DEVELOPMENT OF SYNBIOTICS BASED ON ARCTIC PLANT RAW MATERIALS

Popov Vladimir G.; Aksentyeva Victoria V.; Belina Svetlana A.

УДК 663.051.2

DOI 10.29141/2500-1922-2023-8-2-2 EDN CHCLBA

Development of Synbiotics Based on Arctic Plant Raw Materials

Vladimir G. Popov1 H, Victoria V. Aksentyeva1, Svetlana A. Belina1

1Tyumen Industrial University, Tyumen, Russian Federation

Abstract

The strategic task of the modern food industry is the development of functional food products targeted at the chronic diseases reduction, the life expectancy growth in citizens of the Russian Federation, as well as the replacement of imported food substances widely represented on the Russian market. The research aim is developing a synbiotic that consists of polyphenolic compounds of Arctic phyto raw materials obtained by cultivating callus cells in sterile laboratory conditions, as well as industrially produced probiotic microorganisms. The authors revealed that the glycoside content in the callus tissue was lower than in the plant, the benzoic acid content was also reduced. However, there is an increase in the fla-vonoids amount from 12.6 % to 16.5 % in the callus mass. According to the research results, a man detected the largest biomass growth volumes after 35 days. The complex compound contained a significant amount of fiber up to 18.2 mg/100 g, polyphenolic compounds - up to 515.5 mg/100 g. The resulting product was a light brown powder with a moisture content of 5.8-7.0 %, sent for the subsequent microencapsulation process as a core.

For citation: Vladimir G. Popov, Victoria V. Aksentyeva, Svetlana A. Belina. Development of Synbiotics Based on Arctic Plant Raw Materials. Индустрия питания|Food Industry. 2023. Vol. 8, No. 2. Pp. 13-20. DOI: 10.29141/2500-1922-2023-8-2-2. EDN: CHCLBA.

Paper submitted: March 28, 2023

Проектирование синбиотика на основе арктического растительного сырья

В.Г. Попов1 В.В. Аксентьева1, С.А. Белина1

1Тюменский индустриальный университет, г. Тюмень, Российская Федерация Н [email protected]

Реферат

Стратегической задачей современной пищевой промышленности является разработка функциональных пищевых продуктов, предназначенных для снижения рисков хронических заболеваний, увеличения продолжительности жизни граждан России, а также замена пищевых субстанций импортного производства, широко представленных на российском рынке. Основной целью исследования является проектирование синбиотика, состоящего из полифенольных соединений арктического фи-тосырья, полученного методом культивирования каллусных клеток в стерильных лабораторных условиях, и пробиотических микроорганизмов, выпускаемых промышленностью. Выявлено, что содержание гликозидов в каллусной ткани ниже, чем в растении, содержание бензойной кислоты также понижено, однако наблюдается увеличение содержания флавоноидов от 12,6 до 16,5 % в каллусной массе. По результатам исследований установлено, что наибольший прирост биомассы отмечен через 35 сут. Комплексное соединение содержало в значительном количестве клетчатку -

Н [email protected]

Keywords:

synbiotic;

complex nutritional

supplement;

prebiotic;

probiotic;

dysbacteriosis;

confectionery

Ключевые слова:

синбиотик;

комплексная

пищевая добавка;

пребиотик;

пробиотик;

дисбактериоз;

арктическое сырье

до 18,2 мг/100 г, полифенольные соединения - до 515,5 мг/100 г. Полученная продукция, представляющая собой светло-коричневый порошок с влажностью 5,8-7,0 %, направлялась для последующей микроинкапсуляции в качестве ядра.

Для цитирования: Vladimir G. Popov, Victoria V. Aksentyeva, Svetlana A. Belina. Development of Synbiotics Based on Arctic Plant Raw Materials //Индустрия питания|Food Industry. 2023. Т. 8, № 2. С. 13-20. DOI: 10.2914l/2500-1922-2023-8-2-2. EDN: CHCLBA.

Дата поступления статьи: 28 марта 2023 г.

Introduction

The almost complete absence of food ingredients and substances with experimentally proven physiological properties, evaluation methods and effectiveness confirmation defer the functional food products development in the Russian Federation1.

Professor Yuriy Nikolaev with a group of scientists revealed that wild leaves of cranberries, plantain and hypericum were rich in flavonoids and had an antioxidant effect due to the phenolic compounds content. However, the useful raw materials properties were not equal to the pharmacologically active components sum. The raw materials value increased with the presence of substances forming biological complexes that acted as synergetics [1; 2].

