quantitative analysis of inositol phosphate contents in
TRANSCRIPT
Journal of Cereal Science 96 (2020) 103121
Available online 23 October 20200733-5210/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Quantitative analysis of inositol phosphate contents in oat products using an anion exchange chromatographic method
Noora Makela *, Tuula Sontag-Strohm , Miikka Olin , Vieno Piironen Department of Food and Nutrition, PL 66, Agnes Sjobergin Katu 2, 00790, Helsinki, Finland
A R T I C L E I N F O
Keywords: Anion exchange chromatography Inositol phosphates Phytate Oat
A B S T R A C T
Oat has gained interest due to its high nutritional value. When utilising oat fractions rich in dietary fibre, their inositol phosphate (InsP, including phytate) content is considerably high due to the lack of active phytase in the kilned oat ingredients. The high InsP content is linked to decreased mineral absorption in the gut, but the mineral-binding ability of InsPs also results in antioxidativity and a decrease in starch hydrolysis, thus lowering glycaemic response. This study aimed to further develop an anion exchange liquid chromatographic method for quantification of different InsP forms from oat products and to study the changes in the InsP contents resulting from the differences in the ingredients or processes. The method was applicable for quantifying such InsP forms that can effectively bind minerals. The InsPs were stable at moderate temperatures and in the oat baking process, but a significant degradation occurred during the high-temperature treatments, extrusion, and bacterial fermentation.
1. Introduction
Interest in the use of oat as a food ingredient has increased during the past years. One reason for this is its high nutritional value; when compared to the other major food crops used in Europe, oat has a relatively high protein content, is gluten free, contains a high amount of lipids, and is rich in beta-glucan (Welch, 2011). The health claims approved by the European Food Safety Authority for the cholesterol-lowering and postprandial blood sugar-attenuating effects of oat beta-glucan have also considerably affected the appeal of oat (EFSA, 2010, 2011).
Compared to the other major food crops, oat has a relatively high phytate content. All cereal grains have significant amounts of phytate (Reddy, 2001), but the content of the phytate-cleaving enzyme, phytase, is lower in oat than in wheat, barley or rye (Bartnik and Szafranska, 1987). Also, in oat, a larger part of phytate stays intact as the oat pro-cessing involves kilning where the grain material is moistened and heated to inactivate the lipases. This process is a requirement for the prevention of oat, which is high in lipids, becoming rancid, but it simultaneously inactivates other enzymes, such as phytase.
The structure of phytate consists of a myo-inositol ring with six phos-phate groups that can occur in an acid form (=inositol hexaphosphoric acid) or a salt form (=inositol hexaphosphate, InsP6). In grains, inositol
phosphates (InsPs) occur mainly in the InsP6 form, although low amounts of InsPs with a lower phosphorylation degree are also present (Schlemmer et al., 2009). During the processing of foods, phytate may degrade forming InsPs with varying degrees of phosphorylation. Phytate may extensively degrade by the action of microbial phytases, such as from lactobacilli and yeast, but phytases from grain sources other than oat may also result in the hydrolysis of oat phytate in products with several grain ingredients (Koniezny and Greiner, 2002; Schlemmer et al., 2009). The hydrolysis of phytate by phytase occurs as a stepwise reaction, forming InsPs with a different degree of phosphorylation at different stages of the reaction pathway, and the reaction rate declines with the decreasing degree of phosphorylation.
Phytate is often considered an anti-nutrient due to its ability to bind minerals resulting in their decreased absorption in digestion (Schlem-mer et al., 2009). However, phytate can also have a positive role in the physiological functionality of oat. Knuckles and Betschart (1987) showed a decreased digestion of starch by α-amylase in the presence of InsPs. Phytate was reported to be the most effective InsP form in the inhibition of starch hydrolysis and the efficiency declined with the decreasing phosphorylation degree of the InsP. Also, phytate is one of the antioxidative compounds of oat, and this antioxidant activity is resulting from the capability of phytate to bind iron thus preventing it
* Corresponding author. E-mail addresses: [email protected] (N. Makela), [email protected] (T. Sontag-Strohm), [email protected] (M. Olin), vieno.piironen@
helsinki.fi (V. Piironen).
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Journal of Cereal Science
journal homepage: http://www.elsevier.com/locate/jcs
https://doi.org/10.1016/j.jcs.2020.103121 Received 25 August 2020; Received in revised form 19 October 2020; Accepted 20 October 2020
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from catalysing oxidation reactions (Peterson, 2001; Zhou and Erdman, 1995). Additionally, phytate may chelate minerals that are needed in enzymatic oxidation reactions in vivo. The influence on mineral ab-sorption has been suggested to positively affect cholesterol metabolism by balancing the zinc-to-copper ratio in the body. Thus, the total InsP content has significance in the physiological functionality of oat. How-ever, the InsP form should also be taken into account when evaluating the functionality, as the mineral binding capacity of InsP is affected by the degree of phosphorylation. The InsP6 and InsP5 have been found to bind minerals and affect mineral absorption more than lower InsP forms (InsP4 and InsP3) (Phillippy, 2003).
