Solid-State Fermentation Reduces Phytic Acid Level , Improves the Pro fi le of Myo-Inositol Phosphates and Enhances the Availability of Selected Minerals in Flaxseed Oil Cake

Flaxseed oil cake is the solid remaining aft er pressing the fl axseeds to extract the oil. It is oft en used as a feed component due to its high nutritional value (1). However, fl axseed oil cake, especially aft er cold pressing, is high in soluble fi bre, high-quality protein, plant lignans, minerals and polyunsaturated fatt y acids and may off er benefi ts when used as a food additive (2). Protein isolates obtained from fl axseed 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).


Introducti on
Flaxseed oil cake is the solid remaining aft er pressing the fl axseeds to extract the oil.It is oft en used as a feed component due to its high nutritional value (1).However, fl axseed oil cake, especially aft er cold pressing, is high in soluble fi bre, high-quality protein, plant lignans, minerals and polyunsaturated fatt y acids and may off er benefi ts when used as a food additive (2).Protein isolates obtained from fl axseed 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 ob-tained through solid-state fermentation (SSF) (5), as well as a valuable component of dough in baking bread (6,7).Despite these advantages, fl axseed by-products also contain antinutrients.Depending on the variety and growth conditions of plants, fl axseed 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 fi lamentous 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 fl axseed 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 eff ects of fermentation of fl axseed oil cake with R. oligosporus DSM 1964 and ATCC 64063 strains on the content of phytates, profi le 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 fl axseed 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. Aft er fi ltration (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 fl axseed oil cake
Flaxseed oil cake (45 % moisture content, acidifi ed 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).Aft er sterilisation, the fl axseed oil cake was cooled to 30 °C, mixed with R. oligosporus inoculum (3•10 6 spores added to 100 g of raw fl axseed 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 aft er 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 fl axseed 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 aft er 48 and 96 h of fermentation were treated as described by Stodolak et al. (18).Briefl y, 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 NaH-CO 3 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 buff er (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 fl axseed oil cake and the dialysates from the in vitro procedure were determined by atomic absorption spectrometry with the fl ame atomisation technique (Varian AA 240FS; Agilent Technologies, Santa Clara, CA, USA), using an automatic dispensing sample system (SIPS-20; Agilent).The fl ows 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 HNO 3 in sealed pressure vessels using a microwave oven Mars Xpress (1200 W, 170 °C, 15 min; CEM Corp., Matt hews, NC, USA).The elements were determined using a single sample aspiration via a rapid sequence mode (called fast sequential).Standard solutions of Mg 2+ (100 mg/L) and Ca 2+ (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 profi les 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

Statistical analysis
Experimental data were subjected to the one-way analysis of variance (ANOVA) to detect signifi cant diff erences among mean values and expressed as a mean val-ue±standard deviation (S.D.).Diff erences 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 soft ware.

Profi les of phytate and inositol phosphates
Phytate and inositol phosphates were analysed in fl ax seed oil cake aft er 48 and 96 h of SSF with two diff erent strains of R. oligosporus.The total phytate content on dry mass basis in fl axseed 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).Aft er 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 aft er 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).
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 eff ects (25).The observed changes in the profi les of inositol phosphates could therefore be applied in value--added food products based on fl axseed 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 fl axseed oil cake varied from 3.30 to 3.53 mg/g; these values are comparable to those reported previously of 4.91-5.85mg/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 infl uence signifi cantly the contents of Ca and P. The mass fraction of P in all test-ed samples was much higher (34-36 mg/g) than previously reported (6.43-8.24mg/g) ( 6).

Inositol phosphates in the dialysates aft er in vitro digestions
There was a high correlation between the phytate content in the fl axseed oil cake (unfermented and fermented) and the InsP 6 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.
Irrespective of the sample, in vitro digestions resulted in an increase in the percentage of InsP 3 (31-37 %) in the dialysates as compared to the control (26 %) and a de-  Changes in the profi les of inositol phosphates observed aft er 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.Aff rifah et al. (27) showed that phytases of cowpea seeds retained 95-50 % activity aft er steaming for 2-32 min.High thermal stability of phytases from diff erent fungal sources was described by Simon and Igbasan (28) and Azeke et al. (29).They found that activities of purifi ed intracellular phytases isolated from R. oligosporus decreased by 20 and 80 % aft er 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 purifi ed 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
Signifi cant diff erences were found in the in vitro bioavailability of Ca, Mg and P in fermented and unfermented fl axseed oil cak e (Table 4).Fermentation of fl axseed oil cake by R. oligosporus DSM 1964 improved the bioavailability of Ca, on average by 15.5 %.This is consistent with earlier fi ndings 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.
It is worth stressing out that in our study the fermentation decreased the content of InsP 6-5 , 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 aft er fermentation of fl axseed oil cake with R. oligosporus DSM 1964, from 9.6 % in the control sample to 12.9 % in fermented fl axseed oil cake (Table 4).
The in vitro bioavailability of phosphorus increased in all process ed samples, with the exception of fl axseed oil cake fermented with R. oligosporus ATCC 64063 for 48 h.Phosphorus bioavailability values obtained aft er SSF were 3.8 % higher aft er fermentation with R. oligosporus DSM 1964 and 1.6 % higher aft er 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 effi cient hydrolysis of InsP 6 (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 fl axseed oil cake with Rhizopus oligosporus DSM 1964 strongly reduced the content of antinutritional phytate and generated a favourable profi le of lower inositol phosphates, with a signifi cant amount of inositol triphosphates that have benefi cial physiological activities.The changes in the phytate level and in the profi le of inositol phosphates in the fermented fl axseed oil cake were correlated with the improvement in the in vitro bioavailability of calcium, magnesium and phosphorus.In conclusion, the fermentation of fl axseed by-product with R. oligosporus DSM 1964 can increase its potential application as a food additive.

Table 1 .
Total phytate content, profi le of residual low er inositol phosphates and total myo-inositol mass fraction in fl axseed oil cake subjected to solid-state fermentation with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains Values in columns are mean±S.D. of the sample (N=3), values with diff erent superscripts within columns are signifi cantly diff erent (p<0.05)

Table 3 .
Eff ects of solid-state fermentation of fl axseed oil cake with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains and in vitro simulated gastrointestinal digestion on phytate bioavailability and the profi le of residual lower inositol phosphates in dialysates Values in columns are mean±S.D. of the sample (N=3), values with diff erent superscripts within columns are signifi cantly diff erent (p<0.05)

Table 2 .
The mass fractions of minerals in fl axseed oil cake before and aft er solid-state fermentation with Rhizopus oligosp orus DSM 1964 and ATCC 64063 strains in the percentage of InsP 5-6 .Moreover, in the dialysate obtained from the 96-hour culture of R. oligosporus DSM 1964, the percentage of the InsP 1-2 fraction was the highest (29 %), while that of the InsP 6-5 fraction was the lowest (26 %). crease

Table 4 .
Eff ects of solid-state fermentation of fl axseed oil cake with Rhizopus oligosporus DSM 1964 and ATCC 64063 strains and in vitro simulated gastrointestinal digestion on mineral bioavailability Values in columns are mean±S.D. of the sample (N=3), values with diff erent superscripts within columns are signifi cantly diff erent (p<0.05)