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https://doi.org/10.17113/ftb.55.03.17.4981

Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače

Robert Duliński   ORCID icon orcid.org/0000-0002-0370-2556 ; Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland
Bożena Stodolak ; Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland
Łukasz Byczyński ; Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland
Aleksander Poreda ; Department of Fermentation Technology and Technical Microbiology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland
Anna Starzyńska-Janiszewska ; Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland
Krzysztof Żyła ; Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture in Krakow, ul. Balicka 122, PL-30-149 Kraków, Poland

Fulltext: croatian, pdf (446 KB) pages 413-419 downloads: 60* cite
APA 6th Edition
Duliński, R., Stodolak, B., Byczyński, Ł., Poreda, A., Starzyńska-Janiszewska, A. & Żyła, K. (2017). Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače. Food Technology and Biotechnology, 55 (3), 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
MLA 8th Edition
Duliński, Robert, et al. "Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače." Food Technology and Biotechnology, vol. 55, no. 3, 2017, pp. 413-419. https://doi.org/10.17113/ftb.55.03.17.4981. Accessed 30 Nov. 2020.
Chicago 17th Edition
Duliński, Robert, Bożena Stodolak, Łukasz Byczyński, Aleksander Poreda, Anna Starzyńska-Janiszewska and Krzysztof Żyła. "Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače." Food Technology and Biotechnology 55, no. 3 (2017): 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
Harvard
Duliński, R., et al. (2017). 'Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače', Food Technology and Biotechnology, 55(3), pp. 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
Vancouver
Duliński R, Stodolak B, Byczyński Ł, Poreda A, Starzyńska-Janiszewska A, Żyła K. Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače. Food Technology and Biotechnology [Internet]. 2017 [cited 2020 November 30];55(3):413-419. https://doi.org/10.17113/ftb.55.03.17.4981
IEEE
R. Duliński, B. Stodolak, Ł. Byczyński, A. Poreda, A. Starzyńska-Janiszewska and K. Żyła, "Fermentacija na čvrstoj podlozi smanjuje maseni udjel fitinske kiseline, poboljšava svojstva mioinozitol fosfata i povećava iskoristivost mineralnih tvari iz lanene pogače", Food Technology and Biotechnology, vol.55, no. 3, pp. 413-419, 2017. [Online]. https://doi.org/10.17113/ftb.55.03.17.4981
Fulltext: english, pdf (446 KB) pages 413-419 downloads: 107* cite
APA 6th Edition
Duliński, R., Stodolak, B., Byczyński, Ł., Poreda, A., Starzyńska-Janiszewska, A. & Żyła, K. (2017). Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake. Food Technology and Biotechnology, 55 (3), 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
MLA 8th Edition
Duliński, Robert, et al. "Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake." Food Technology and Biotechnology, vol. 55, no. 3, 2017, pp. 413-419. https://doi.org/10.17113/ftb.55.03.17.4981. Accessed 30 Nov. 2020.
Chicago 17th Edition
Duliński, Robert, Bożena Stodolak, Łukasz Byczyński, Aleksander Poreda, Anna Starzyńska-Janiszewska and Krzysztof Żyła. "Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake." Food Technology and Biotechnology 55, no. 3 (2017): 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
Harvard
Duliński, R., et al. (2017). 'Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake', Food Technology and Biotechnology, 55(3), pp. 413-419. https://doi.org/10.17113/ftb.55.03.17.4981
Vancouver
Duliński R, Stodolak B, Byczyński Ł, Poreda A, Starzyńska-Janiszewska A, Żyła K. Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake. Food Technology and Biotechnology [Internet]. 2017 [cited 2020 November 30];55(3):413-419. https://doi.org/10.17113/ftb.55.03.17.4981
IEEE
R. Duliński, B. Stodolak, Ł. Byczyński, A. Poreda, A. Starzyńska-Janiszewska and K. Żyła, "Solid-State Fermentation Reduces Phytic Acid Level, Improves the Profile of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake", Food Technology and Biotechnology, vol.55, no. 3, pp. 413-419, 2017. [Online]. https://doi.org/10.17113/ftb.55.03.17.4981

