 g of freshly picked ovaries contained 1±0.02 g (p<0.05) of total lipids. The resulting product had a characteristic odor and an oily texture. The shelf life of the resulting product at 5-7°C is 12 months.

The resulting bioproduct weighing 1 g was dissolved in 10 ml of a 10% oil solution of vitamin E. The mixture was defended for 72 hours in the dark at a temperature of 5-7°C. As a result, 10 ml of a fatty acid-saturated lemon-colored vitamin E oil solution were obtained. An oily solution of vitamin E was used as a preservative to prevent rapid degradation of PUFAs. Storage of the obtained extract - 24 months.

Thus, lipid extracts from the rapana's egg cells containing valuable PUFAs are promising for use in the food and cosmetic industries. Combining PUFAs with oily solutions, such as 10% vitamin E oily solution, results in a product with antioxidant properties. Oxidative processes in such a composition are minimized, which increases its shelf life.

Literature sources taken into account

1. Chukhchin V.D. Ecology of gastropods in the Black Sea. Kyiv: Naukova Dumka, 1984. 176 p.

2. Pirkova A.V., Ladygina L.V. Embryonic and Larval Intracapsular Development of the Rapa Whelk Rapana venosa (Valenciennes, 1846) (Gastropoda, Muricidae) // Russian Journal of Biological Invasions. 2019 Vol. 10, no. 1. P. 39-47.

3. Saglam H, Duzgunes E. Deposition of capsule egg and larval development of Rapana venosa (Gastropoda: Muricidae) from the south-eastern Black Sea Mar // Journal of the Marine Biological Association of the United Kingdom. 2007 Vol. 87. P. 953-957. https://doi.org/10.1017/S0025315407056330

4. Chukhchin V.D. Reproduction of Rapana (Rapana bezoar L.) in the Black Sea // In: Proceedings of the Sevastopol Biological Station / Ed. ed. V.A. Vodyanitsky. Sevastopol. 1961a. T. 14. S. 163-168.

5. Cesari P., Mizzan L. Osservazioni su Rapana venosa (Valenciennes, 1846) in cattivita (Gastropoda: Muricidae, Thaidinae // Bollettino del Museo Civico di Storia Naturalea di Venezia. 1993. No. 42. P. 9-21.

6. Harding J.M., Mann R., Kilduff C.W. Influence of environmental factors and female size on reproductive output in an invasive temperate marine gastropod Rapana venosa (Muricidae) // Marine Biology. 2008 Vol. 155. No. 6. P. 571-581.

7. Chung E.Y., Kim S.Y., Kim Y.G. Korean J. Reproductive ecology of the purple shell Rapana venosa (Gastropoda: Muricidae), with special reference to the reproductive cycle, deposits of egg capsules and hatchings of larvae // Malacology. 1993 No. 9. P. 1-15.

8. Makhutova O.N., Gladyshev M.I. Essential polyunsaturated fatty acids in the physiology and metabolism of fish and humans: significance, needs, sources // Russian Journal of Physiology. THEM. Sechenov. 2020. Vol. 105, No. 5. pp. 601-621. https://doi.org/l0.31857/S0869813920050040

9. Levachev M.M. Fish fats in the dietology of hyperlipoproteinemia and hypertension / M.M. Levachev. Moscow: Medicine, 1988. 150 p.

10. Gavrisyuk V.K. The use of Omega-3 polyunsaturated fatty acids in medicine / V.K. Gavrisyuk // Ukrainian journal of pulmonology. 2001. No. 3. pp. 5-10.

11. Podzolkov V.I. The role of omega-3 polyunsaturated fatty acids in the management of cardiovascular risk / V.I. Podzolkov, M.V. Pisarev // Cardiovascular therapy and prevention. 2020. Vol. 19, no. 3:2589. https://doi.org/10.15829/1728-8800-2020-2589

12. Dernekbaşl S., Öksüz A., Dernekbasi S., Oksuz A., Celik M.Y., Karayiicel L, Karayucel S. The fatty acid composition of cultured mussels (M galloprovincialis Lamarck 1819) in offshore longline system in the Black Sea // Journal of Aquaculture & Marine Biology. 2015. Vol. 2, iss. 6. Art. no. 00049 (5 p.). https://doi.org/10.15406/jamb.2015.02.00049.

Claims (4)

1. A method for obtaining biological preparations from Rapana venosa Val. for 72 hours, then by adding distilled water in a ratio of 1:1.5 (spiro-chloroform extract : distilled water), the water-immiscible chloroform layer containing PUFA is separated, which is filtered and distilled off using a rotary evaporator, and the residue is combined with 10 % oil solution of vitamin E in a ratio of 1:10, moreover, intravital obtaining of rapana mollusks includes the collection of rapana mollusks from their natural habitat during the breeding season, transplanting into aerated plastic cages filled with sea water with a temperature of 20 ± 5 ° C, in which one once every 24 hours they change the water, and from the walls of the cages they collect rapana's sheep. 2. The method according to p. 1, characterized in that for the extraction of PUFA, an alcohol-chloroform mixture is used in a ratio of 1:1 (96% ethanol: chloroform). 3. The method according to p. 1, characterized in that molluscs of rapana with a shell height of up to 115 mm, aged 2+ years, are subject to collection. 4. The method according to claim 1, characterized in that the collected mollusks are fed with live mussels with a valve size of 40 mm or more.