67 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 67–71, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.9 Drahomíra Sopková*, Radoslava Vlčková, Zuzana Andrejčáková, Soňa Gancarčíková Department of Anatomy, Histology and Physiology, Institute of Physiology, University of Veterinary Medicine and Pharmacy, Košice, Slovak Republic, *drahomira.sopkova@uvlf.sk Diet supplementation with flaxseed stimulates gut metabolism in mice Introduction �e close relationship between intestinal microbiota and health and diseases has aroused a great interest in the use of probiotics and prebiotics in the nutrition of humans and animals. �e main reason is the modulation of digestive processes via increased counts of bene�cial bacteria and their enzymatic activity (Axling et al., 2012; Borovská et al., 2013). One of the possibilities of increasing the e�ect of bene- �cial bacteria is combining them with polyunsaturated fatty acids (PUFAs) that are an integral component of cellular membranes and are able to modify the adherence of bacteria to intestinal epithelium (Borovská et al., 2013). Under our conditions, one of the richest plant sources of ω-3 PUFAs is the �axseed (Linum usitatissimum L.). Of total fatty acids (FA) found in �axseed oil, 9% are saturated, 18% monoun- saturated, and 73% polyunsaturated FA containing 50−61% of ω-3 PUFA α-linolen- ic acid. At the same time, �axseed is a rich source of �bres and the soluble (slimy) �brous component is considered functional (Borovská et al., 2013; Martinez et al., 2013). In the digestive tract, it acts as a prebiotic and creates a suitable environment for bacteria bene�cial to health. �e aim of the study was to investigate the e�ects of the forti�cation of the diet with �axseed in a model experiment on mice with a focus on intestinal metabolism. Material and methods Plant material �e experiment was approved by the State Veterinary and Food Administration of the Slovak Republic (No 1177/14-22) and carried out on 24 mice of BALB/c line, 5 weeks old, allocated to two groups: control group (K-; n = 12) fed a standard diet for mice D ra ho m íra S op ko vá , R ad os la va V lč ko vá , Z uz an a A nd re jč ák ov á, S oň a G an ca rč ík ov á 68 ST–1 (Altromin 1311; Velaz; Czechia) at a dose of 1.75 g/head/day; and the experi- mental group (MK; n = 12) fed diet supplemented with crushed �axseed variety Flan- ders for the period of 35 days, at 5% concentration in feed. Faeces of animals were collected and examined during the experiment and before euthanasia. �e animals were killed by cervical dislocation and caecum samples were collected for the deter- mination of the concentration of organic acids using the method of capillary isotac- hophoresis (ITP). Statistical analysis �e results obtained were evaluated statistically using so�ware GraphPad Prism 3.0 for Windows (GraphPad So�ware, San Diego, California, USA). �e dynamics of changes in individual parameters were evaluated by Repeated Measures ANOVA and Tukey post hoc tests and the di�erences between the groups by means of t-tests. �e signi�cance level was set to p < 0.05. Fig. 1. Level of organic acids in the caecum and faeces of groups K and MK; the level of organic acids in mice caecum a�er 35 days of supplementation of �axseed and the dynamics of concentration of aceto- acetic, lactic, succinic, acetic, propionic, butyric, and valeric acids in mice faeces; K – group of mice fed exclusively the standard diet; MK – group of mice fed standard diet supplemented with crushed �axseed (5% added to the feed); mean values (Mean ± SEM) are presented in mmol.l-1; a,b,c,x,y,z di�erences between columns with the same superscript are signi�cant (a,x = p < 0.05; b,y = p < 0.01; c,z = p < 0.001); the su- perscripts a,b,c indicate intergroup di�erences while superscripts x,y,z indicate di�erences between groups D iet supplem entation w ith flaxseed stim ulates gut m etabolism in m ice 69 Results Supplementation of diet with �axseed (FAs) caused a signi�cant increase in the level of organic acids in mice caecum (acetic acid, p < 