Paper in XML format

Abstracts
U radu su ispitani in vitro sljedeći parametri: udjel fitata (InsP6), svojstva mioinozitol fosfata i iskoristivost esencijalnih mineralnih tvari iz lanene pogače fermentirane s pomoću gljivice Rhizopus oligosporus (DSM 1964 i ATCC 64063). Maseni udjel fitata u lanenoj pogači bio je 13,9 mg/g, nakon 96 sati fermentacije s pomoću R. oligosporus DSM 1964 smanjen je za
48 %, a s pomoću R. oligosporus ATCC 64063 za 33 %. Fermentacijom s pomoću soja DSM 1964 postignuti su bolji rezultati, jer je dobiven dominantni mioinozitol InsP3-5, dok je fermentacijom s pomoću soja ATCC 64603 dobiven uglavnom InsP5-6. Fermentacijom na čvrstoj podlozi od lanene pogače poboljšana je in vitro iskoristivost kalcija za 14 %, magnezija za 3,3 % i fosfora za 2-4 %.

Keywords
lanena pogača; fermentacija na čvrstoj podlozi; fitati; mioinozitol fosfati; iskoristivost mineralnih tvari

Hrčak ID: 186558

URI
https://hrcak.srce.hr/186558

▼ Article Information



Introduction

Flaxseed oil cake is the solid remaining after pressing the flaxseeds to extract the oil. It is often used as a feed component due to its high nutritional value (1). However, flaxseed oil cake, especially after cold pressing, is high in soluble fibre, high-quality protein, plant lignans, minerals and polyunsaturated fatty acids and may offer benefits when used as a food additive (2). Protein isolates obtained from flaxseed oil cake have functional value (3) and prohealth and therapeutic properties (4). Flaxseed oil cake has also been used as a component of a food product obtained through solid-state fermentation (SSF) (5), as well as a valuable component of dough in baking bread (6, 7).

Despite these advantages, flaxseed by-products also contain antinutrients. Depending on the variety and growth conditions of plants, flaxseed meal contains 2.3–3.3% of phytic acid (myo-inositol-(1,2,3,4,5,6)-hexakisphosphate) (8). Phytates are the main storage forms of phosphorus in plants. A highly charged phytic acid molecule electrostatically binds minerals and proteins in complexes, decreasing their bioavailability (9, 10). Phytate dephosphorylation may be performed by means of thermal hydrolysis, e.g. during cooking, or be the result of the action of phytases. Phytases are produced endogenously in plant seeds during soaking and sprouting (11), and are also secreted by various bacteria (12) and filamentous fungi (13). Feed supplementation with phytases is a commonly used practice in the poultry nutrition and can also be applied during dough processing in wholemeal bread manufacturing (14, 15).

Rhizopus oligosporus has already been proven to reduce the phytate level during SSF of plant substrates (16). Rhizopus sp. are used in the production of tempeh, an Indonesian fermented food with interesting organoleptic characteristics and a high nutritional value. This kind of processing is an old and well-known treatment of soybeans, other legumes and legume-grain mixtures. It can also be applied as an alternative method for the utilisation of plant by-products. Our previous research showed that the application of R. oligosporus DSM 1964 and ATCC 64063 cultures in the processing of flaxseed oil cake may result in products with enhanced soluble phenolic content and increased antioxidant activity, suitable as functional food additives (17).

The aim of these investigations is to study the effects of fermentation of flaxseed oil cake with R. oligosporus DSM 1964 and ATCC 64063 strains on the content of phytates, profile of lower inositol phosphates, many of which are known for their active metabolic functions, and on the availability of essential minerals, assessed using multistep digestion in vitro procedure.

Material and Methods

The substrate for the fermentation was a cold-pressed flaxseed oil cake, supplied by the Przedsiębiorstwo Nasienne Centrala Nasienna in Sanok (Poland).