Thus, not only is the total content of InsPs in the consumed oat products of interest, but also the content of the forms that have the mineral-binding capacity. For routine analysis of phytic acid, the mea-surement is usually done by measuring the phosphorus in hydrolysed InsPs and calculating the phytic acid content from the phosphorus amount (McKie and McCleary, 2016). However, these kinds of calcula-tions assume that all the phosphorus originates from phytic acid, which results in overestimation of InsP6 in samples with lower InsP forms (InsP1-InsP5). Especially in processed foods, the separation and quanti-fication of the different InsPs are needed for accurate measurement. Cooper et al. (2007) reviewed different analytical methods for InsP analysis and concluded that the ion exchange was the most suitable separation method for InsPs. In several analysis methods, the pre-treatments and purification steps are laborious or the instruments and equipment costly. However, in the method reported by Rounds and Nielsen (1993) the detection was based on a ligand exchange reaction of Fe3+-sulfosalicylic acid complex and the InsPs, and required no purifi-cation steps other than centrifugation of the InsP extract. The method was concluded to be simple and executable with a rather simple liquid chromatography system and suggested to be applicable for quantifica-tion of different InsP forms. More recently, also liquid chromatographic separation coupled with mass spectrometric (MS) detection has been utilised in the analysis of InsPs (Duong et al., 2017; Kaleda et al., 2020). However, these methods require the access to the costly instruments, which may prevent the usage in more routine analysis of InsPs. Sec-ondly, MS detection of InsPs often utilises electron spray ionisation (ESI) and as Zhou et al. (2017) describes, ESI is especially sensitive for matrix effects. Thus, this makes it somewhat complicated to utilise MS methods for analysing InsPs from wide variety of matrices.
In this study, an anion exchange chromatographic method by Rounds and Nielsen (1993) was further developed for quantification of InsPs and applied for studying the inositol phosphate contents of varying oat products. The aim was to evaluate the impact of different ingredients and processing treatments on the inositol phosphate content and composition of oat products. The ingredients containing active phytases and processes with harsh temperature treatments were hypothesised to result in significant degradation of InsPs.
2. Materials and methods
2.1. Anion exchange chromatographic method for quantification of inositol phosphates
2.1.1. Preparation of reference sample An in-house reference (IHR) was prepared to evaluate the stability of
the system by analysing this same reference sample in the beginning and end of every sample set. Preparation of the IHR with different inositol phosphate forms was based on Blaabjerg et al. (2010) with some mod-ifications. 1.5 g of sodium phytate (Phytic acid sodium salt hydrate from rice, Sigma-Aldrich, Switzerland) was hydrolysed with 100 ml of 0.5 M HCl by boiling the solution with reflux for 12 h. The pH was increased to 4.5 b y adding solid NaOH crystals. The reference sample was evapo-rated to dryness with a vacuum concentrator (SpeedVac Plus, SC110A combined with Refrigerated Condensation Trap RT100, Savant, Savant Instruments Inc. U.S.), after which the dried sample was dissolved in
100 ml of MQ water and divided into several aliquots, which were stored at − 20 ◦C until analysis. The presence of all six InsP forms in this IHR sample was ensured with a mass spectrometer. The IHR was diluted with 20% isopropanol containing 1% triethylamine (≥99%, Sigma-Aldrich, USA) to enhance ionisation, and direct injection without chromato-graphic separation was used. The MS system (Thermo Finnigan LXQ, Thermo Electron Corporation, USA) consisted of an electron spray ion-isation and linear trap, and the ions were detected in negative mode. The sheath gas and auxiliary gas flow rates were 10 (arb) and 5 (arb), respectively, and spray voltage was set to 3.5 kV, capillary voltage to − 40 V, tube lens to − 110 V and capillary temperature to 350 ◦C.