0.01; propionic, butyric and valeric acids p < 0.001) in comparison with group K (Fig. 1a). Examination of faeces of �axseed-fed mice showed increased concentrations of lactic acid on days 7 and 28 (p < 0.01), acetic acid on days 14 (p < 0.05), 21 and 28 (p < 0.01) and butyric acid on days 14 (p < 0.001), 21 and 28 (p < 0.01) of supplementation in comparison with control group K (Fig. 1b-d). �e most pronounced e�ect of the supplementation of �axseed on the level of all investigated organic acids was observed on day 28 of the feeding experiment (lactic, acetic, and butyric acids p < 0.01). On day 35 of the experiment, we observed a pro- nounced decrease in the concentration of acetic (p < 0.001), lactic (p < 0.01), and butyric (p < 0.05) acids in the forti�ed group. Discussion A positive in�uence on microbiocenosis of the intestinal tract and thus also on the resistance of the macro-organism has been ascribed inter alia also to the prebiotic component of the additive, the �axseed (Smith et al., 2006; Hekmatdoost et al., 2008). Polyunsaturated fatty acids found in �axseed are capable of a�ecting the adherence of bacteria by the modi�cation of the lipid composition of the intestinal wall or the bacterial cell wall. For example, (Yu et al., 2014) reported the stimulation of growth and the adherence of lactobacilli in the digestive tract of mice and (Nemcová et al., 2012) found similar results in pigs. �e ω-3 fatty acids reduce intestinal permeability by bacteria and the number of apoptotic cells in ileal mucosa (Generoso et al., 2015). �e slimy �brous material in �axseed can serve as a speci�c growth substrate for ben- e�cial bacteria that slow down the production or absorption of toxic products of me- tabolism, and additionally, by their action, the �brous material decomposes to volatile FA that ful�l important protective role against infections and maintain the integrity of intestinal mucosa (Smith et al., 2006; Wong, Jenkins, 2007). One of the mechanisms of the inhibition of pathogenic microorganisms by bene�- cial micro�ora involves the production of antibacterial substances, such as organic ac- ids. �e concentrations of these acids were signi�cantly increased in the caecum (ace- toacetic, acetic, lactic, propionic, and butyric) and faeces (lactic, acetic, butyric) of ex- perimental animals in our study. �e bene�cial bacteria are able to reduce the produc- tion of harmful nitrogenous compounds that cause damage to intestinal mucosa; and, in this way, they exert a positive e�ect on intestinal function, improve digestive pro- cesses, and subsequently increase the weight gain of animals (Serban, 2014). Adhesion D ra ho m íra S op ko vá , R ad os la va V lč ko vá , Z uz an a A nd re jč ák ov á, S oň a G an ca rč ík ov á 70 of probiotics to intestinal mucosa is important for the promotion of health and the ac- tion of bacteria bene�cial to intestinal health can be positively a�ected by ω-3 PUFAs. �e 35-day forti�cation of mice diet with �axseed stimulated intestinal metabo- lism and fermentation activity of bene�cial autochtonous intestinal bacteria in mice caecum, which indicates its prospective use for pronounced improvement and protec- tion of animal health. References Axling, U., Olsson, C., Xu, J., Fernandez, C., Larsson, S., Ström, Ahrné, S., Holm, C., Molin, G., Berger, K. (2012). Green tea powder and Lactobacillus plantarum a�ect gut microbiota, lipid metabolism and in�am- mation in high-fat fed C57BL/6J mice. Nutrition & Metabolism, 9, 105. DOI: 10.1186/1743-7075-9-105 Borovská, D., Nemcová, R., Gancarčíková, S., Koščová, J. (2013). �e synbiotic e�ect of Lactobacilli and �axseed on selected intestinal micro�ora and organic acid levels in weaned piglets. Microbiology, 2(10), 82–86. Generoso de Vasconcelos, S., Rodrigues, N.M., Trindale, L.M., Paiva, N.C., Cardoso, V.N., Carneiro, C.M., Ferreira, De Matos, A.D., Faria, A.M.C., Maioli, T.U. (2015). Dietary supplementation with omega-3 fatty acid attenuates 5-�uorouracil induced mucositis in mice. Lipids in Health and Disease, 14(54), 1–10. DOI: 10.1186/s12944-015-0052-z