Inoculum

The spores of Rhizopus oligosporus DSM 1964 and ATCC 64063 strains (twelve-day cultures on potato extract agar slants) were harvested with sterile 0.85% NaCl supplemented with 0.01 g/L of peptone and 0.1 mL/L of Tween 80. After filtration (d=11 µm, Nylon Net Filtres; Merck Millipore, Cork, Ireland), the spore density in the suspension was measured using Thoma chamber under an optical microscope (model NK4; PZO Sp. z o.o., Warsaw, Poland).

Fermentation of flaxseed oil cake

Flaxseed oil cake (45% moisture content, acidified to pH=4–5 with 5%, by mass per volume, lactic acid) was autoclaved at 121 °C for 20 min (ELMI ESS-207; SMS Sp. z o.o., Warsaw, Poland). After sterilisation, the flaxseed oil cake was cooled to 30 °C, mixed with R. oligosporus inoculum (3·106 spores added to 100 g of raw flaxseed oil cake) and packed into Petri dishes (d=11 cm, three replications in each fermentation stage). Inoculated material was incubated at 37 °C for 2 h (spore germination induction) followed by incubation at 30 °C. The plates were removed after 48 and 96 h of fermentation. The fermentation was stopped by steaming (10 min). The lyophilised products were stored at 4 °C for later analysis. A sample of flaxseed oil cake that was prepared for fermentation, but was not inoculated, was also kept for analysis.

In vitro digesting procedure

The bioavailability of selected compounds was estimated by an in vitro method. Flaxseed oil cake samples prepared for inoculation and after 48 and 96 h of fermentation were treated as described by Stodolak et al. (18). Briefly, 0.5 g of material was mixed with 1.7 mg of pepsin (4750 U/mg; Sigma-Aldrich, Steinheim, Germany) dissolved in 0.1 mol/L of HCl and incubated at 37 °C, pH=2.0, for 2 h. Next, 2.5 mg of pancreatin (from porcine pancreas, 8×USP (United States Pharmacopeia); Sigma-Aldrich, St. Louis, MO, USA) and 31 mg of bile extract porcine (Sigma-Aldrich, St. Louis) dissolved in 0.1 mol/L of NaHCO3 were added. The sample was put into a dialysis tube (cellulose membrane 25 mm×16 mm; Sigma-Aldrich, St. Louis) and incubated at 37 °C for 4 h in 50 mL of imidazole buffer (pH=7.0). The dialysates were collected and the levels of phytate, inositol phosphates, phosphorus, myo- -inositol and minerals were determined (as described below). The amounts of phytate and minerals released into the dialysate divided by their total content expressed in percentages were used as a bioavailability indicator.

Determination of metal ions

The contents of metals in the samples of fermented flaxseed oil cake and the dialysates from the in vitro procedure were determined by atomic absorption spectrometry with the flame atomisation technique (Varian AA 240FS; Agilent Technologies, Santa Clara, CA, USA), using an automatic dispensing sample system (SIPS–20; Agilent). The flows of gas (acetylene) and air were 3.5 and 14 L/min, respectively. Before analysis, the samples were subjected to a process of wet mineralisation, with the addition of 4 mL of concentrated HNO3 in sealed pressure vessels using a microwave oven Mars Xpress (1200 W, 170 °C, 15 min; CEM Corp., Matthews, NC, USA). The elements were determined using a single sample aspiration via a rapid sequence mode (called fast sequential). Standard solutions of Mg2+ (100 mg/L) and Ca2+ (40 mg/L) were prepared from 1000 mg/L of stock solutions (Merck, Bilerica, MA, USA).

Phosphorus determination

The Fiske-Subbarow method (19) was used to determine phosphorus content on dry mass basis (mg/g) in dialysates and in raw samples (total content) previously mineralised in the Hach Digesdahl® digestion aparatus at 280 °C (Hach Company, Loveland, CO, USA).

