RU2750150C1 - Method for producing stable lutein paste based on vegetable cryo raw materials with increased carotenoid content - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a method for producing stable lutein paste from annual oats (Avena sativa L.). A method for obtaining stable lutein paste from annual plant oats (Avena sativa L.), characterized by the use of green raw materials - annual plant oats (Avena sativa L.), harvested after exposure to natural cold, which is crushed by grinding in liquid nitrogen, obtained frozen the powder is subjected to freeze-drying on a lyophilizer, the finished lyophilisate is extracted with a 96% solution of ethyl alcohol to obtain carotenoid pigments containing a lutein complex, at the rate of 8 ml of alcohol per 1 g of lyophilisate, after which the solution is evaporated under vacuum until the ethyl alcohol is completely removed.

EFFECT: allows to obtain a stable lutein paste containing lutein and zeaxanthin (lutein complex) and β-carotene, with a high content of carotenoids in the paste.

1 cl, 6 dwg, 5 tbl

Description

The invention relates to biotechnology, namely to methods for producing stable lutein paste with a high content of biologically active substances - chlorophylls, carotenoids, including chlorophyll a and b, xanthophylls, including lutein and zeaxanthin (lutein complex), as well as β-carotene (provitamin A) used as feed, production of medical and veterinary drugs.

Animals and humans are unable to synthesize carotenoids, important metabolic regulators, the intake of which is directly related to nutrition. Plants are the main and sometimes the only source of these compounds for animals and humans. Of these, the most famous is β-carotene - provitamin A (see Karnaukhov V.N.Biological functions of carotenoids. - M, 1988. - 197 p.). Vitamin A synthesized on its basis (retinol and its derivatives) is recognized as an essential food component for combating infections and a high mortality rate (see Mayev I.V., Kazyulin A.N., Bely P.A. Vitamins. - M .: MEDpress-inform, 2011 .-- 544 p.).

Modern research makes it possible to classify carotenoids, including xanthophylls, as the leading compounds responsible for maintaining visual functions. Xanthophylls practically do not possess A-provitamin activity, but they are effective as antioxidants and are the only pigments transported to the retina and forming a yellow macular pigment or macular xanthophyll, while stabilizing and compensating for degenerative processes in the retina (see Wenzel AJ, Fuld K., Stringham JM Light exposure and macular pigment optical density // Invest. Ophthalmol. Vis. Sci. - 2003. - V. 44 (1). - P. 306-309).

In this regard, the search for natural sources of carotenoids (lutein and zeaxanthin) is quite relevant at the present time. If for a person, in particular, sources of vitamin A are animal products - liver, oil, chicken eggs, and lutein and zeaxanthin (lutein complex) - egg yolks, as well as spinach and corn (see Mayev I.V., Kazyulin A .N., Bely P.A. Vitamins. - M .: MED press-inform, 2011. - 544 p.), Then in herbivorous farm animals, they are exclusively vegetable feed. For medical purposes, luteins are also used, extracted by essential oil extraction from marigold plants (Calendula sp.) And a number of marigold varieties (Tagetes sp.). The main plant species grown for the extraction of the luteal complex are thermophilic, therefore, on an industrial scale, the production of carotenoids is concentrated in tropical and subtropical countries.

There is a known method for the production of carotene for balancing feed rations and a means for its implementation (see RU No. 2355156, class A01D91 / 04, A01F29 / 00, B02C13 / 04, publ. 20.05.2009), including mowing the grass with crimping and raking wilted to 40% grass moisture in the windrows.

The disadvantages of this method are the lack of conditions for the timing of sowing (for annual species) and collection of grass, in which the optimal content of carotene is achieved. In this case, only the parameters are known that are necessary for harvesting and subsequent processing of harvested herbs in order to minimize the loss of carotene in them. In addition, the known solution provides for the production of only one of the individual carotenoids, namely carotene, and not a complex of all important carotenoids contained in plants (lutein, zeaxanthin, etc.).