2.1.2. Preparation of standards The following standards were purchased from Cayman Chemicals
(USA): D-myo-inositol-1-phosphate (sodium salt); D-myo-inositol-1,4- diphosphate (sodium salt); D-myo-inositol-1,3,5-triphosphate (sodium salt); D-myo-inositol-1,3,4,5-tetraphosphate (sodium salt); D-myo- inositol-1,3,4,5,6-pentaphosphate (sodium salt); and D-chiro-inositol- 1,2,3,4,5,6-hexakisphosphate (sodium salt). All standards were dis-solved at a ratio of 0.5 mg of standard per 1 ml of MQ to make a stock solution. From these stock solutions, 1:1, 1:4, 1:9 and 1:19 dilutions were made to prepare standard curves with four concentrations. The concentrations of the standards were calculated, taking into account the sodium and calculating the concentration of the InsP alone. Due to the differences in the mass ratio of sodium to InsP in the different standards, the final concentrations differed. However, from the actual samples, each InsP form was quantified at such a concentration, which was within the range of the standard curve of the specific InsP form.
2.1.3. Chromatographic method The separation and detection of InsPs were conducted according to a
method by Rounds and Nielsen (1993) with some modifications. The liquid chromatography system consisted of Agilent 1200 series in-struments (Agilent Technologies, USA) with a binary pump module, degasser, autosampler, column oven and diode array detector. The in-jection volume of the samples was 50 μl and, for some samples, also 100 μl, as described in 2.2.2. The analytes were separated using a PL-SAX anion exchange column (1000 Å, 8 μm, 50 × 4.6 mm, Agilent Tech-nologies, UK) with the column oven temperature set to 25 ◦C.
The elution was conducted using two eluents, and the total flow rate through the column was kept at 1 ml/min. Eluent A was 0.01 M 1-methyl piperazine (99%, Sigma-Aldrich, China), which was adjusted to pH 4 with 3 M hydrochloric acid, and eluent B was 0.5 M sodium nitrate (NaNO3, ReagentPlus®, ≥99%, Sigma-Aldrich, India) in eluent A. Elution was performed with a linear gradient from eluent A to eluent B over the 20-min run time, followed by a 5-min ramp back from eluent B to eluent A. For the detection of the InsPs, a post-column reaction was conducted. The post-column reagent, 0.015% iron (III) chloride hexa-hydrate (FeCl3 × 6H2O, ≥99%, Sigma-Aldrich, Germany) in 0.15% 5- sulfosalicylic acid dehydrate (ReagentPlus®, ≥99%, Sigma-Aldrich, Germany), was pumped with an external pump (515 HPLC Pump, Wa-ters, USA) at a flow rate of 1 ml/min and mixed with the flow from the column in a PEEK tubing (3 m × 0.508 mm, Waters, USA). The overall flow rate to the detector was, thus, 2 ml/min. All eluents were filtered using nylon 66 filter membranes (0.45 μm).
The detection was conducted using a diode-array detector (1200 Series, G1315B, Agilent Technologies, USA), and the InsPs were detec-ted at 500 nm as negative peaks as the reaction of the InsPs with the post-column reagent resulted in a decrease in absorbance. The standards contained only one isomer each, but the samples and IHR may have contained several. All isomers of each InsP were integrated as one peak, and the peak area was used to quantify the content of the specific InsP form using an external standard method with standards described in 2.1.2. The stability of the system and detector response were checked at the beginning and end of each run set using IHR, and the need for new standard curves was evaluated based on the results of the IHR.
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2.2. Analysing the content of different inositol phosphate forms from oat products
2.2.1. Materials The contents of different InsP forms were analysed from different oat
products (Table 1). The same samples were analysed for the state of beta-glucan in an in vitro small intestine phase simulation by Makela et al. (2020). The samples were bought from local supermarkets in the Helsinki area, and the products were selected from different manufac-turers. The samples were pre-treated in the previous study and were stored frozen (− 20 ◦C) until they were melted for the extraction of InsPs. The samples in powder form had been stored as such without pre-treatments. The solid samples (including breads, crisps, extruded breakfast cereals and meat analogues) had been homogenised as, described by Makela et al. (2020), and air-dried prior to the frozen storage. The oat flakes had been cooked to porridge and the spoonable products had been freeze-dried. In addition to the commercial oat products listed in the table, one oat control flour was added to the set to see the InsP level in a minimally processed product. These oat groats had been kilned prior to the milling to inactivate the endogenous enzymes.
2.2.2. Extraction of inositol phosphates The InsPs were extracted from the oat products after the pre-
treatments and frozen storage, which were described in 2.2.1. The extraction was conducted by weighing 0.3–0.5 g of each sample (the amount optimised for each sample) and adding 5 ml of 0.66 M
hydrochloric acid to each. The suspensions were shaken overnight to extract InsPs. The extracts were filtered by centrifuging the solution using filter tubes (Amicon® Ultra centrifugal filter units, 0.5 ml, 10 K, Regenerated cellulose, Merck Millipore, Ireland). All the solutions were analysed at the concentration obtained after extraction. Additionally, most of the extracts were diluted after centrifugal filtration by mixing 100 μl of the extract with 300 μl of 0.66 M hydrochloric acid, and a few samples with lower total InsP content were analysed using an additional injection volume of 100 μl. Analysis at two different aliquot amounts was used to quantify each InsP form from such injection where the concentration fitted into the standard curve. The samples were all filtered (0.45 μm, Acrodisc, Pall, UK) prior to the analysis of InsP forms according to the method described in 2.1.2. The results are reported as mg/g dry weight.
