PaPer 424 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: antioxidant capacity, bioactive compounds, buckwheat groats, Rhizopus oligosporus, solid-state fermentation - EffEct of solid-statE fErmEntation with Rhizopus oligospoRus on bioactivE compounds and antioxidant capacity of raw and roastEd buckwhEat groats wronkowska małgorzata*, honkE Joanna and piskuła mariusz konrad Department of Chemistry and Biodynamics of Food, Division of Food Science, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland *Corresponding author: m.wronkowska@pan.olsztyn.pl AbstrAct the effect of solid-state fermentation with Rhizopus oligosporus on the changes in the total phe- nolic compounds, rutin, vitamin b and c, tocopherol, phytic acid and antioxidant capacity of raw and roasted buckwheat groats was studied. the roasted groats contained reduced level of stud- ied bioactive compounds as compared to raw groats. In this study was evidenced that the solid- state fermentation with Rhizopus oligosporus enhanced water soluble vitamins (thiamine, pyri- doxine and L-ascorbic acid) as well as tocopherols contents. In contrast the decrease of the inosi- tol hexaphosphate, phenolic compounds, the rutin content and antioxidant capacity determined by AcL and Abts methods was noticed. mailto:m.wronkowska%40pan.olsztyn.pl?subject= Ital. J. Food Sci., vol. 27 - 2015 425 IntroductIon Globally, there is a growing interest in buck- wheat products as healthy foods. the major pro- ducers of buckwheat are china, russia, ukraine and Kazakhstan, but it is also cultivated in slo- venia, Poland, Hungary, brazil and Austria (bon- AFAccIA et al., 2003). rutin (quercetin-3-ruti- noside) is the main buckwheat flavonoid, which poses antioxidant, anti-inflammatory and anti- carcinogenic properties. buckwheat is also rich in other antioxidant compounds such as phe- nolic acids, tocopherols, reduced glutathione, inositol phosphates and melatonin (WIJnGAArd and ArEndt, 2006). this pseudocereal is char- acterised also in a high content of thiamine (vi- tamin b1) and riboflavin (vitamin b2), proteins with a well balance amino acid composition, in- cluding a high lysine content, phytosterols, sol- uble carbohydrates, d-chiro-inositol, fagopyri- tols and thiamin-binding proteins. Its fatty acid composition is superior to that of cereal grains, with typically 80% unsaturated fatty acids, in- cluding more than 40% of linoleic acid, an es- sential polyunsaturated fatty acid (stEAdMAn et al., 2001). raw and roasted buckwheat groats are par- ticularly popular in central and Eastern Europe. roasted buckwheat groats are usually served like rice after cooking, while raw buckwheat groat or flour is used as a substitute for wheat flour in products for people with allergy to glu- ten and can be a valuable ingredient in diets or food products for coeliac patients (WronKoWsKA et al., 2010). roasting affects the chemical com- position and functional properties of buckwheat groats. the reduction of parent antioxidants as well as the formation of Maillard reaction prod- ucts after roasting was observed (ZIELIŃsKA et al., 2007a). tempeh, or “tempe”, if we use authentic Indo- nesian spelling, a traditional product originat- ing from Indonesia, is usually made from soy- beans. the traditional tempeh process involves soaking and cooking, cooling and dehulling of the beans, followed by 20-30 hours of solid-state fermentation with Rhizopus oligosporus. temp- eh products have high protein contents of 40- 50% and can be serve as tasty protein comple- ments to starchy staples, and can substitute meat or fish (nout and roMbouts, 1990). the solid-state fermentation with Rhizopus oligospo- rus has several beneficial effects, for example en- hances antioxidant properties such as free-rad- ical scavenging activity. HAndoyo et al. (2006) used fermentation with R. oligosporus to produce a fermented buckwheat