In US patent No. 5648564 (class C07C 35/00, C07C 35/21, C07C 35/08, publ. 15.07.1997) discloses a method of saponification of a lutein ester and purification of free lutein. In this case, saponification is carried out at a temperature of 65-80 ° C, which is high enough to cause the destruction of lutein. In addition, the saponification process takes at least 3 hours. This long processing time, combined with the high temperature, also contributes to the additional breakdown of lutein. The reaction is carried out in propylene glycol, ethanol and aqueous alkali, and the resulting reaction mixture, due to the presence of water in it, is a viscous soapy material in which lutein crystals are dispersed, from which it is technologically difficult to efficiently isolate all free lutein.

There is a known method of isolation and purification of mixed carotenoids with a high concentration of specific compounds (see US No. 6380442, class С07С 35/21, С07С 35/00, publ. 30.04.2002), including hydrolysis of essential oil of marigolds using isopropyl alcohol, water and alkali at a temperature of 60-65 ° C for 90 minutes. And there is also a known method for purifying essential oil with alkali and acid (see US No. 6504067, class А23L 1/275, А23L 1/27, С07С 29/00, С07С 29/76, С07С 29/88, С07С 29/86, С09В 61/00, C07C 35/21, publ. 07.01.2003), in which the purified essential oil is subjected to aqueous-alkaline hydrolysis at a temperature of 90 ° C for 8 hours in the presence of some emulsifiers that provide effective contact.

The disadvantages of the known solutions are also the high temperature and long processing time, which cause the destruction of lutein. In addition, the known approaches do not solve the problem of efficient separation of precipitated crystals of free lutein from a dense, silty and soapy mixture of alkaline solutions.

A known method of obtaining plant materials with an increased content of carotenoids from annual and perennial species of herbaceous plants with a high content of biologically active substances - carotenoids (see RU No. 2649338, class A23K 10/30, publ. 02.04.2018), including xanthophylls , including lutein and zeaxanthin (lutein complex), as well as β-carotene, used as feed and in the production of medical and veterinary drugs.

However, the known technical solution does not provide for the production of a stable lutein paste with a high content of carotenoids, including on the basis of vegetable cryopreparations containing the lutein complex in preparative quantities.

As a prototype, a method for producing a stable lutein paste from essential oil was chosen (see RU No. 2321582, class С07С 403/24, С07С 35/21, publ. 10.04.2008), in which the resulting lutein paste is mixed with 4-5 volumes of alcohol a solvent from the group consisting of normal aliphatic alcohols containing from one to five carbon atoms (the alcohol is selected from the group consisting of: methanol, ethanol, propanol, butanol and pentanol).

The disadvantages of the closest analogue are the permitted use of methanol as a solvent, which is unsafe for the consumer (ingestion of about 10 ml of methanol can lead to severe poisoning (see Vale A (2007). "Methanol". Medicine. 35 (12): 633-4 . DOI: 10.1016 / j.mpmed.2007.09.014)). In addition, lutein is extracted by essential oil extraction from marigold plants (Calendula sp.), The cultivation of which in cold climates (down to -10 ° C) is problematic.

At the same time, alternative sources of xanthophyll production can be high-yielding one- and perennial herbaceous plant species that are zoned in cold climates: oats, wheatgrass, rump and some others, capable of accumulating significant amounts of lutein and zeaxanthin under conditions of low-temperature stress using proper technology (3 , 5-4.0 mg / g of crude residue of the alcoholic extract, see RU No. 2649338, class A23K 10/30, publ. 02.04.2018).

The problem to be solved by the claimed invention is expressed in expanding the range of lutein pastes of plant origin, methods for their production and increasing the content of carotenoids in the paste.

The technical result is a method for obtaining a stable lutein paste, including lutein and zeaxanthin (lutein complex) and β-carotene, based on herbaceous plants, adapted to cold climates. In addition, the claimed solution uses natural, climatic cold to obtain carotenoids and their subsequent use as feed and in the production of drugs for medical and veterinary purposes, which generally reduces the cost of technological processes.