The prebiotics are known to be carbohydrates not fermented in the upper gastrointestinal tract, while being a nutrient source for the normal intestinal microflora. In the high arctic conditions, the intestinal absorption of carbohydrates from plant foods seems to be difficult. Thus, the "Arctic nutrition" improvement aims at providing a body with fiber and vitamins in the necessary volumes [1].

In the studies, a man has obtained complex preb-iotics with the "nutrient medium" function for beneficial intestinal bacteria types, which have an increased preventive effect. E. Distrutti and other Italian scientists revealed that when taking a preb-iotic mixture consisting of galacto-oligosaccharides daily for four weeks, anxiety through the patients with the irritable bowel syndrome suffered from severe anxiety decreased, which influenced the life quality positively [3].

Probiotics are beneficial microorganisms with immunostimulating activity contributing to the protection against gastrointestinal infections [4].

There is no general consensus about the probi-otics use benefits for the gastrointestinal disease treatment among specialists and nutritionists yet, despite numerous studies with positive effects noted.

1 Strategy for the Arctic Zone Development of the Russian Federation and Ensuring National Security for the Period up to 2035. Approved by Decree of the President of the Russian Federation of October 26, 2020. No. 465.

For example, in type 2 diabetes, the eight weeks probiotic use prevents a rise in FPG, reduces the hs-CRP level in the serum and increases the total GSH in plasma [5].

In diarrhea, live probiotics of high concentration (1010 CFU/ml) inhibit the colon muscles contraction [6].

In rheumatoid arthritis, probiotic supplements intake for eight weeks reduces the disease activity, the insulin level and its release [7].

Beneficial bacteria produce short-chain fatty acids, which strengthen the connections between intestinal cells preventing the toxic compounds hit into the bloodstream [8]. When the normal microbial flora number decreases, the mucin (intestinal mucus) composition changes. Therefore, the following symptoms reveals: protective barrier loss, so-called "leaky gut syndrome" development - toxins from the digestive system enter the bloodstream triggering systemic inflammation [9]. Probiotics and prebiotics prevent the colitis recurrence according to Chinese scientists [10].

Specialists correlate antioxidant protection and microecological balance violations in the intestinal biocenosium encouraging the dysbiosis development [11].

Wild cranberry leaves are full of arbutin phenolic glycoside, as well as ursolic acid and phytoncides. Leaf phytoncides inhibit the Staphylococcus aureus growth, have a demineralizing effect, increase the antibiotics effectiveness, stimulate phagocytosis and other body defenses [12].

The flavonoid composition of the leaves of V. vi-tis-idaeae L. includes: kaempferol, quercitrin, rutin, luteolin with high antioxidant activity triggered by the 3',4'-dihydroxyphenyl group of the B ring [13]. The flavonoid levels in different V. vitis-idaea L. parts are the following: leaves of the current year (28.0 mg/g) > perennial leaves (24.1 mg/g) > shoots (19.2 mg/g) > roots (12.0 mg/g). The increased content of flavonoids in young V. vitis-idaea parts is due to the biosynthesis peculiarities (more intensive metabolism in young tissues).

The chemical composition of V. vitis-idaea L. changes significantly with increasing leaf age. The

concentrations of cellulose, lignin and stoichiometric parameters (lignin/cellulose, lignin/N) decrease, while the phenolic compounds and condensed tannins content increases significantly. A man determined the content of lignin and cellulose by treating the sample with 72 % sulfuric acid, after preliminary boiling in the cetyl trimethyl ammonium bromide solution (10 g of cetyltrimethylammonium bromide in 1 liter of 0.5 M H2SO4) [14].

Wild plantain leaves (Plantago major L.) contain, g/100 g: proteins - 4.0, carbohydrates - 15.2, dietary fiber - 2.1, phenolic glycoside aucubin, flavo-noids (baikalin, scutelarin), polysaccharides, tannins, mucus, carotenoids (xanthophyll), silicic, oleic, citric and ascorbic acids, vitamin K [15].

Wild St. John's wort (Hypericiherba) leaves contain flavonoids (1.5-5 %), tannins (10-13 %), essential oil (0.1-0.33 %). Flavonoids consist of such compounds as hyperoside 1.2 %, rutin, quercitrin, isoquercitrin, quercetin, anthocyanins (5-6 %) [16].