flour that not only had a higher content of amino acids and minerals than non-fermented buckwheat samples, but also a lower content of allergic proteins. However, the production of tempeh-like products from buck- wheat groats themselves has not been investi- gated. therefore, in order to promote the utiliza- tion of raw or roasted buckwheat groats as well as to create a new type of healthy food, the ef- fects of solid-state fermentation with Rhizopus oligosporus on the changes in the total phenol- ic compounds, rutin, vitamin b and c, tocoph- erol, phytic acid and antioxidant capacity of raw and roasted buckwheat groats was addressed in this study. MAtErIALs And MEtHods Chemicals Acetonitrile and methanol (HPLc-grade) were provided by Merck (darmstad, Germany). ru- tin (quercetin-3-rutinoside), L-ascorbic acid, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfon- ic acid) diammonium salt (Abts), 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), thiamine, riboflavin, pyridoxine, taka- diastase from Aspergillus oryzae (Ec no 232- 588-1) and inositol hexaphosphoric acid (dode- casodium salt) from corn were purchased from sigma (sigma chemical co., st. Louis, Mo, u.s.A.). other reagents of reagent-grade quali- ty were from Poch, Gliwice, Poland. Water was purified with a Milli-Q-system (Milipore, bed- ford, usA). All solutions prepared for HPLc were passed through a 0.45 µm nylon filter before use. Solid-state fermentation (SSF) the raw (b) and roasted common (Fagopyrum esculentum Moench) buckwheat groats (rb) were purchased from a local healthy food shop (olsz- tyn, Poland). the ssF was performed according to the modified method of HAndoyo et al. (2006). briefly, 50 grams of raw or roasted buckwheat groats were cooked with 200 mL of deionized water for 5 min at 100ºc. After cooling to room temperature, the excess of water was discard- ed. the drained samples were then inoculated with Rhizopus oligosporus nrrL 2710 (approx. 104 spores/g, northern regional research Lab- oratory, Peoria, Illinois, usA) and incubated at 37ºc for 24 h. the fermented raw groats (ssF-b) and fermented roasted groats (ssF-rb), as well as unfermented materials, were lyophilized us- ing a Labconco co. (Kansas city, Missouri, usA) laboratory freeze-dryer at a pressure of 13 Pa at -60°c for 24 h. Sample preparation for measurement of rutin, total phenolic compounds and antioxidant capacity the lyophilized buckwheat samples were ex- tracted in triplicate with 80% aqueous metha- nol (1/10; w/v) for 2 h, with shaking at 37oc (1400rpm, comfort r, Eppendorf, Germany). samples were then centrifuged at 2600xg at 4oc for 15 min in a beckman Gs-15r centri- 426 Ital. J. Food Sci., vol. 27 - 2015 fuge (beckman Instruments, Inc., Palo Alto, cL, usA). the samples were preserved at -20oc pri- or to further analysis. Preparation of hydrophilic and lipophilic extracts for the measurement of antioxidant capacity by photo-induced chemiluminescence (PCL) assay For the hydrophilic extract, about 100 mg of lyophilized buckwheat samples were extracted for 3 min with 1 mL of HPLc-grade water using a Genie-2 type vortex (scientific Industries, usA). next, samples were centrifuged for 5 min at 4°c, at 16100xg (5415 r, Eppendorf, Germany) and the fresh supernatants were directly used to de- termine the antioxidant capacity formed by wa- ter-soluble antioxidants (AcW). For the lipophilic extracts, about 100 mg of lyophilized buckwheat samples were extracted for 3 min with a mixture of 200 µL of n-hexane and 800 µL of methanol using a Genie-2 type vortex (scientific Indus- tries, usA). next, samples were centrifuged for 5 min at 4°c, at 16100xg (5415 r, Eppendorf, Germany) and the fresh supernatants were di- rectly used to determine the antioxidant capac- ity formed by lipid-soluble antioxidants (AcL). Determination of total phenolic compounds the content of total phenolic compounds (tPc) was determined according to sHAHIdI and nAc- ZK (1995). buckwheat extracts (0.25 mL) were mixed with 0.25 mL of Folin-ciocalteu reagent (previously diluted with water, 1:1 v/v) and 0.5 mL of na 2 co 3 solution, and 4 mL of water. the mixture was allowed to stand at room tempera- ture for 25 min and then centrifuged at 2000xg for 10 min. the absorbance of the supernatant was measured at 725 nm using a spectropho- tometer (uV-160 1Pc, shimadzu, Japan). the data were calculated as rutin equivalents. Determination of rutin content by HPLC rutin content was analyzed in a HPLc sys- tem (shimadzu, Kyoto, Japan) comprising two pumps (Lc-10 Ad), a uV detector (sPd-10A) set at 330 nm, an autosampler set for 5 μL injection (sIL-10 AdVP), a column oven (cto-10 AsVP), and a system controller (sIL-10 AdVP) according to method described by ZIELIŃsKA et al. (2010). Determination of thiamine (B1) and riboflavin (B2) by HPLC to determine the content of thiamine and ri- boflavin, the modified method of ProdAnoV et al. (1997) was used. the 500 mg of lyophi- lized buckwheat were extracted by acid hydrol- ysis with 15 mL of 0.3 M Hcl in autoclave (15 min at 120°c) and, after cooling, the pH was ad- justed to 5-5.4 using 4 M sodium acetate. then 5 mL of an aqueous solution of taka-diastase enzyme (100 u/mg) was added to the samples and incubated for 3 h at 45°c, in a water bath with shaking (120rpm, Hs-b20, IKA Labortech- nik, Germany). After enzymatic hydrolysis, the extracts were filtered by Whatman no. 40 filters and water was added to complete the volume to 25 mL. the samples were preserved at -20oc pri- or to HPLc analysis. Determination of pyridoxine (B6) and L-ascorbic acid by HPLC Analysis of pyridoxine and L-ascorbic acid content was made by the modified method of EstEVE et al. (1997). Approximately 100 mg of lyophilized buckwheat samples were added to 1 mL of 5% aqueous solution of metaphosphoric acid. the samples were extracted in triplicate, and then centrifuged at 12000xg for 10 min at 4oc (Gs-15 r beckman Instruments, Inc., Palo Alto, cL, usA). the supernatant were mixed with 100 µL of dithiotreitol (dtt), incubated for 1 h (without light) and then centrifuged at 13000xg for 5 min at 4oc. the samples were preserved at -20oc prior to further HPLc analysis. Determination of tocopherol content tocopherol (α-t, β-t, γ-t, δ-t) was extract- ed with methanol (0.5 g of sample/7 mL). Af- ter evaporation, extracts were redissolved in n- hexane. the HPLc analysis was run with a shi- madzu system (Lichrospher® si-60, 5-µm par- ticle size, 4 x 250-mm column) according to the method described by PEGG and AMAroWIcZ (2009). the tocopherols contents were calculat- ed from the peak areas using standard curves of tocopherols. Determination of inositol hexaphosphates and its lower forms the analysis of the content of tri-, tetra-, pen- ta-, and hexaphosphate inositols was made ac- cording to the method of HonKE et al. (1999). Inositol hexaphosphate was determined as fol- lows: exactly 0.5 g of the buckwheat samples were extracted with 20 mL 0.5 M Hcl for 5 h us- ing a bM1 magnetic stirrer (IKA, staufen, Ger- many). the extract was centrifuged at 3500xg for 40 min (centrifuge MPW-360, Factory of Pre- cise Mechanics, Warsaw, Poland) and the super- natants were decanted, frozen overnight (-20°c), thawed at room temperature and recentrifuged at 3500xg for 40 min. the supernatants (15 mL) were evaporated under reduced pressure to dry- ness at 40°c and dissolved in 15 mL of 0.025 M Hcl. the samples were transferred to mini- columns filled with dowex AG 1-X8 resin, from which the inositol phosphates were eluted us- ing 2 M Hcl (5 x 4 mL). After removal of the sol- Ital. J. Food Sci., vol. 27 - 2015 427 vent by evaporation with an air stream, the dry residue was dissolved in a mobile phase. then the samples were analysed by HPLc. the ino- sitol hexaphosphates contents