Determination of inositol phosphates

Inositol phosphates were extracted from samples according to Duliński et al. (20). The profiles of the myo-inositol phosphates were determined by the analytical system using high-performance anion-exchange chromatography (HPAEC) with postcolumn derivatisation and UV/Vis detection (21). A reference sample was prepared by dissolving 2.3 g of sodium phytate in deionised water (50 mL) and adjusting the pH to 4.0 with 2 M HCl. Next, the solution was autoclaved for 40 min at 121 °C under 101 kPa (autoclave ELMI ESS-207; SMS Sp. z o.o.). The elution sequence of InsP6-2 isomers was established according to the work of Blaabjerg et al. (21) mentioned above, using appropriate standard solution, sodium phytate (InsP6), Ins(1,2, 4,5,6)P5, Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,4,5)P3, Ins(1,3,4)P3, and myo-inositol 2-monophosphate (all purchased from Sigma-Aldrich, Steinheim).

Phytate analysis

Ion chromatography system (Dionex UltiMate 3000) coupled with a ED50a electrochemical detector and conductivity cell (Dionex, Sunnyvale, CA, USA) was used for the analysis. Briefly, samples extracted according to Duliński et al. (20) were separated on Omnipac Pax-100 anion exchange column (250 mm×4 mm i.d.) connected in series with Omnipac Pax-100 (8 mm×1 mm) guard column (Dionex). A mobile phase using a mixture of 200 mM sodium hydroxide (A), deionised water (B), and water-isopropanol (50:50, by volume) (C) was applied. An anion micromembrane suppressor AMMS 300 4-mm (Dionex) system was used to suppress the mobile phase conductivity before entering the conductivity cell (regenerant 0.25 M sulfuric acid) according to Dionex Application Note 65 (22).

Myo-inositol analysis

The concentration of total and free myo-inositol in samples and in dialysates was measured by HPLC assay according to Duliński et al. (20), using Rezex™ RCM Ca2+ column (375 mm×4 mm i.d., Phenomenex, Torrance, CA, USA).

Statistical analysis

Experimental data were subjected to the one-way analysis of variance (ANOVA) to detect significant differences among mean values and expressed as a mean value±standard deviation (S.D.). Differences among mean values were checked by the Tukey’s test at p<0.05 using Statistica for Windows, v. 12.5 (StatSoft Inc., Tulsa, OK, USA) statistical software.

Results and Discussion

Profiles of phytate and inositol phosphates

Phytate and inositol phosphates were analysed in flaxseed oil cake after 48 and 96 h of SSF with two different strains of R. oligosporus. The total phytate content on dry mass basis in flaxseed oil cake prepared for the inoculation was 13.9 mg/g (Table 1). R. oligosporus DSM 1964 decreased the phytate level to 7.8 mg/g in 48 h (a 44% reduction). After 96 h, the phytate content was reduced to 7.2 mg/g, but this decrease was not statistically significant. R. oligosporus ATCC 64063, on the other hand, decreased the phytate level to 9.3 mg/g after 96 h, yielding 35% reduction. The decrease in phytate levels was similar to the 32–42% decrease reported during the fermentation of sorghum grains with lactic acid bacteria (23), but lower than the 74–89% decrease reported during the fermentation of oat and barley grains with R. oligosporus (24).

Table 1 Total phytate content, profile of residual lower inositol phosphates and total myo-inositol mass fraction in flaxseed oil cake subjected to solid-state fermentation with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains
Samplet(fermentation)
h
m(phytate InsP6)
mg/g
Phytate reduction
%
Average relative peak area/%m(myo-inositol
InsP0)
mg/g
InsP6InsP5InsP4InsP3InsP2-1
Flaxseed oil cake0(13.9±1.9)a074.517.21.31.15.9(6.98±0.04)
DSM 196448(7.8±0.2)c–4442.622.113.311.011.0(6.96±0.01)
96(7.2±0.5)c–4837.817.112.018.713.7(6.97±0.07)
ATCC 6406348(9.1±0.1)b–3553.722.13.44.116.7(6.88±0.05)
96(9.3±0.5)b–3348.618.83.01.927.7(6.84±0.04)