A dry squeeze of pumpkin fruit contains no more than 6.0% water. The dry residual consists of 4.615 % sugars, 24.5 % fiber, 3.54 % pectins, 0.18 % crude fat, 0.56 % acids, 0.52 % ash, 1.41 mg per 100 g of ascorbic acid, 68.3 mg per 100 g of retinol [17].

The callus culture obtained on Anderson Medium is a product with a high content of vitamins and gly-cosides capable of adsorbing water and swelling, causing a laxative action [18].

The research aims at developing a complex food additive in the form of a synbiotic based on Arctic plant raw materials obtained from a culture of callus cells under sterile conditions; containing a significant number of polyphenolic compounds, oligo-and polysaccharides, as well as fiber; performing prebiotic [19] and probiotic functions; representing living non-pathogenic microorganisms capable to restore the intestinal mucosa.

Research Objects and Methods

The research objects are the cell cultures of the leaves of cranberry (vulgaris Vaccinium vitis-idaea L.) of the variety Kostromichka, plantain (Plantago major), hypericum (Hypericum perforatum L.) collected in the Arctic territory of the Yamalo-Nenets Autonomous District and dried pumpkin pulp produced by the company LLC "Yagody Sibiry" (Tyumen).

A man determined the glycosides percentage, the polyphenolic compounds number, the amount of benzoic acid and vitamins B9 by highly effective liquid chromatography; the water-soluble tannins content - by the titrimetric method; the amount of lycopene - by the spectrophotometric method; the content of fiber, carotene - by the content of crude fiber according to the method of Henneberg and Shtoman; the percentage flavonoids -

by the photocolorimetric method, the content of lignin - by the Folin-Denis colorimetric method; the amount of vitamin C - according to the GOST 24556-89;vitamin PP (NE) - according to the GOST R 50479-9; vitamin B2 - by the Beit-Smith method.

The main stage of the research was the tissues and cells cultivation in vitro in the culture room obtained from the cell culture of young Arctic plants leaves collected in May aimed at the complex food additives production use. Explants were sterile plant leaves, pre-soaked in 75 % ethanol for 2 minutes under the laminar box conditions, then in 15 % hydrogen peroxide for 3 minutes, followed by washing in sterile distilled water [20]. To obtain biomass the authors used the surface cultivation method on a semi-solid agarized medium for 5 weeks. After receiving BAS, they subjected it to drying by the spray drying method for 25 seconds. After the drying and grinding process, a man obtained powdery dry substances with a moisture content of no more than 6.0 % and a size of no more than 0.4 microns. The researchers combined concentrates acquired from the culture of cranberry, plantain, hypericum leaf cells, as well as pumpkin seeds presented in powdered form in a biochemical reactor of the Unik-150-2 brand at a temperature of 65 °C in a ratio of 40:10:30:15, using rapeseed lecithin in the amount of 5.0% of the total mass as an emulsifier to obtain a complex compound. Complied physical and chemical BAS cultivation conditions enable to achieve the maximum secondary metabolites content.

A man run the polyphenolic compounds concentration in confectionery samples on a high-performance liquid chromatograph GILSTON, model 305, followed by computer processing of the research results. The quercetin content was (2.15 ± 0.21) %, dehydroquercetin - (2.75 ± 0.21) %. Using the photocolorimetric method at a wavelength of 650 nm enabled to determine the flavonoids quantity in the samples. The authors calculated the flavonoids and phenolic compounds quantity according to calibration graphs based on quercetin. The flavonoids sum content in the finished marmalade filling was (750.5 ± 8.50) mg per 100 g.

Cupcakes cooking was according to the GOST 15052-2014; while filling - according to the GOST 32741-2014.

Research Results and Its Discussion

After purification of the target product from a complex mixture, a man run comparative studies to determine the chemical composition, nutritional value and content of secondary metabolites in callus cultures and wild native leaves.

The phenols concentration for perennial leaves and leaves of the current year, collected in the Arc-

tic territory of the Yamalo-Nenets Autonomous District and in the south of the Tyumen Region, differs slightly and averages 32.5 mg/g and 28.2 mg/g. The young shoots of Arctic raw materials contain 38.1.1 mg/g phenols, and the roots - 26.5 mg/g.