were calculated from the peak areas using standard curves of inositol hexaphosphates. Measurement of the antioxidant capacity of buckwheat products against ABTS+• and O 2 -• the antioxidant capacities of the 80% aque- ous methanol extracts from lyophilized buck- wheat samples were determined against Abts+• radical cation using a spectrophotometric assay. the Abts+• radical cation was prepared by mix- ing Abts+• stock solution (7 mM in water) with 2.45 mM potassium persulfate. this mixture re- mained for 12-24 h until the reaction was com- plete and the absorbance was stable. Antioxi- dant capacity was determined following the pro- cedure described by rE et al. (1999) with a mi- nor modification. the Abts+• solution was dilut- ed with 80% methanol to an absorbance of 0.700 ± 0.020 at 734 nm. For the photometric assay, 1.48 mL of the Abts+• solution and 20 µL of the buckwheat extracts or trolox standards were mixed and measured immediately and again after 6 min at 30°c and 734 nm using a spec- trophotometer (uV-160 1Pc, shimadzu, Kyoto, Japan). Appropriate solvent blanks were run in each assay. the antioxidant capacity was cal- culated on the basis of percentage inhibition of absorbance at 734 nm using a trolox standard curve and was expressed as μmol trolox/g of dry matter (d.m.). the Photo-Induced chemiluminescence As- say (PcL), carried out according to the method of PoPoV and LEWIn (1999), was used to meas- ure the antioxidant capacity of unfermented and fermented groat extracts against superox- ide anion radicals (o 2 −•), which were generated from luminol, a photosensitizer, under exposure to uV light. the antioxidant capacity of buck- wheat water or methanol extracts were deter- mined using AcW (antioxidative capacity in wa- ter-soluble substances) and AcL (antioxidative capacity in lipid-soluble substances) kits (Ana- lytik Jena, Leipzig, Germany) reported in details by ZIELIŃsKA et al. (2010). Antioxidant capacity was expressed in terms of μmol trolox/g d.m. Statistical analysis the measurement were performed in tripli- cate for each of two independent fermentation batches. the data are the mean results with the standard deviation. the effects of the two param- eters, type of product (P) and fermentation pro- cess (F) or their interactions (P x F) were test- ed using a two-way AnoVA (statistica, ver. 7.1, usA). Fisher’s Least significant difference test at a significance level of p<0.05 was performed for post-hoc comparison. rEsuLts And dIscussIon Total phenolic compounds (TPC) and rutin (Ru) contents table 1 shows total phenolic compounds (tPc) and rutin (ru) contents of unfermented and fer- mented raw and roasted buckwheat groats. raw buckwheat groats (7.2 mg ru equiv/g d.m.) was almost two times richer in phenolic compounds table 1 - the phenolic, vitamin b1, b2, b6, c and α-, γ- and δ-tocopherol contents of ssF raw and roasted buckwheat groats. B SSF-B RB SSF-RB ANOVA P F P x F The phenolic contents rutin [µg/g d.m.] 205.84±0.94a 164.54±24.59b 158.84±3.42b 142.09±1.81b ns *** *** total phenolic compounds 7.19±0.84a 5.15±0.23b 3.54±0.02c 3.41±0.16c *** *** *** [mg Ru equiv/g d.m.] Content of vitamin B1, B2, B6 and C thiamine (B1) [µg/g d.m.] 9.70±0.29b 10.91±0.50a 5.41±0.13d 7.56±0.29c *** *** *** riboflavin (B2) [µg/g d.m.] 1.65±0.12a 1.70±0.08a 1.29±0.09b 1.18±0.01c *** *** *** pyridoxin (B6) [mg/g d.m.] 0.12±0.01b 0.33±0.01a 0.11±0.02b 0.12±0.01b *** *** *** L-ascorbic acid [mg/g d.m.] 0.05±0.01c 0.11±0.02a 0.05±0.01c 0.09±0.01b *** *** *** Content of α-, γ- and δ-tocopherol α-tocopherol 0.73±0.06b 1.10±0.12a 0.27±0.17c 0.64±0.07b *** *** *** γ-tocopherol 106.20±1.97b 126.88±3.18a 60.40±0.59d 95.08±1.01c *** *** *** δ-tocopherol 2.93±0.30b 3.45±0.28a 3.78±0.32a 2.10±0.07c ns *** *** Control buckwheat groats: raw (B) and roasted (RB); fermented buckwheat groats: raw (SSF-B) and roasted (SSF-RB). Data expressed as mean±standard de- viation (n=6). Different letters within the same line indicate statistically significant differences at p<0.05 in NIR Fisher test (for interactions PxF). *** significant effects by kind of products (P), fermentation process (F) or their interactions (PxF) at p<0.05; ns, not significant. 