To solve this problem, the method of obtaining stable luteal paste from annual plant oats (Avena sativa L.) is characterized by the use of green raw materials - annual plant oats (Avena sativa L.), collected after exposure to natural cold, which is crushed by grinding in liquid nitrogen , the resulting frozen powder is subjected to freeze drying on a lyophilizer, the finished lyophilisate is extracted with a 96% solution of ethyl alcohol to obtain carotenoid pigments containing a lutein complex, at the rate of 8 ml of alcohol per 1 g of lyophilisate, after which the solution is evaporated under vacuum until the ethyl alcohol is completely removed.

The claimed technical result is achieved due to the fact that, for example, plants of the bluegrass family (Poaceae Barnhart) are used as plant raw materials with a high content of carotenoids: annual oats (Avena sativa L.).

An analysis of the features of the declared solution testifies to the compliance of the declared solution with the "novelty" criterion.

The combination of essential features provides a solution to the stated technical problem, namely, the production of lutein paste with a high content of carotenoids.

The proposed solution consists in the fact that the basis is taken vegetable cryoscopy with a high content of carotenoids (lutein + zeaxanthin).

Thus, the plant material (cryo raw material) collected after exposure to natural cold is crushed, then placed in a porcelain cup to obtain a frozen powder. For this, the crushed cryo-raw materials are poured with liquid nitrogen and quickly ground with a porcelain pestle until a frozen powder is obtained, if necessary, adding liquid nitrogen as it evaporates.

The frozen plant material is transferred into pre-prepared flasks for subsequent freeze drying using a lyophilizer, for example, brand "VirTis" (USA).

Further, 96% ethyl alcohol is added to the finished lyophilisate of cryo-raw materials of herbaceous plants for the extraction of carotenoid pigments containing the lutein complex (lutein + zeaxanthin). In this case, the calculation is used: for 1 g of lyophilisate - 8 ml of ethanol. After that, the solutions are evaporated under vacuum in porcelain cups until the ethanol is completely removed. The result is a lutein paste containing a lutein complex (lutein + zeaxanthin).

The claimed solution is illustrated in the drawing, where figure 1 shows a scheme for obtaining a stable lutein paste in preparative quantities from green cryo raw material A. sativa; figure 2 - chromatographic image of pigments of freshly harvested leaves of Avena sativa L. after chromatographic separation, where 1 is the starting line; 2 - neoxanthine; 3 - violaxanthin; 4 - lutein + zeaxanthin; 5-chlorophyll b; 6 - chlorophyll a; 7 - β-carotene; figure 3 - chromatographic image of pigments of lyophilized leaves of Avena sativa L. after chromatographic separation, where 1 is the start line; 2 - neoxanthine; 3 - violaxanthin; 4 - lutein + zeaxanthin; 5-chlorophyll b; 6 - chlorophyll a; 7 - β-carotene; figure 4 is a chromatographic picture of the pigments of the obtained lutein paste Avena sativa L. after chromatographic separation, where 1 is the start line; 2 - neoxanthine; 3 - violaxanthin; 4 - lutein + zeaxanthin; 5- chlorophyll b; 6 - chlorophyll a; 7 - β-carotene; figure 5 - the results of gradient multistep-HPTLC fractions from the paste A. sativa (reverse shooting mode, I-XII - gradient solvent system hexane-acetone (100: 0 → 50: 50)); 6 - HPTLC densitogram of A. sativa paste (SiO2; CHCl3-MeOH-H2O 14: 7: 1.1; detector - anisic aldehyde / H ₂ SO ₄ ), ES (+) - MS spectrum of zone A (avenacoside B) and structural formulas of triterpene glycosides), where the positions of compounds A are avenacoside B, B is avenacoside A, C is 26-desglucoavenacoside B, D is 26-desglucoavenacoside A.