Table 1 demonstrates the results of the chemical composition studies of native wild-growing Arctic raw materials and callus masses obtained by synthesis from cell cultures.

As shown in Table 1, the glycosides content in callus tissue is lower than in plants, as well as the ben-zoic acid content. However, there is an increase in the flavonoids content from 12.6 % to 16.5 % in the callus mass. The research results enable to confirm the effectiveness of using the culturing callus cell method in order to obtain callus masses containing a significant amount of biologically active substances, primarily polyphenolic compounds. The technology can be a potential source of raw materials for the biologically active substances production.

The temperature and pH of the medium influence the biomass growth. The optimal pH level is (5.7 ± 0.3), the temperature is within 25-26 °C.

Table 2 presents the vitamin content in the natural plant and in the callus masses.

The table shows that the content of vitamin C, carotene, and B vitamins in the callus is higher than in the native plant.

The complex compound formation provides a more complete minerals and secondary metabolites from plant matter absorption in the human body, compared with a mixture of similar concentrates [23]. The Figure 1 demonstrates an increase in callus cell biomass during 35 days.

According to the research results, the greatest increase in biomass was between 30 and 40 days. The Figure shows the measurement control points results. The largest volumes of biomass growth were detected after 35 days. The complex compound contained a significant amount of fiber up to

Table 1. Chemical Composition of Native Wild-Growing Arctic Raw Materials and Callus Messes Obtained

by Synthesis from Cell Cultures Таблица 1. Химический состав нативного дикорастущего арктического сырья и каллусныхмасс,

полученных синтезом из культур клеток

Content in 100 g of Raw Materials

Indicators Cranberry Leaves Plantain Leaves Hypericum Leaves

Natural Callus Natural Callus Natural Callus

Glycosides, % 9.0 i 0.1 8.40 i 0.08 12.20 i 0.09 7.50 i 0.08 7.2 i 0.1 8.б0 i 0.08

Water-Soluble Tanins, % 5.10 i 0.01 7.90 i 0.02 4.3 i 0.1 б.30 i 0.02 9.80 i 0.09 11.70 i 0.01

Benzoic Acid, mg 95.5 i 0.1 75.2 i 1.2 б4.1 i 1.5 58.7 i 0.9 72.5 i 1.0 б0.1 i 0.1

Lycopene, mg 1.50 i 0.08 2.1 i 0.1 0.5 i 1.0 0.7 i 0.5 1.20 i 0.01 0.80 i 0.08

Fiber, mg 1.б0 i 0.01 1.8 i 0.1 7.40 i 0.01 8.20 i 0.01 1.20 i 0.01 0.90 i 0.01

Flavonoids, % 12.3 i 1.5 15.5 i 1.б 5.7 i 0.1 7.2 i 0.9 14.8 i 0.9 17.2 i 1.3

Polyphenolic Compounds, mg 1б2.5 i 0.1 184.10 i 0.09 215.50 i 0.01 243.1 i 0.5 480.40 i 0.09 415.7 i 0.1

Lignin, % 3.20 i 0.05 2.50 i 0.05 1.70 i 0.02 1.50 i 0.01 2.70 i 0.05 2.10 i 0.02

Table 2. Vitamins and Minerals Content in Native Plants and Callus Masses Таблица 2. Содержание витаминов и минеральных веществ в нативных растениях и каллусных массах

Content in 100 g of Raw Materials

Indicators Cranberry Leaves Plantain Leaves Hypericum Leaves

Natural Callus Natural I Callus Natural Callus

Vitamin C, mg 170.0 i 0.1 205.2 i 0.1 85.0 i 0.1 98.2 i 0.1 150.0 i 0.1 125.0 i 0.1

Vitamin PP, (Niacin Equivalent) mg 8.5 i 0.8 7.2 i 0.8 б.3 i 0.б 8.2 i 0.б 4.2 i 0.9 5.1 i 0.б

Carotene, mg 5.2 i 0.4 7.3 i 0.4 4.3 i 0.5 б.1 i 0.5 2.7 i 0.9 3.4 i 0.9

Vitamin B2, mg 0.б i 0.1 0.9 i 0.1 0.20 i 0.02 0.5 i 0.2 0.3 i 0.1 0.3 i 0.1

Vitamin B9, mg 0.05 i 0.01 0.09 i 0.01 0.02 i 0.01 0.03 i 0.1 0.07 i 0.02 0.04 i 0.01

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- Cranberry Cell Cultures

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

- Plantain Cell Cultures

Hypericum Cell Cultures

14

21

28

35

42

Cultivation Period, Days

Increase in the Callus Cells Biomass during Cultivation for 42 Days Прирост биомассы каллусных клеток в процессе культивирования в течение 42 сут

18.2 mg per 100 g, polyphenolic compounds - up to 515.5 mg per 100 g and their secondary metabolites (resveratrol, hesperidin, anthocyanins, phenolic acids). The fiber and polyphenolic compounds content in a complex food additive is an essential component for the food products enrichment used in the diets of the population living and working in the Arctic territories.