428 Ital. J. Food Sci., vol. 27 - 2015 than the roasted groat (3.5 mg ru equiv/g d.m.). our results confirms results of ZIELIŃsKA et al. (2007b). ssF led to a statistically significant decrease in the rutin content of raw groats (by 20%) but did not affect the rutin content of roasted groats. Performed two-way AnoVA analysis showed that the content of rutin was closely associated with the used ssF process. It is well known that buckwheat groats con- tain mainly rutin and a little amounts of iso- vitexin, depending on the cultivar and growth conditions (WIJnGAArd and ArEndt, 2006). Isovitexin was not detected in our buckwheat samples. the rutin content of raw buckwheat groats was 205.8 µg/g d.m. and 23% lower (p<0.05) for roasted groats. It is connected with the hydro-thermal processes used for roasting. dIEtrIcH-sZostAK and oLEsZEK (1999) found that rutin content in buckwheat groat was af- fected by temperature and heating time ad- versely. WAnG et al. (2011) found the reduc- tion of rutin content after the fermentation of peanut flour with the different strains of lac- tic acid bacteria. ssF led to a statistically significant decrease in tPc for raw groats (by 28%), but did not af- fect the tPc of ssF roasted groats. dordEVIc et al. (2010) showed that the fermentation of buckwheat by S. cerevisiae and L. rhamnosus caused the increasing of tPc compared to non- fermented samples. duEñAs et al. (2012) found significant increase of phenolic acid content in the soybean fermented with different microor- ganisms (Aspergillus oryzae, Rhizopus oryzae and Bacillus subtilis). Vitamins B and C contents the contents of vitamins b1, b2, b6 and c before and after fermentation of raw and roast- ed buckwheat groats are presented in table 1. the raw and roasted groats used for fermenta- tion had different thiamine and riboflavin con- tents, but the levels of pyridoxine and L-ascorbic acid were similar. the level of vitamin c in whole buckwheat flour ranges from 3.9 to 7.3 mg/ l00 g, our results are similar to those present- ed by other authors (WIJnGAArd and ArEndt, 2006). the concentrations of b1 and b2 in the raw groats were 80 and 28%, higher, respective- ly, than those of roasted groats. this finding is in accordance with reports regarding the pres- ence of vitamins b in buckwheat (WIJnGAArd and ArEndt, 2006). the ssF of raw groats caused a statistically significant increase of thiamine, pyridoxine and L-ascorbic acid (p<0.05) compared to the con- trol samples. the pyridoxine content increased almost three-fold, the L-ascorbic acid content more than two-fold, whereas the thiamine con- tent by 12%. Fermentation did not change the riboflavin content of raw groats. ssF of roasted groats caused the increasing of thiamine con- tent by 40%, the L-ascorbic acid content almost doubled, the pyridoxine contents did not change and riboflavin content decreased. the increase of the contents of some b-group vitamins obtained in this study is similar to those of previous reported by other authors. the increases in thiamine content that we ob- tained after fermentation of the raw and roast- ed buckwheat groats are in contrast to results reported for ssF of other substrates. nout and roMbouts (1990) found that tempeh fer- mentation of soya caused the increase of con- tent of vitamins, except for thiamine for which decrease of the content was observed. FAdA- HunsI (2009) showed the effect of Rhizopus ol- igosporus on the vitamin content in the flour obtained from bambara nut. After the 24 h fer- mentation this author found the significant re- duction in the thiamine