2.2.3. The moisture content analyses The InsP contents were calculated on a dry weight basis, and thus
moisture contents of the samples were analysed. Two different methods were used: an air oven method according to the AACC 44–15.02 was used for solid samples, and a quartz and sand method according to the AACC 44–60.01 was used for semi-solid samples (porridges).
2.2.4. Statistical analyses All analyses were conducted in three replicates, and the results are
reported as an average ± standard error of mean (SEM). Statistical Package for the Social Science (SPSS Statistics version 25, IBM, USA) was used to carry out ANOVA with a post-hoc Tukey test. Differences were considered significant if P < 0.05.
3. Results and discussion
3.1. Quantification of inositol phosphates using an anion exchange chromatographic method
The method reported by Rounds and Nielsen (1993) was originally used only for qualitative analysis of different InsP forms. Thus, this study aimed to further develop the method to be applicable for quantification of InsPs from oat products. The separation efficacy was tested with the IHR, where a mixture of different InsPs was produced in acid hydrolysis. The MS confirmed the presence of all six InsP forms with different phosphorylation degrees in the IHR (Fig. 1). The IHR contained all six inositol phosphates in their sodium salt form, detected with MS in negative mode as anions where one sodium had been cleaved. Thus, this sample was applicable for ensuring the stability of both the elution and detector response by monitoring retention times and peak areas. In the anion exchange chromatography system, five forms (InsP2-InsP6) were separated (Fig. 2) and identified by comparing to the commercial stan-dard compounds. The IHR contained several isomers of InsP forms, which were not fully separated with this method. However, the sepa-ration of different isomers was not needed since all isomers were to be calculated together. Thus, the run program was optimised to gain enough resolution for different InsP forms but simultaneously minimise the separation of different isomers. A similar method has been previ-ously used by Rounds and Nielsen (1993) for separation of InsP forms in a sample prepared by degrading sodium phytate by phytase. The peaks seemed to be narrower in the phytase-degraded phytate compared to the acid hydrolysed IHR in this study, which is likely resulting from the more specific degradation mechanisms when using phytase instead of random cleavage in acid hydrolysis. Thus, the differences in the peak forms are caused not by the dissimilar resolution, but by the IHR in this study containing a wider isomer distribution. Carlsson et al. (2001) stated that when using ion exchange chromatography, the separation of different isomers may actually lead to decreased sensitivity for some lower InsPs, a factor that should be acknowledged. However, as Carlsson et al. (2001) concluded, this is often not a problem for analysis of InsPs in food products, since the degradation in those most often occurs due to
Table 1 Ingredients of the samples described and the samples categorised (Adapted from Makela et al., 2020).
Sample name Sample description
Bread 1 100% of the grain ingredients from oat. Includes added hydrocolloids.
Bread 2 100% of the grain ingredients from oat. Includes added hydrocolloids and seeds.
Bread 3 100% of the grain ingredients from oat. Includes added hydrocolloids.
Bread 4 100% of the grain ingredients from oat. Includes added hydrocolloids.
Bread 5 100% of the grain ingredients from oat. Bread 6 100% of the grain ingredients from oat, except for rye flour used
as flour on the belt conveyor. Crisps and chips 1 Also contains grain ingredients other than oat. Crisps and chips 2 100% of the grain ingredients from oat. Includes added
hydrocolloids. Crisps and chips 3 100% of the grain ingredients from oat. Includes added
hydrocolloids. Porridge 1 Regular oat flakes. 100% of the grain ingredients from oat. Porridge 2 Regular oat flakes and bran. 100% of the grain ingredients from
oat. Porridge 3 Regular oat flakes and muesli flakes. 100% of the grain
ingredients from oat. Extruded product
1 Also contains grain ingredients other than oat.
Extruded product 2
Other grain ingredients from oat, except added starch. Includes added hydrocolloids.
Extruded product 3
Other grain ingredients from oat, except added starch. Includes added hydrocolloids.
Extruded product 4
100% of the grain ingredients from oat.
Spoonable product 1
100% of the grain ingredients from oat. Includes added hydrocolloids. Fermented.
Spoonable product 2
Other grain ingredients from oat, except added starch. Includes added hydrocolloids. Fermented.