The research results enable to use the studied plant raw materials for growing callus crops, in order to obtain sterile plant concentrates suitable for developing a natural prebiotic capable of distributing active substances to the lower gastrointestinal tract, for the most effective absorption of the active plants substances by the mucous membrane cells.

At the next stage, a man enriched the resulting mixture with dry probiotic of industrial manufacture, containing the bacteria Lactobacillus acidophilus and Streptococcus salivarius, the most thermola-bile, withstanding a maximum temperature of up to +75 °C. After intensive mixing, the researchers sprayed the enriched mixture dried at an incoming air temperature of 120 °C for 15 seconds, then cooled to a temperature of +25 °C and crushed to a size of 10-9 microns. The resulting product was a light brown powder with a moisture content of 5.8-7.0 %, sent for the microencapsulation process as a core.

Capsule shells were as following: modified potato starch, lecithin from Siberian rapeseed and malto-dextrin in a ratio of 7:2:1. A man obtained microcapsules of 10-6 microns in size with a ratio of shell:core as 40:60 in a laboratory installation by spraying in a fluidized bed; stored the resulting microcapsules in a closed vessel at a temperature of -18 °C.

The main microencapsulation purpose is the ability to protect the active substances enclosed in capsules from the destructive effects of the stomach, to deliver this substance to the lower gastrointestinal tract, where it is most effectively absorbed by

the mucous membrane cells ensuring stable release of the active substance.

A man intends the resulting complex food additive in the form of a synbiotic to be include in the formulation of flour confectionery for the functional properties purpose in order to implement preventive gastrointestinal dysbiosis measures.

The conducted experimental studies showed weak thermal lability of probiotics, a component of synbiotic, when included in the confectionery sample formulation during dough kneading. It demonstrated a reduction till 60-65 % under the following conditions: temperature 120 °C, duration 15 minutes.

Research on the survival rate increase of probiotic microorganisms contained in synbiotics during thermal treatment is continuing to rise the preventive effectiveness of the functional product. The developed synbiotic should be an additive in the marmalade filling used for the confectionery production after thermal exposure. Berry marmalade filling has the least negative effect on the gastrointestinal tract diseases, unlike chocolate or milk filling. As the filling is not a subject of heat treatment in the confectionery production, so it is advisable to insert the synbiotic into it. When the flour semifinished product reached 30 °C, a man stuffed it with marmalade filling. The filling contains cranberry puree, sugar 2:1 and agar-agar 100:1 in relation to the puree, enriched with synbiotic in an amount of 12 per 100 g.

Conclusion

The conducted studies enable to develop a syn-biotic for the flour confectionery products enrichment, consisting of polyphenolic compounds of Arctic phytochemicals obtained from cell cultures, as well as probiotics containing the bacteria Lacto-bacillus acidophilus and Streptococcus salivarius. The research results demonstrate the effectiveness of obtaining callus masses in terms of nutritional val-

ue and biologically active substances, showing the feasibility of year-round scarce plant ingredients production, and developing complex food mixtures with probiotics.

The practical significance of developed synbiotics is due to the prevention of gastrointestinal dysbiosis among the adult population, primarily working on a

shift basis in the Arctic territories. Physiological efficiency is due to the formation of synergy obtained by increasing the beneficial bacteria reproduction rate by 2 times, according to the probiotics ability to be fixed in the intestine among with prebiotics. These increase the probiotics delivery speed to the destination due to its transition on prebiotics [21].

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Библиографический список

1. Николаев Ю.А., Пальцев А.И., Кузнецова Т.В. Особенности клинического течения заболеваний органов пищеварения у пришлого населения на Севере // Бюллетень Сибирского отделения Российской академии медицинских наук. 2006. Т. 26, № 3. С. 122-126. EDN: HVGAWD.