content, while ribofla- vin, folacin, niacin and biotin content increased significantly. In traditional turkish fermented wheat-flour-yoghurt mixture (tarhana) EKIncI (2005) found no significant differences in thia- mine and pyridoxine contents with the increase of fermentation period. While, he observed sig- nificant increases of riboflavin, niacin, panto- thenic acid, ascorbic acid and folic acid during the fermentation. Tocopherol content table 1 shows the tocopherol content in raw and roasted buckwheat groats before and after fermentation. In buckwheat, γ-tocopherol is the major tocopherol homologue and the β-form is present in only trace amounts. In our study α-t, γ-t, δ-t were found in the raw and fermented buckwheat materials, whilst β-t was not detect- ed. the raw groats had almost three- and two- fold higher contents of α-t and γ-t, respective- ly, compared to the roasted groats. GEMrot et al. (2006) found for gourd seeds the decrease of tocopherol content under the influence of roast- ing process. In the literature data there are no information concerning the tocopherol level in fermented buckwheat groats. the ssF of raw and roasted groats caused a statistically significant increase of the α-t and γ-t. the content of δ-tocopherol was significantly associated with the used fermentation process, but not with the type of the product. In contrast to results obtained in this study the literature data showed the decrease of tocopherols con- tent under the influence of fermentation. For soya fermented with A. oryzae decreased of α-t content was observed by EsAKI et al. (1994), but they do not observed a modification of tocopher- ol content for soya fermented by B. subtilis or R. oligosporus. dEntEr et al. (1998) found that in soybean tempeh the tocopherols content slight- ly decreased as a consequence of fermentation with 14 varieties of Rhizopus studied. Ital. J. Food Sci., vol. 27 - 2015 429 Contents of inositol hexaphosphate and its lower forms table 2 shows the contents of inositol hexaphosphate (IP-6) and its lower forms (IP- 5, IP-4 and IP-3) in raw and roasted buckwheat groasts before and after fermentation. Phytic acid may be classified as a prohealthy or anti- nutritional compound, depending on its action. It can form an iron chelate that inhibits iron-me- diated oxidative reactions and limits site specific dnA damage. Phytic acid inhibits tumor growth by suppressing the formation of damaging hy- droxide free radicals and other reactive oxygen species (VucEnIK and sHAMsuddIny, 2003). the raw groats contained 18.3 mg/g d.m. of IP-6. After roasting, a decrease by 43% was not- ed. this finding was in accordance with the pre- viously reported by other authors (ZIELIŃsKI et al., 2006). In our study, IP-5, IP-4 or IP-3 were not found in raw groats, but they were detect- ed after roasting. After 24 h fermentation with Rhizopus oli- gosporus, the formation of IP-3, IP-4, and IP-5 was noted in fermented raw groats. Moreover, ssF caused the significant increase of IP-3 and IP-4 in fermented roasted groats (table 2). these findings were related to the three- and two-fold decreased content of IP-6 in fermented raw and roasted groats. Fermentation, steaming and extrusion cooking were identified as process- es causing the degradation of IP-6 to the lower forms (ZIELIŃsKI et al., 2006). EGounLEty and AWorH (2003) showed that fermentation with R. oligosporus reduced the phytic content by 30.7% in soybean, 32.6% in cowpea and 29.1% in ground bean. Antioxidant capacity In this study, the antioxidant capacity of raw and roasted groats before and after fermenta- tion was measured against the 2,2’-azinobis-(3- ethylbenzothiazoline-6-sulfonate) radical cati- on (Abts+•) and by the photo-induced chemilu- minescence assay (PcL) against the superoxide anion radical (o 2 -•). the PcL method can be conducted by two dif- ferent protocols, AcW and AcL, which