Values in columns are mean±S.D. of the sample (N=3), values with different superscripts within columns are significantly different (p<0.05)

The intermediate products of phytate degradation during the fermentation of flaxseed oil cake were: Ins(1,2,- 4,5,6)P5, with lower amounts of Ins(1,2,3,4,5)P5, Ins(1,2,3,- 4,6)P5, Ins(1,4,5,6)P4 and Ins(1,2,4,5)P4 (B and C in Fig. 1). The profiles indicate that the phytases of R. oligosporus initially removed D-3 phosphate residue from the myo- -inositol ring, suggesting that they were 3-phytases (EC 3.1.3.8). However, the two R. oligosporus strains differed significantly in phytase activity as proven by the differences in the magnitude of InsP6 degradation and by the spectrum of lower inositol phosphates formed. R. oligosporus DSM 1964 produced more InsP3 isomers, mainly Ins(1,2,6)/(1,4,5)P3 and Ins(2,4,6)P3 than R. oligosporus ATCC 64603 did (B and C in Fig. 1). The fermentation of flaxseed oil cake with R. oligosporus DSM 1964 produced a more advantageous profile of inositol phosphates in the products, with predominance of inositol phosphates having 3–5 phosphate moieties after 96 h, with levels of these isomers being reasonably similar (Table 1). In the case of R. oligosporus ATCC 64603 fermentation, InsP5 (18–22%) and InsP1-2 (17–28%) were the dominant inositol phosphates found in the product.

Fig. 1 Overlaid chromatograms of myo-inositol phosphate analysis in raw and fermented flaxseed oil cake after 48 h of fermentation. Myo-inositol phosphate in-house reference standard acquired after thermal hydrolysis of phytic acid (A), flaxseed oil cake fermented by R. oligosporus ATCC 64063 (B) and by R. oligosporus DSM 1964 (C), and raw flaxseed oil cake (D). Peaks: 1=inorganic phosphate, 2–4=InsP1-2, 5=Ins(1,3,5)P3, 6=Ins(2,4,6)P3, 7=Ins(1,2,6)P3, 8=Ins(x,y,z)P3, 9=dl-Ins(1,5,6)P3, 10=dl-Ins(4,5,6)P3, 11=dl-Ins(1,2,4,6)P4, 12=dl-Ins(1,2,- 3,4)P4, 13=dl-Ins(1,2,4,5)P4, 14=InsP4, 15=dl-Ins(1,2,5,6)P4, 16=Ins(2,4,5,6)P4, 17=Ins(1,4,5,6)P4, 18=Ins(1,2, 3,4,6)P5, 19=Ins(1,2,3,4,5)P5, 20=dl-Ins(1,2,4,5,6)P5, 21=Ins(v,w,x,y,z)P5, 22=Ins(1,3,4,5,6)P5 and 23=Ins(1,2,3,4,5,6)P6
FTB-55-413-f1

Inositol triphosphates and particularly those with conserved 1,2,3 or 2,4,5 conformation of phosphate moieties are known for their antioxidant and immunostimulating effects (25). The observed changes in the profiles of inositol phosphates could therefore be applied in value- -added food products based on flaxseed oil cake, since the mixtures of phytate with lower inositol phosphates have proven anticancerogenic properties (25).

Mineral content

The amount of magnesium in the initial flaxseed oil cake varied from 3.30 to 3.53 mg/g; these values are comparable to those reported previously of 4.91–5.85 mg/g (6) and 5.8–6 mg/g (26). Fermentation with both R. oligosporus strains caused a slight increase (7% in 96 h) of the mass fraction of Mg (Table 2) but did not influence significantly the contents of Ca and P. The mass fraction of P in all tested samples was much higher (34–36 mg/g) than previously reported (6.43–8.24 mg/g) (6).