Spoonable product 3
100% of the grain ingredients from oat. Includes added hydrocolloids. Fermented.
Meat analogue 1 100% of the grain ingredients from oat. Includes legume ingredients.
Meat analogue 2 100% of the grain ingredients from oat. Includes legume ingredients.
Meat analogue 3 100% of the grain ingredients from oat. Includes legume ingredients.
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enzymatic hydrolysis, which produces smaller amounts of isomers than other, more random degradation mechanisms.
The standard curves of the InsPs indicated a strong decrease in the sensitivity of this system when the degree of phosphorylation decreased (Fig. 3). Thus, the method is most applicable for the higher InsP forms, including InsP6, InsP5 and InsP4, but less sensitive for the lower InsP forms. InsP1 was not detected at the concentrations used, which is logical, as the monophosphate form cannot efficiently bind multivalent cations. Therefore, this method is not optimal for analysing the total InsP distribution in sample materials, but is here suggested to be used for the analysis of the so-called phytate-active forms, which include those forms that can significantly bind multivalent cations. Based on the standard curves, InsP4, InsP5 and InsP6 were considered to be efficiently binding iron, and thus, in this study, these three InsP forms are considered phytate active. Thus, it is to be borne in mind that when the quantified total InsP contents are reported, those might be slightly underestimating the actual total InsP content, since the lower forms are not necessarily detected. However, for IHR no underestimation was
observed. The calculated concentration of phytate in sodium salt hy-drate form in the IHR was 14.6 mg/ml. This phytate product contained unknown amount of sodium and water, and thus the actual amount of inositol phosphates in the IHR (= hydrolysed sodium phytate hydrate) was not known, but was expected to be somewhat less than the content of sodium salt hydrate form of phytate. The quantified total InsP content of IHR was 13.3 mg/ml, which seems reasonable when comparing to the known content, which includes the sodium and water. The contents of InsP2, InsP3, InsP4, InsP5, and InsP6 in IHR were 1.1 ±0.01 mg/ml, 3.6 ±0.01 mg/ml, 3.5 ±0.2 mg/ml, 4.8 ±0.3 mg/ml, and 0.24 ± 0.01 mg/ml, respectively.
Rounds and Nielsen (1993) used a similar method for the separation of different InsPs in a sample where a mixture of different InsPs was produced from sodium phytate using phytase. They showed good reso-lution for forms from InsP2 to InsP6 but concluded that myo-inositol and InsP1 remained unretained and thus eluted in the void peak. Since they did not conduct quantification, significant differences in the sensitivity of this detection method for different InsP forms were not shown. However, the present study indicated large differences in the sensitivity between the InsP forms, which must be borne in mind when using the method for either qualitative or quantitative purposes.
3.2. Inositol phosphate contents in oat products
The InsP contents of the consumed food products are of interest, as InsPs have several functional properties that are linked to their ability to bind minerals. In this study, the InsP contents were analysed from 23 oat products, including a non-commercial oat flour that was included in the sample set to see the InsP level in a minimally processed oat ingredient. Overall, the total InsP contents ranged from 1.34 mg/g (dw) to 13.6 mg/g (dw). In all products, the main InsP form was InsP6, indicating that in any product, phytate had not totally degraded to lower InsPs (Fig. 4). A significant amount of InsP5 was also found in all the analysed oat products, and a small amount of InsP4 was detected in many of the products. The results confirm the hypothesis, according to which the possibility of degradation of InsPs due to the endogenous oat phytases was considered minimal in all products. This hypothesis relied on the fact that oat phytase is efficiently deactivated by heating at a
Fig. 1. All six inositol phosphate forms identified in the in-house reference sample using mass spectrometric detection.
Fig. 2. Inositol diphosphate (InsP2), inositol triphosphate (InsP3), inositol tet-raphosphate (InsP4), inositol pentaphosphate (InsP5) and inositol hex-aphosphate (InsP6) separated in the anion exchange chromatography. Inositol monophosphate was not detected with this method.
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temperature ≥60 ◦C (Greiner and Alminger, 1999), and the oat pro-cessing in Finland includes a kilning treatment, which uses tempera-tures higher than that.
3.2.1. Inositol phosphates in minimally processed oat products remain rather intact
The oat flour that had been made from the dehulled and kilned groats contained, in total, 8.6 mg/g (dw) of InsPs. This is in accordance with the phytate contents reported for oat, as the reviewed variation was 4.2–11.6 mg/g (dw) (Reddy, 2001). The used analysis method varies in different studies, and in the analyses based on the measurement of phosphorus released in the enzyme hydrolysis, some overestimation of the phytate content is likely to occur, since all the phosphorus is
calculated to originate from InsP6. However, in minimally processed oat ingredients, such as oat groats and oat flours, the error is assumed to be rather minimal due to the low amount of phytate degradation.