allowed for measurement of antioxidant capacity of the water- and lipid-soluble components, respective- ly. Finally, it was possible to calculate the total antoxidant capacity as a sum of AcW and AcL values. the results are compiled in table 3. the antioxidant capacity of raw groats against Abts+• and o 2 -•, expressed as AcW+AcL, was 34 and 20% higher compared to the roasted groats. It should be also noted that lipophilic antioxidants (AcL) were the main contributor (up to 80%) to the total antioxidant capacity of raw and roast- ed groats. ZIELIŃsKA et al. (2007a) observed de- crease by 27% of Abts value for buckwheat groats after using roasting process. Also ZHAnG et al. (2010) found that the scavenging activity of tartary buckwheat flour against o 2 -• was re- duced by roasting. the ssF of raw and roasted groats caused statistically significant decreases (p<0.05), by 32 and 15%, respectively, of antioxidant ca- pacity measured against Abts+•. Also decrease of total antioxidant capacity evaluated by PcL (AcW+AcL) was noticed under the influence of ssF. similar finding were presented by bErG- HoFEr et al. (1998) for the fermented product obtained from faba bean, soybean and oat. In our study lipophilic antioxidants were significant- ly reduced after fermentation of raw and roast- ed groats (by 35 and 13%). on the other hand, these lipophilic antioxidants highly contribut- ed, up to 75%, to the total antioxidant capaci- ty of the both fermented buckwheat products. the observed decrease in antioxidant capaci- ty of fermented raw and roasted groats could be connected with the increasing of water soluble table 2 - content of inositol phosphates of ssF buckwheat groats. Sample Inositol phosphates [mg/g d.m.] IP-3 IP-4 IP-5 IP-6 B nd nd nd 18.33±0.24a SSF-B 0.33±0.04b 1.65±0.15b 2.01±0.18b 5.65±0.19c RB 0.29±0.01b 2.22±0.07a 3.80±0.08a 10.51±0.07b SSF-RB 0.44±0.05a 1.86±0.18b 3.62±0.09a 5.66±0.10c ANOVA effects P *** *** *** *** F *** *** *** *** P x F *** *** *** *** Control buckwheat groats: raw (B) and roasted (RB); fermented buckwheat groats: raw (SSF-B) and roasted (SSF-RB). Data expressed as mean±standard de- viation (n=6). Different letters within the same column indicate statistically significant differences at p<0.05 in NIR Fisher test (for interactions PxF). *** significant effects by kind of products (P), fermentation process (F) or their interactions (PxF) at p<0.05; ns, not significant; nd, non detected. 430 Ital. J. Food Sci., vol. 27 - 2015 antioxidants (e.g. vitamins b, L-ascorbic acid) and lipid-soluble antioxidant (e.g. increased to- coperols level) and decreasing of the phenolic compounds, including rutin. In our study, the total content of vitamins b was positively corre- lated with the antioxidant capacity of buckwheat groats before and after fermentation, when eval- uated by Abts test (r = 0.43) and by AcW as- say (r = 0.86). the level of ascorbic acid was also positively correlated with antioxidant capacity, as measured by AcW assay (r = 0.74). Moreo- ver, a very high correlation was calculated be- tween rutin contents and antioxidant capacity, as determined by the Abts test (r = 0.99). the same observation was made in relation to tPc contents and values provided by Abts. Phenolic compounds, including rutin, could be extracted by medium used for the AcL assay since a high correlation was also noted between ru and tPc vs AcL (r = 0.90 and r = 0.77, respectively). In our study, no correlation existed between total tocopherols and AcL values, what could indicate that this group of compounds has no impact on the formation of antioxidant capacity of non-fer- mented and fermented buckwheat groats, both raw and roasted. therefore, the antioxidant ca- pacity of fermented groats clearly depended on the antioxidant activity of vitamins b, vitamin c and rutin. It is a known that vitamins b have little or no antioxidant activity (GLIsZcZyŃsKA- ŚWIGŁo, 2006). on the other hand, the antiox- idant activity of rutin provided by Abts and PcL AcL assay (1.16±0.02 and 1.38±0.07 mmol trolox, respectively) as reported by ZIELIŃsKA et al. (2010), was higher than antioxidant activi- ty of L-ascorbic acid (1.05±0.02 mmol trolox by Abts and 1.0 mmol trolox in PLc AcW) (rE et al., 1999). therefore, the noted decrease of the antioxidant capacity evaluated by Abts and PcL assay in ssF raw and roasted groats was mainly related to the decreased rutin content and could not be ameliorated by increased content of both vitamins b and c. General remarks two-way AnoVA used for statistical analy- sis of the obtained data indicated that both an- alysed parameters: type of product (raw and roasted), fermentation process (fermented and unfermented) and their interactions significant- ly influenced on the obtained results. only in the case of rutin and δ-tocopherol content, the type of product had not significant effect. Also principal component analysis (PcA) was per- formed on the covariance matrix of the samples with no rotation (data not showed). two princi- pal components were extracted (Pc1 and Pc2) and together explained 83.66% of the total var- iance. the Pc1 was differentiated by almost all investigated compounds except rutin, IP-5, IP- 6, δ-tocopherol and antioxidant capacity deter- mined by AcL and Abts methods. concLusIons solid-state fermentation with Rhizopus oli- gosporus was used to obtain tempeh-type prod- ucts from raw and roasted buckwheat groats. the used ssF enhanced water soluble vitamins (thiamine, pyridoxine and L-ascorbic acid), as well as and α-, δ- and γ-tocopherol contents. After fermentation, a decrease in total phenol- ic compounds as well as rutin contents was ob- served. these changes had an impact on Abts•+ radical cation and superoxide anion radical (o 2 −•) scavenging activity of fermented raw and roast- ed buckwheat groats. based on the correlation studies and knowledge on the antioxidant activ- table 3 - the antioxidant capacity of ssF buckwheat groats provided by Abts and PcL assay. Sample Antioxidant capacity (μmol Trolox/g d.m.) against ABTS+• against O 2 -• ACW ACL ACW + ACL B 22.93±1.24a 3.97±0.59b 17.32±0.59a 21.29±0.11 SSF-B 15.68±0.94b 4.57±0.59a 11.18±0.59d 15.75±0.12 RB 15.15±2.34b 3.76±0.59c 13.19±0.59b 16.95±0.74 SSF-RB 12.82±0.51c 3.84±0.59c 11.50±0.59c 15.34±0.20 ANOVA effects P *** *** *** *** F *** *** *** *** P x F *** *** *** *** Control buckwheat groats: raw (B) and roasted (RB); fermented buckwheat groats: raw (SSF-B) and roasted (SSF-RB). Data expressed as mean±standard de- viation (n=6). Different letters within the same column indicate statistically significant differences at p<0.05 in NIR Fisher test (for interactions PxF). *** significant effects by kind of products (P), fermentation process (F) or their interactions (PxF) at p<0.05; ns, not significant. Ital. J. Food Sci., vol. 27 - 2015 431 ity of analysed bioactive components it should be noted that rutin content was the main factor responsible for the antioxidant capacity of fer- mented products. on the other hand, the fer- mented products were rich source of vitamins b and c and therefore ssF with Rhizopus oligospo- rus can be recommended for production of tem- peh-like functional buckwheat-based foods with reduced antinutritional factor. 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Paper Received July 16, 2014 Accepted December 1,2014 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Berghofer%2C+E.%29 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Grzeskowiak%2C+B.%29 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Mundigler%2C+N.%29 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Sentall%2C+W.+B.%29 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Sentall%2C+W.+B.%29 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Walcak%2C+J.%29