Table 2 The mass fractions of minerals in flaxseed oil cake before and after solid-state fermentation with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains
Samplet(fermen-
tation)
h
w/(mg/g)
CaMgP
Flaxseed
oil cake
0(1.01±0.07)(3.30 ±0.07)a(35.8 ±2.4)
DSM 196448(0.96±0.04)(3.53 ±0.08)b(34.0 ±1.5)
96(1.02±0.07)(3.55 ±0.04)b(36.1 ±2.5)
ATCC 6406348(1.01±0.03)(3.45 ±0.11)ab(36.0 ±1.2)
96(1.01±0.07)(3.55 ±0.04)b(35.9 ±2.6)
Mean value(1.00±0.06)(3.47 ±0.07)(35.5 ±2.0)

Values in columns are mean±S.D. of the sample (N=3), values with different superscripts within columns are significantly different (p<0.05)

Inositol phosphates in the dialysates after in vitro digestions

There was a high correlation between the phytate content in the flaxseed oil cake (unfermented and fermented) and the InsP6 level detected in dialysates obtained from the in vitro digestion (R=0.91). The availability of phytate in the product of the 48–hour fermentation with R. oligosporus was higher (33 and 34%) than that of the control sample (28%) (Table 3). However, it was lower than the values reported for cooked buckwheat groats (39%) and buckwheat tempeh (69–62%) (16) as well as for rye bread (50%) (14). The increase of the phytate bioavailability in fermented samples could result from the metabolic activity of the mould that loosened plant tissues, which, in turn, facilitated the release (leaching) of this low-molecular compound during the in vitro digestion.

Table 3 Effects of solid-state fermentation of flaxseed oil cake with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains and in vitro simulated gastrointestinal digestion on phytate bioavailability and the profile of residual lower inositol phosphates in dialysates
Samplet(fermentation)
h
w(phytate)
mg/g
Phytate availability
%
Average relative peak area/%m(myo-inositol
InsP0)
mg/g
InsP6InsP5InsP4InsP3InsP2-1
Flaxseed oil cake0(3.9±0.4)a2845.416.610.725.81.5n.d.
DSM 196448(2.6±0.4)b3430.118.813.332.99.6n.d.
96(2.5±0.1)b3513.712.48.636.528.8n.d.
ATCC 6406348(3.0±0.2)c3329.826.97.035.12.2n.d.
96(3.5±0.1)a3730.827.27.630.85.2n.d.

Values in columns are mean±S.D. of the sample (N=3), values with different superscripts within columns are significantly different (p<0.05)

Irrespective of the sample, in vitro digestions resulted in an increase in the percentage of InsP3 (31–37%) in the dialysates as compared to the control (26%) and a decrease in the percentage of InsP5-6. Moreover, in the dialysate obtained from the 96-hour culture of R. oligosporus DSM 1964, the percentage of the InsP1-2 fraction was the highest (29%), while that of the InsP6-5 fraction was the lowest (26%).

Changes in the profiles of inositol phosphates observed after in vitro digestion could result from the residual activity of plant and mould phytases and phosphatases that acted against higher phosphorylated forms of myo-inositol in previously fermented samples. Such phosphatases could be active in both the substrate and products of the fermentation despite the high temperature treatments during processing, like autoclaving before inoculation and steaming at the end of the fermentation. Affrifah et al. (27) showed that phytases of cowpea seeds retained 95–50% activity after steaming for 2–32 min. High thermal stability of phytases from different fungal sources was described by Simon and Igbasan (28) and Azeke et al. (29). They found that activities of purified intracellular phytases isolated from R. oligosporus decreased by 20 and 80% after treatment at 70 and 80 °C for 10 min, respectively. It has been proven that phytases present in the food matrix within the tempeh structure are more heat resistant than the purified enzymes (30).

In our previous study on rye bread (20), the supplementation of dough with an exogenous phytase resulted in almost complete dephosphorylation of phytic acid. As a consequence, a release of myo-inositol into dialysates was observed during in vitro digestion. This phenomenon was not observed in the present research. Free myo-inositol was not detected in the dialysates measured by an appropriate HPLC method (Table 3).