Hekmatdoost, A., Feizabadi, M.M., Djazayery, A., Mirsha�ey, A., Eshraghian, M.R., Yeganeh, S.M., Se- daghat, R., Jacobson, K. (2008). �e e�ect of dietary oils on caecal micro�ora in experimental colitis in mice. Indian Journal of Gastroenterology, 27, 186–189 Kiarie, E., Nyachoti, C.M., Slominski, B.A., Blank, G. (2007). Growth performance, gastrointestinal mi- crobial activity and nutrient digestibility in early-weaned pigs fed diets containing �axseed and car- bohydrase enzyme. Journal of Animal Science, 85, 2982–2993. DOI: 10.2527/jas.2006-481 Martinez, F.A.C., Balciunas, E.M., Converti, A., Cotter, P.D., Oliveira, R.P.S. (2013). Bacteriocin pro- duction by Bi�dobacterium spp.: A review. Biotechnology Advances, 31, 482–488. DOI: 10.1016/j. biotechadv. 2013.01.010 Nemcová, R., Borovská, D., Koščová, J., Gancarčíková, S., Mudroňová, D., Buleca, V., Pistl, J. (2012). �e e�ect of supplementation of �ax-seed oil on interaction of Lactobacillus plantarum – Biocenol™ LP96 and Escherichia coli O8:K88ab:H9 in the gut of germ-free piglets. Research in Veterinary Science, 93, 39–41. DOI: 10.1016/j.rvsc.2011.07.031 Serban, D.E. (2014). Gastro-intestinal cancers: in�uence of gut microbiota, probiotics and prebiotics. Cancer Letters, 345(2), 258–270. DOI: 10.1016/j.canlet.2013.08.013 Wong, J.M., Jenkins, D.J. (2007). Carbohydrate digestibility and metabolic e�ects. Journal of Nutrition, 137, 2539–2546. DOI: 10.1093/jn/137.11.2539S Yu, H-N., Zhu, J., Wen-Sheng, Pan, W-S., Shen, S-R., Shan, W-G., Das, U.N. (2014). E�ects of �sh oil with a high content of n-3 polyunsaturated fatty acids on mouse gut microbiota. Archives of Medicinal Research, 45(3), 195–202. DOI: 10.1016/j.arcmed.2014.03.008 Acknowledgement �e study was supported by the project VEGA No. 1/0476/16. Abstract Essential polyunsaturated fatty acids (PUFAs) in the feed may a�ect the gastrointestinal microbiota. �e present study investigated the e�ect of 35-day supplementation of mice diet with 5% concentration of high-ω-3 PUFAs in �axseed with a focus on the intestinal metabolism of mice. �e capillary isotachopho- D iet supplem entation w ith flaxseed stim ulates gut m etabolism in m ice 71 resis method was used for the assessment of the level of organic acids in the gut material and faeces. Sup- plementation of �axseed increased the level of organic acids in the caecum (acetic, propionic, butyric, and valeric acids) and faeces (lactic, acetic, butyric acids). �e most signi�cant e�ect was observed on day 28 of �axseed supplementation. �e investigated additive had a stimulatory e�ect on the intestinal metabolism and fermentation activity of bene�cial bacteria. Key words: �ax seed, intestine, mice, microbiota, organic acids Received: [2018.05.30] Accepted: [2018.12.11] Suplementacja diety siemieniem lnianym stymuluje metabolizm jelit u myszy Streszczenie Niezbędne w  pożywieniu długołańcuchowe wielonienasycone kwasy tłuszczowe (ang. PUFAs) wpływają na mikro�orę jelitową. Celem niniejszej pracy było zbadanie wpływu 35-cio dniowej suplementacji diety siemieniem lnianym o 5% stężeniu ω-3 PUFAs na metabolizm jelitowy myszy. Przy użyciu techniki izota- choforezy kapilarnej (ang. CITP) oznaczono stężenie kwasów organicznych w jelicie i kale. Suplementacja siemieniem lnianym powodowała wzrost poziomu kwasów organicznych w  kątnicy (kwas octowy, kwas propionowy, kwas masłowy, kwas walerianowy) i kale (kwas mlekowy, kwas octowy, kwas masłowy). Naj- większy istotny wpływ obserwowano w 28 dniu suplementacji siemieniem lnianym. Badany dodatek sie- mienia miał stymulujące działanie na metabolizm jelitowy i aktywność procesów fermentacyjnych prowa- dzonych przez pożyteczne bakterie jelitowe. Słowa kluczowe: siemię lniane, jelita, myszy, mikro�ora, kwasy Information on the authors Drahomíra Sopková Her scienti�c �eld is aimed at lipid metabolism. Radoslava Vlčková Her scienti�c �eld is focused on animal reproduction. Soňa Gancarčíková Her research is focused on the metabolism of gut environment. Zuzana Andrejčáková Her scienti�c �eld is aimed at animal physiology and biochemistry.