The results, however, also showed some degradation in the mini-mally processed oat flour, since in this sample the proportions of InsP6, InsP5 and InsP4 were 72%, 26% and 2%, respectively. This differed from the results of Kasim and Edwards (1998), who reported that only InsP6 was detectable with a liquid chromatography method using a reverse-phase column and refractive index (RI) detector. However, Lehrfeld (1994) also used RI for detection of InsPs and showed the presence of InsP6, InsP5, InsP4 and InsP3 with the relative amounts being 81%, 16%, 2% and 1%, respectively. In a study by Sandberg and Svanberg (1991), InsP5 and InsP4, in addition to InsP6, were detected
Fig. 3. Standard curves for inositol diphosphate (InsP2), inositol triphosphate (InsP3), inositol tetraphosphate (InsP4), inositol pentaphosphate (InsP5) and inositol hexaphosphate (InsP6), indicating the relation of the chromatogram peak area and concentration of the specific InsP form (mg/ml). The coefficient of determination (R2) is shown for each InsP form.
Fig. 4. Inositol phosphate (InsP) contents (mg/g dry weight) of oat products from different product categories. The detected forms included inositol triphosphate (InsP3), inositol tetraphosphate (InsP4), inositol pentaphosphate (InsP5) and inositol hexaphosphate (InsP6). Error bars show the standard error of mean for each InsP form.
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from the whole meal oat flour, where the phytase had been deactivated by heat treatment. In the non-heated whole meal oat flour, 96% of the InsPs were in InsP6 form, but during the phytase-deactivation, they showed some degradation of InsPs: In the whole meal flour made from the phytase-deactivated oat, 88% of the InsPs were in InsP6 form and the portions of InsP5 and InsP4 were about 12% and 2%, respectively. The deactivation process involved autoclaving the samples at 120 ◦C for 6 min, and the authors suspected some degradation during this process. The difference in the observed proportion of different InsP forms in these two studies may result from the differently conducted heat treatment. This result for the oat flour suggests that the phytate does not remain fully intact during the enzyme-inactivation step. However, without that step, degradation would be more severe due to the active phytase in the products. This was seen in a study by Sandberg and Svanberg (1991), where a 61% reduction in the InsP6 content was shown during the 17-h incubation at 55 ◦C and pH 5 for non-heated whole meal oat flour where the endogenous enzymes were not inactivated. Thus, in all oat products, likely some level of degradation occurs, either during the heat treatment of the oat ingredient to inactivate the enzymes or during food produc-tion due to phytase or processing-induced degradation.
The oat porridges were cooked using oat flakes, and since these flakes contain the whole dehulled oat groat, the InsP contents were assumed to be at similar level as in oat groats or oat flours generally. The total InsP contents of oat porridges were 9.4–10.2 mg/g (dw), and thus the values obtained were in the range (4.2–11.6 mg/g [dw]) reported for phytate content of oat groats by Reddy (2001). None of the porridge samples contained other InsP forms than InsP6 and InsP5, which in-dicates a low extent of degradation. However, the proportion of InsP5 was still significant, especially in porridge 3, where about 40% of InsPs were InsP5. Since the endogenous phytase was expected to be inacti-vated in the kilning step, the degradation degree varied most likely due to the differences in the flaking process. Grüner et al. (1996) studied phytic acid content before and after flaking treatments and showed some differences. The dehulled oat grains were shown to contain 12.07 mg/g (dw) of phytic acid, but the amount decreased to 11.17 mg/g (dw) after the hydrothermal treatment, which was done prior to the actual flaking. The flaking step was conducted at 70 ◦C, and during that, the phytic acid content further decreased to 10.46 mg/g (dw). Although the degrada-tion during the flaking process was not considered substantial, the re-sults indicate a possibility of some degradation. The method used by Grüner et al. (1996) did not analyse the InsP forms but calculated the determined phytic acid phosphorus to phytic acid. Thus, potential degradation of InsP6 to InsP5 would indeed be seen only as a slightly lowered total phytic acid content. The differences seen in the proportion of InsP6 of the total InsP content in porridge samples may be caused by the possible differences in the flaking process, since there are differences in the diameter and thickness of the flakes and the amount of fine ground material, both of which may affect the amount of degradation in the material.
3.2.2. Phytase activities in the fermentation processes in production of breads and spoonable products result in differing extents of InsP degradation
The possibility of degradation of InsPs resulting from the phytase secreted by the microbes was to be considered in such sample categories where the production involved fermentation. All samples in the bread category contained yeast, and thus in these sample the effect of yeast fermentation on the phytate degradation was investigated. Additionally, the effect of bacterial phytases was studied in the category of spoonable products, as their production had included bacterial fermentation.