Mineral availability

Significant differences were found in the in vitro bioavailability of Ca, Mg and P in fermented and unfermented flaxseed oil cake (Table 4). Fermentation of flaxseed oil cake by R. oligosporus DSM 1964 improved the bioavailability of Ca, on average by 15.5%. This is consistent with earlier findings of many researchers concerning the preparation of dephytinised wheat (31), rye–wheat bread (32) and bread with the addition of pseudocereal grains (33), where the treatment of samples with microbial phytases increased the bioavailability of minerals, especially Ca, Zn and Fe. Recent paper by Abid et al. (34) demonstrates that transgenic expression of phytase in wheat endosperm increases Zn and Fe bioavailability, thus enhancing its nutritional value.

Table 4 Effects of solid-state fermentation of flaxseed oil cake with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains and in vitro simulated gastrointestinal digestion on mineral bioavailability
Samplet(fermen-
tation)
h
In vitro bioavailability/%
CaMgP
Flaxseed
oil cake
0(20.9±3.2)a(9.6±0.2)a(4.5±0.9)a
DSM 196448(35.02±5.1)b(12.9±1.6)b(8.4±0.8)c
96(37.80±6.1)b(12.6±1.3)b(8.2±0.2)c
ATCC 6406348(28.6±7.3)ab(11.5±0.5)ab(4.2±0.3)a
96(34.0±2.2)ab(11.8±0.4)ab(6.1±0.3)b
Mean value(1.00±0.06)(3.47±0.07)(35.5±2.0)

Values in columns are mean±S.D. of the sample (N=3), values with different superscripts within columns are significantly different (p<0.05)

It is worth stressing out that in our study the fermentation decreased the content of InsP6-5, the compounds that have a strong capacity for chelating minerals. This effect was most evident when R. oligosporus DSM 1964 strain was used (Table 1).

The in vitro bioavailability of Mg also slightly increased after fermentation of flaxseed oil cake with R. oligosporus DSM 1964, from 9.6% in the control sample to 12.9% in fermented flaxseed oil cake (Table 4).

The in vitro bioavailability of phosphorus increased in all processed samples, with the exception of flaxseed oil cake fermented with R. oligosporus ATCC 64063 for 48 h. Phosphorus bioavailability values obtained after SSF were 3.8% higher after fermentation with R. oligosporus DSM 1964 and 1.6% higher after fermentation with R. oligosporus ATCC 64063 than in control. The phosphorus level in dialysates from the in vitro digestion was strongly negatively correlated with total (R=–0.78) and dialysable 
(R=–0.7) phytate contents. Thus, it can be assumed that the increase in the phosphorus bioavailability was the consequence of the release of this mineral from the phytate. The efficient hydrolysis of InsP6 (Table 1) observed in our study is the result of the action of fungal phytases and phosphatases (35). The increase in phosphorus bioavailability by 4.5% was also reported for Aspergillus niger phytase added to corn-soy feed in a broiler diet (36).

Conclusions

Solid-state fermentation of flaxseed oil cake with Rhizopus oligosporus DSM 1964 strongly reduced the content of antinutritional phytate and generated a favourable profile of lower inositol phosphates, with a significant amount of inositol triphosphates that have beneficial physiological activities. The changes in the phytate level and in the profile of inositol phosphates in the fermented flaxseed oil cake were correlated with the improvement in the in vitro bioavailability of calcium, magnesium and phosphorus. In conclusion, the fermentation of flaxseed by-product with R. oligosporus DSM 1964 can increase its potential application as a food additive.

Acknowledgements

The authors would like to thank for the financial support from the Ministry of Science and Higher Education of Poland (grant number DS 3700/WTŻ/2015).

Notes

[1] Conflicts of interest There were no conflicts of interest expressed by the authors. The authors confirm their responsibility for the content and writing of the paper.

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