Thus, for the experimental work, the preparation of stable lutein paste in preparative quantities from the green cryo raw material of A. sativa was carried out according to the scheme shown in figure 1. At the same time, constant monitoring of the qualitative and quantitative composition of carotenoids, including lutein + zeaxanthin, was carried out in the initial raw material, lyophilized leaves and in plant paste using analytical thin layer chromatography (see Fig. 2-4).

From the data obtained, it follows that as a result of the concentration of pigments in the final product (lutein paste), the content of total and individual carotenoids increases by an order of magnitude (see table 1).

In addition to studying the pigment complex obtained from oats, the authors studied a number of perennial herbaceous plants (Elytrigia repens L., Poa pratensis L.), which have a wide distribution area under the conditions of Yakutia (see Tables 2, 3). It was shown that in creeping wheatgrass the total content of carotenoids in lutein paste is almost two times lower than in meadow bluegrass paste and twice as high as in oat paste.

In addition, studies of the effect of the storage time of the obtained lutein paste at low positive temperatures on the preservation of carotenoids in A. sativa showed that the content of carotenoids does not change significantly (see Table 4).

Thus, studies in laboratory conditions revealed the presence of at least 20 components in the resulting A. sativa luteal paste, including 6 compounds of the chlorophyll group and 11 compounds of the carotenoid series. For identification, comparative data on chromatographic mobility (HPTLC, HPLC), UV-, IR- and MS-spectrometry data were used (see figure 5).

As a result of identification in A. sativa paste, the presence of six chlorophylls was established:

- chlorophylls a and b;

- pheofetins a and b;

- pheophorbides a and b.

11 compounds were found in the composition of carotenoids:

- hydrocarbons - α-carotene, β-carotene, γ-carotene;

- monoles of carotenes - α-cryptoxanthin, β-cryptoxanthin;

- monol epoxies - cryptoxanthin epoxides (mixture of isomers);

- carotene diols - lutein, zeaxanthin;

- epoxides of diols - antheraxanthin (mixture of isomers);

- diols of diepoxides - violaxanthin (mixture of isomers);

- carotene polyols - neoxanthine (mixture of isomers).

A number of methods were used to quantify the content of each of the identified carotenoids, incl. HPLC and HPTLC stationary phase densitometry magnesium oxide / diatomaceous earth (see Schwartz, Patroni-Killam, 1985). The dominant carotenoids are lutein and β-carotene (see table 5).

It is known that typical representatives of the sterols of the aerial part of A. sativa are avenacosides A and B and their deglucosylated derivatives (see Laudenbach and Kesselmeier, 1982). Their presence is found in the plant during the entire growing season. The total content of sterols in the biomass of A. sativa from Yakutia was 0.8%, in the paste - 1.4%. To identify the qualitative composition, HPTLC-densitometric method was used, which made it possible to establish the presence of four compounds A-D. Identification was performed based on chromatographic mobility and mass spectra (ES (+) - MS) with valid terpene samples (see FIG. 6).

The identified compounds were identified as avenacoside A (B), avenacoside B (A), 26-desglucoavenacoside A (D), and 26-desglucoavenacoside B (C). According to the data of quantitative analysis, the ratio of compounds in A. sativa paste was 2.2: 3.4: 1: 2.9, respectively, for avenacoside A, avenacoside B, 26-desglucoavenacoside A, and 26-desglucoavenacoside B.

The use of the present invention makes it possible to obtain a stable lutein paste containing lutein and zeaxanthin (on average 3.5 ± 0.2 mg / g paste) (lutein complex) and β-carotene, based on herbaceous plants, adapted to cold climates. In addition, the claimed solution uses natural, climatic cold to obtain carotenoids and their subsequent use as feed and in the production of drugs for medical and veterinary purposes, which, in general, makes the technological processes cheaper.