In the bread products, the detected total InsP contents ranged from 7.30 to 8.90 mg/g (dw), and thus were similar to the InsP content of oat flour (8.61 mg/g [dw]). In all breads, oat was the only grain ingredient and the breads contained only minor amounts of components other than those from oat, and thus the phytases from grain ingredients were not expected to be active. For this reason, the total content of InsPs was hypothesised to be at similar level as in oat flour or slightly higher due to
the inclusion of other oat fractions (e.g. oat bran) in the ingredients. The proportion of InsP6 of the total content varied only slightly within the bread category, and overall, the contents of different InsP forms seemed to be rather similar as in the oat flour. In five of the bread samples, the InsP6 accounted for 64–72% of the total InsP content, but in bread 1, the percentage was higher (80%). This difference was not explained by the ingredients, since there were no ingredient variations that would affect the extent of the InsP degradation. Instead, the added bran was expected to increase the total InsP content, since InsPs locate in the outer layers of the grain. Larsson and Sandberg (1991) reported almost six times higher phytate content in oat bran than in oat flour, as the contents were 38.5 and 6.6 μM/g (dw), respectively. However, here, the inclusion of oat bran in the ingredients of the breads was not reflected in the total InsP contents, which might be due to the relatively small proportion of oat ingredients being bran. Overall, the effect of ingredients is difficult to estimate in these bread products, since the proportional amounts of different oat ingredients in the recipes and the InsP contents of the original ingredients used for the breads are not known. Considering the results of these different oat bread products, however, the variation in the full oat bread category seems to be small.
Drastic degradation of InsPs during the bread-baking process has been previously reported (García-Estepa et al., 1999; Sandberg et al., 1999), but in those studies, the breads contained wheat ingredients. Since in this study, the breads contained no other grain ingredients than oat, the endogenous enzymes were inactivated by the kilning of the oat groats prior to processing them further. However, the influence of the phytase activity of the added yeast was not known, and hence, the possibility of degradation was considered. The results, though, indicate that no drastic degradation occurred during baking. Nakamura et al. (2000) studied the phytase secretion of 300 yeast strains and found significant phytase activity in 35 strains, one being the yeast commonly used in baking, Saccharomyces cerevisiae. All the phytases secreted by the yeasts were found to have an optimal pH in the acidic range (at pH ≤ 5), and the optimal temperature ranges were relatively high (≥60 ◦C, often even 75 ◦C). Thus, even if there was yeast-secreted phytase activity during the fermentation step of the bread production, the low pH opti-mum and high temperature optimum of the yeast phytases are probably resulting in a quite low degree of degradation during this process.
All the studied spoonable products contained low amounts of InsPs ranging from 1.3 to 2.7 mg/g (dw). The total InsP contents were significantly lower in the spoonable products compared to the other product categories, which indicates drastic degradation of InsPs during their production. The production of all of these spoonable products included bacterial fermentation, and the bacteria used in the process are most likely similar to those used for yogurt production. The common yogurt bacteria include lactic acid bacteria, which in many cases do not have phytase activity, as reported by Koniezny and Greiner (2002). They suggested that the decreased phytate content in plant-based foods fer-mented with lactic acid bacteria could result from the precipitation of phytate with protein. However, Sreeramulu et al. (1996) reported phytase activity for some bacteria that can be used in yogurt production: Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus plantarum and Streptococcus lactis. When comparing the total InsP contents of the bread samples and spoonable products, the results imply that the bac-teria used in the fermentation in spoonable products either have higher phytase activity than the yeast in the bread making or that the condi-tions during the bacterial fermentation are more favourable for the phytases present.
