Effect of heat treatment and storage on anthocyanins levels in food plants

Anthocyanins constitute a vast group of polyphenolic water-soluble plant pigments that give flowers, fruits, leaves, and root crops a reddish-blue to purplish-black coloration. C hemically, anthocyanins are a glycosylated form of hydroxy- and methoxy-substituted derivatives of 2-phenylchromene or anthocyanidins. Anthocyanins are widespread in nature, mainly in flowers, vegetables and fruits, in which more than 600 derivatives of the six main anthocyanidins – cyanidin (C y), delphinidin (Dp), pelargonidin (Pg), peonidin (Pn), malvidin (Mv) and petunidin (Pt) – were found [1].

Anthocyanins exhibit significant antioxidant activity (AOA) [2, 3], due to which, they counteract oxidative stress in the human body, the main factor in the development of m any pathological conditions [4, 5]. Vegetable and fruit supplements rich in anthocyanins reduce the risks of cardiovascular, cancer and degenerative disorders [6]. Recently, anthocyanins that prevent functional disorders of the body have received more attention [7]: international programs of their in-depth study are being introduced [8], target regulations for anthocyanin-containing foods and special berry-based diets are being developed [9].

However, despite their potential, the widespread introduction of natural anthocyanin-containing supplements and components is limited by high lability of anthocyanins, their degradation during the processing and storage of plant raw m aterials, which significantly reduces their effectiveness [10].

Anthocyanins stability varies within significant limits, depends both on external factors and to a large extent on the chemical nature and the ratio of anthocyanins in different tissues [11].

Selection of the most promising plant varieties with high content of resistant anthocyanins for industrial cultivation is relevant [12], as well as development of express methods to determine their resistance. Among the existing methods for analyzing anthocyanins, the fastest and relatively simple is spectrophotometry [13], which is advisable to choose as an express method to determine resistance. As model external influencing factors of interest is the use of heat treatment and storage of plants, usually used in the technology of processing of raw materials and having a destructive effect on anthocyanins.

The objective of the work was to experimentally study the anthocyanins stability by spectrophotometric method during heat treatment and storage of berry and vegetable plants.

W hen planning the research, it was quite natural to focus not only on blue onion, red cabbage, red raspberry, and blue honeysuckle, which are familiar to Russians in their diets, but also on “exotic” purple carrots and purple potatoes not yet widespread in Russia.

STUDY OBJECTS AND METHODS

Plant material was used as parts of vegetable and berry plants (Table 1) grown by the authors at the territory of the O cean Horticultural Noncom mercial Partnership in Kiparisovo plot, Nadezhinsky District, Primorsky Krai, RF in the summer of 2021 (43˚27 ʹ37 ʺ, 131˚58 ʹ3ʺ).

Plant m aterial was selected by the random number method [14], from 3 to 5 PM , in dry sunny weather. The collected material was examined immediately fresh (raw), as well as after heat treatment (boiled) or during storage. During heat treatment, samples of plant m aterial were covered with boiling water 1:10, placed in a boiling water bath for 20 min, removed and dried with filter paper after cooling to room temperature. The collected plant parts were stored in a domestic refrigerator FR-415W (Ocean,

Table 1. Plant materials Таблица 1. Растительный материал

Item No.	Plant			Supplier of planting material
	Name, genus, family	Cultivar	Part and coloration
1	Potato (calamus sticks) – Solanum tuberosum L., Solanum L., Solanaceae Juss.	Purple	Dark purple flesh, skin	A.G. Lorch Federal Potato Research Centre
2		Lobella	Red skin	Ovoshchevod Co., Ltd., Artyom city
3	Eggplant – Solanum melongena L., Solanum L., Solanaceae Juss.	Diamond	Purple-black skin
4	Cabbage – Brassica oleracea L. , Brassica L., Brassicaceae Burnett	Red Jewel	Purple-red leaf
5		Mikhnevskaya		Scientific Production Association Gardens
6	Garden carrot – Daucus carota subsp. sativus Hoffm., Daucus L., Apiaceae Lindl.	Purple Haze	Purple root	of Russia, Chelyabinsk city
7	Common raspberry – Rubus idaeus L., Rubus L, Rosaceae Juss.	Monomakh Cap	Red berry
8	Onions – Allium cepa L., Allium L., Amaryllidaceae J.St.-Hlt	Carmen	Purple bulb
9	Blue honeysuckle – Lonicera caerulea L., Lonicera L., Caprifoliaceae Juss.	Dlinnoplodnaya	Purple-blue berry	Omskiy Sadovod Co., Ltd., Omsk city

Russia) at (4 ± 2) ˚C for 1.5–3 months. Every 15 days, the anthocyanin content was recorded, which was expressed as a percentage of the initial value taken as 100%.

To prepare all extracts, we ground a sample of plant material to 1.5 to 2 mm, placed a sample of about 0.5 g (precise sample weight) in a dark glass vial, added 5 ml of 95% ethanol (Constanta Farm Co., Ltd., Russia) acidified with hydrochloric acid (NevaReactiv, Russia) in the 100:1 ratio (pH = 1–1.2), kept it for 5–6 hours and filtered through a 0.45 μm PTFE-H membrane filter (Hyundai micro, South Korea).

The extracts’ absorption spectra (AS) were recorded on a U V-2501PC digital spectrophotometer (Shimadzu, Japan) in the wavelength range of 200–650 nm in 1 nm increments and were adjusted to 1% extract. The given AS were processed according to the previously described method [14], and the coordinates of the maximums and inflection points of the spectral line contours were determined. The integral absorption intensity, numerically equal to the area under the spectral line, was calculated by the Simpson's formula [15], the wavelengths of the spectral line contour inflection points in the vicinity of the “anthocyanin” m aximum were taken as the integration limits. For example, for the potato skin extract, the integral absorption intensity corresponds to the area S (Figure 1, oblique shading) within the X b , X_c inflection points b and c wavelengths.

The stability factor ( SF ) was calculated using the original formula:

SF = [AM2xAM1]/[S2xSi], where AM1, AM2 are absorptions of “anthocyanin” maxima, S1, S2 are areas under the spectral line, index 1 refers to raw samples, and index 2 to cooked samples.

The total amount of anthocyanins ( TA ) in 100 g of the sample was determined spectrophotometrically, calculated by the formula:

TA= 100x[AMxKxV1x(V2+V3)]/[V2xM], where AM is the absorption of the “anthocyanin” maximum in the spectrum reduced to 1% extract, rel. un.; K is the conversion factor of 0.004, mg/ml·unit; V1 is the volume of extractant used to prepare the extract, ml; V2 is the extract volume taken into the cuvette, ml; V3 is the extractant volume added to the cuvette, ml; M is the sample weight, g.

Five independent randomized samples were taken in the study of each part of the plant. The experimental material was statistically processed using the small sample and correlation analysis methods [16], the differences were considered statistically significant or reliable at the null hypothesis significance level p < 0.05.

RESULTS AND DISCUSSION