Table 1

Comparative analysis of the content of individual carotenoids in the leaves of common oat (Avena sativa L.) and lutein paste obtained from cryopreserved raw materials

Option Carotenoids, μg / g fr wt The amount of carotenoids, μg / g fr wt Xantho phyll sum, μg / g fr wt Neo Vio Lute + Zea β-car Fresh
leaves 72.03 ± 2.00 40.80 ± 1.87 241.28 ± 4.43 88.50 ± 3.63 442.61 354.11 Lyof. dry. leaves 83.26 ± 6.67 47.20 ± 9.28 345.70 ± 6.38 110.86 ± 12.05 587.02 476.12 Cryo-raw paste 635.95 ± 18.40 300.00 ± 12.36 3562.79 ± 40.73 1159.36 ± 21.77 5658.10 4498.74

table 2

Comparative analysis of the content of individual carotenoids in the leaves of creeping wheatgrass (Elytrigia repens L.) and lutein paste obtained from cryo raw materials

Option Carotenoids, μg / g fr wt The amount of carotenoids, μg / g fr wt The amount of xanthophylls, μg / g fr wt Neo Vio Lute + Zea β-car Fresh
leaves 75.46 ± 3.21 69.07 ± 0.69 252.09 ± 3.50 133.91 ± 3.16 530.53 396.62 Lyof. dry. leaves 103.08 ± 1.29 140.40 ± 0.80 441.98 ± 2.88 191.45 ± 2.99 876.91 685.46 Cryo-raw paste 499.56 ± 18.31 258.00 ± 12.00 1571.51 ± 38.37 184.09 ± 19.48 2513.16 2329.07

Table 3

Comparative analysis of the content of individual carotenoids in the leaves of meadow bluegrass (Poa pratensis L.) and lutein paste obtained from cryo-raw materials

Option Carotenoids, μg / g fr wt The amount of carotenoids, μg / g fr wt The amount of xanthophylls,
μg / g fr wt Neo Vio Lute + Zea β-car Fresh
leaves 90.40 ± 1.83 87.87 ± 2.93 383.14 ± 2.59 144.41 ± 1.50 705.81 561.40 Lyof. dry. leaves 124.10 ± 2.38 128.40 ± 14.12 498.84 ± 15.03 213.55 ± 10.94 964.88 751.33 Cryo-raw paste 3764.51 ± 98.43 1584.00 ± 87.25 4861.92 ± 105.54 833.52 ± 59.36 11043.98 not def.

Table 4

The content of carotenoids (lutein + zeaxanthin) of vegetable paste from cryo raw material Avena sativa L. for different storage periods at low positive temperatures (+ 5 ° С)

Carotenoid content (lutein + zeaxanthin), mg / g paste 02/26/13 04/18/13 07/17/13 08/15/13 09/17/13 3.5 ± 0.2 3.4 ± 0.1 3.6 ± 0.1 3.8 ± 0.1 3.5 ± 0.2

Table 5

The content of individual carotenoids in the paste of Avena sativa L.

Compound Content,%, of the amount of carotenoids Content,
mg%, in paste α-carotene 1.7 14.25 β-carotene 24.9 208.64 γ-carotene 0,4 3.35 α-cryptoxanthin 0.3 2.51 β-cryptoxanthin 0.8 6.70 cryptoxanthin epoxides 0.6 5.06 lutein 50.4 422.31 zeaxanthin 1.6 13.41 antheraxanthin 0,4 3.35 violaxanthin 3.5 29.33 neoxanthin 9,7 81.30 Sum of identified compounds 94.3 790.21

Claims (1)

A method of obtaining stable lutein paste from annual plant oats (Avena sativa L.), characterized by the use of green raw materials - annual plant oats (Avena sativa L.), harvested after exposure to natural cold, which is crushed by grinding in liquid nitrogen, obtained frozen the powder is subjected to freeze-drying on a lyophilizer, the finished lyophilisate is extracted with a 96% solution of ethyl alcohol to obtain carotenoid pigments containing a lutein complex, at the rate of 8 ml of alcohol per 1 g of lyophilisate, after which the solution is evaporated under vacuum until the ethyl alcohol is completely removed.

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