3.2.3. High variation in the inositol phosphate contents of products processed with high temperatures or extrusion
The production of crisps and chips includes high temperatures and possibly even extrusion where the high temperature is further combined with shear forces and pressure. Most of the samples in both the cate-gories of crisps/chips and extruded products had lower total InsP con-tents (3.5–6.5 mg/g [dw] and 5.3–7.6 mg/g [dw], respectively) than
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products in the flour, bread and porridge categories, which indicates that the processes including high temperature or pressure may degrade InsPs. However, when comparing these values to the total InsP contents of the minimally processed oat products, the degradation of InsPs was not as drastic as one might expect when considering the harsh conditions during these thermal treatments. Schlemmer et al. (2009) reviewed the effect of different processes on phytate degradation and concluded that InsPs are rather heat stable. At temperatures above 100 ◦C, however, some degradation was reported to occur, and the amount depended on the combination of the temperature and time during the treatment. Some degradation of InsPs was, however, shown in the categories of crisps/chips and extruded products, since they contained rather low relative amounts of InsP6: 57–71% in six samples, the seventh being an exception with 95% of InsPs being InsP6. These results are similar to the values reported for other similar products, the production of which has contained high-temperature treatments. For example, during the pro-cessing of breakfast cereals, different InsP forms have been reported since the proportions of InsP6, InsP5, InsP4, and InsP3 were analysed to be 66%, 25%, 8% and 1%, respectively, as reviewed by Phillippy (2003). Also, Lehrfeld (1994) reported toasted oat cereal to have the following InsP distribution: 51% InsP6, 32% InsP5, 13% InsP4 and 5% InsP3. The variation in the processing conditions may be the reason for the observed differences in the total contents and InsP forms of the samples in the crisps/chips category. Also, crisps/chips 1 contained other grain ingredients in addition to oat and thus endogenous enzymes from those grain materials. This resulted in a rather high InsP3 content (0.6 mg/g [dw], which accounts for 16% of the total InsP content), indicating se-vere degradation.
The category of extruded products contains products with low moisture content. In the extruded products, the InsP contents were slightly higher than in crisps and chips, and this is in accordance with a review by Schlemmer et al. (2009), which reported rather mild degra-dation during extrusion. However, the efficacy of extrusion to hydrolyse phytate was stated to depend on the extrusion conditions. In a study by Gualberto et al. (1997), extrusion was shown to decrease the phytate content of oat bran from the original 1.42%–1.05% when the mass temperature during the extrusion gained 194 ◦C, but the change was statistically insignificant. In the present study, some degradation, though rather mild, was observed in the extruded products. The results of crisps/chips and extruded products suggest that, even when using high temperature treatments, sufficient treatment time is required to reduce the InsP content in oat products significantly.
Quite recently, the interest towards the production of meat ana-logues has raised, at least partly due to the need for sustainable plant- based alternatives for meat products and also to be able to provide protein sources for the growing population. The production of meat analogues can occur through several processes, but extrusion cooking with the higher moisture content is one of the known processes (Kaleda et al., 2020; Liu and Hsieh, 2008). However, instead of the decrease in the total InsP content observed in the extruded products’ category, in meat analogues, the contents were similar or even higher to the cate-gories with the minimally processed products. This is explained by the inclusion of legume ingredients in all the meat analogues, since several legumes have higher phytate contents (e.g. 6.1–23.8 mg/g (dw), 5.1–17.7 mg/g (dw) and 2.8–16.0 mg/g (dw) in kidney beans, faba beans and chickpeas, respectively) compared to the 4.2–11.6 mg/g (dw) in oat (reviewed by Reddy, 2001). In the category of meat analogues, the variation in the total InsP contents was high, with the contents varying from 8.5 to 13.6 mg/g (dw). InsP6 accounted for only 56–61% of the total InsP contents, which indicates that regardless of the high total InsP contents, some degradation had occurred. This might result from the phytase activity, since kilning-like heat treatment is not routinely done to the legume ingredients and active phytase is likely to occur in these products during the processing steps prior to the heating in extrusion. Additionally, the extrusion process in the meat analogue production has been shown to degrade phytate, since Kaleda et al. (2020) reported 18%
reduction in InsP6 when meat-like textures were produced from pea-oat powder by extrusion. Altogether, the products in the meat analogue category probably had significant degradation, probably both due to the phytase acitivity and the processing conditions, although the total amounts remained high in these products.
4. Conclusions
Anion exchange chromatography was shown to be a suitable method for quantifying inositol phosphate forms that possess the mineral bind-ing ability close to phytate and thus were called phytate-active forms. In oat products with mild processing, no severe degradation of InsPs occurred. For the thermal degradation of InsPs, the process time has an essential role, and the short exposure to the elevated temperatures does not result in a significant decrease in the InsP content. In oat baking, the InsPs remained rather intact, indicating that no drastic degradation was caused by the yeast phytases during dough fermentation. However, in spoonable products the InsP contents were significantly lower than in other products and this was most likely due to the bacterial fermentation during the production of these products. This suggests that for degra-dation of inositol phosphates by phytases, the processing parameters must be favourable for the enzyme. According to the results, inositol phosphates are rather stable in oat products, while endogenous phytases have been inactivated in the kilning treatment of the grain.
Funding
This work was supported by the Business Finland [129/31/2017].
CRediT authorship contribution statement
Noora Makela: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Tuula Sontag-Strohm: Conceptualization, Writing - review & editing, Funding acquisition. Miikka Olin: Meth-odology, Writing - review & editing. Vieno Piironen: Conceptualiza-tion, Writing - review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2020.103121.
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