Digestibility of protein and iron bioavailability from enriched sprouts

Digestibility of protein and iron bioavailability from enriched sprouts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Digestibility of protein and iron bioavailability from enriched sprouts Magdalena Zielińska-Dawidziak, Wojciech Białas, Dorota Piasecka-Kwiatkowska, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2084755/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Plant ferritin is suggested as a good source of iron for human. Usually present in trace amounts, it was induced in legumes seeds by their sprouting in FeSO 4 solution. Fortified sprouts were digested in the in vitro model of the human gastrointestinal tract. ~49% of lupine and ~ 45% of soy proteins were extracted into gastric fluid and next ~ 12% and only ~ 1% into intestine fluid from lupine and soybean, respectively. Gastric digestion released mainly ferrous iron (~ 85% from lupine and ~ 95% in soybean sprouts). Complexed iron constituted ~ 43% of total iron in intestine after lupine digestion and ~ 55% after soybean digestion. Intestine digestion doubled the total iron released from lupine sprouts (from ~ 21% up to 38%), while in soybean it increase from ~ 16% up to ~ 23%. Ferritin presence was confirmed by the specific antibodies in digestive fluids, but it is only partially extracted from sprouts during in vitro digestion. ferritin digestibility soybean lupine fortified sprouts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Iron deficiency anemia (IDA) is the most common type of anemia (about 50% incidence) affecting ~ 1.48 billion people in 2015 and more than 1.2 billion in 2016, particularly infants, children, adolescents, pregnant women, the elderly or people suffering from eating disorders [ 1 , 2 ]. To treat IDA, diet modification and supplements intake are recommended. However, iron supplementation (both as pills application as well as enriched food consumption) may bring numerous side effects, such as constipation, diarrhea, abdominal pain and nausea [ 3 ]. An alternative to this type of strategy may be the use of food biofortified in iron, such as legumes sprouts enriched in ferritin [ 4 , 5 ]. Ferritin is a protein able to contain up to 4,500 Fe ions, and it also protects iron from chelating agents present in the diet. However, studies on the bioavailability of ferritin iron are contradictory [ 6 – 9 ]. I n vivo results suggest higher stability of ferritin than in vitro studies on the ferritin iron bioavailability [ 6 , 9 , 10 ]. This may be due to the fact that pure, isolated ferritin is usually used in in vitro studies, whereas food rich in ferritin is often part of the human diet in in vivo studies. Clinical studies, despite the large development of in vitro methods, are still considered a "gold standard" of nutritional research [ 11 ]. However, in vitro methods are characterized by a significantly shorter time, the ease of sampling a large number of samples, lower costs and labor consumption, a guarantee of repeatability and no ethical restrictions. That is why still in vitro methods simulating digestive processes are widely used in research on the behavior of food and pharmaceutical products in the digestive tract [ 11 ]. The aim of the presented research was to study the bioavailability of iron and proteins from the sprouts enriched in iron at individual stages of digestion in a simplified in vitro digestion model. It is hypothesised that food constituents may have a protective effect on ferritin during digestion and increase its potential as a source of bioavailable iron. 2. Material And Methods 2.1. Sprouts preparation Soy ( Glycine max , Augusta variety) and lupine seeds ( Lupinus luteus , Lord var.) from the Poznan University of Life Sciences cultivations were used for the preparation of iron-enriched sprouts. The sprouting process was carried out according to the procedure presented previously [ 4 , 12 ]. The seeds were cultured for seven days under controlled conditions and watered with 25 mM FeSO 4 from the third day of cultivation. Then the sprouts were dried, until their moisture was decreased to below 14%, and milled. Two manners of material drying were applied: continuous drying in 35 o C, or two-stage drying: 5 hours in 80 o C, followed by drying in 35 o C. As a control sample the sprouts not fortified in iron (watered with tap water only), prepared in the same conditions, were applied. The sprouts were cultivated and dried in 7-fold repetition, mixed, and stored in an airtight glass container prior to further analysis. 2.2. In vitro digestion experiment The digestion of the prepared sprouts was done by an in vitro method, simulating two-stage multi-enzymatic (gastric and intestinal) digestion [ 13 , 14 ]. The digestion in the oral cavity was not considered, because it has no effect on their digestibility (the studied material is rich in protein, poor in starch). The large intestine stage was also omitted, as not important in iron bioavailability studies. A studied sample was introduced into distilled water containing pepsin (60,000 U) (Sigma) and pH of the mixture was lowered to 2.0 with 1 M HCl. Gastric digestion was carried out for 2 hours, at 37 o C. Then, the pH of the solution was adjusted to 7.4 and a solution containing pancreatic-intestine extract (0.005 g, Sigma) and bile salts (0.03 g, Sigma) in 5 ml 0.1 M NaHCO 3 was added. The digestion was again performed at 37°C for 2 hours. The solution was centrifuged and the remaining extracted and not digested proteins were precipitated with trichloroacetic acid. In the sample prepared in this way, protein nitrogen was determined using the Kjeldahl [ 15 ] method and was related in percentage to the amount of protein nitrogen introduced with the sample into the digestion test. Additionally, the concentration of protein released from the tested material into digestive fluids at individual stages of digestion (i.e. stomach and intestine) before precipitation by TCA was determined with the Bradford method [ 16 ]. 2.3. Iron determination The iron released during digestion from the sprouted seeds was determined as total iron and iron in ionic form. The total iron determination was conducted with atomic absorption spectrometry with air-acetylene flame atomization, while the ionic iron was determined as Fe(II) and Fe(III) forms by the colorimetric procedure. Fe(II) ions content was determined in reaction with 2,2′-bipirydyl in the environment of acetate buffer (pH 4.5), while Fe(III) content was determined in reaction with thiocyanate in the environment of hydrochloric acid (pH < 2) using photometry (wavelength 470 nm). For each sample, a blank sample was prepared and the obtained value was used to correct the interference of the sample color [ 17 ]. The complexed iron content (in the studied preparation considered as iron bound mainly in ferritin form) was calculated from the difference between the total iron content obtained with the atomic absorption spectrometry and ionic iron, which was the sum of Fe(II) and Fe(III) contents obtained during the photometric analyses. 2.4. Lupine ferritin standard preparation Purification of lupine ferritin was made using Korcz and Twardowski method [ 18 ]. The procedure included two-step salting out of protein from the homogenized sprouts (in the buffer containing 50 mM Tris-HCI, pH 8,0, and 30 mM NaCl). Next, the ferritin from the isolated protein was purified by fourfold ultracentrifugation (100000 x g/120 min). 2.5. FPLC separation The chromatographic studies were performed with an AKTA Explorer 100 Air System (Amersham Pharmacia Biotech, Uppsala, Sweden). A HiLoad 26/60 Superdex 200 column from Amersham Pharmacia Biotech (Uppsala, Sweden) was used. Assays on digested samples were performed at room temperature, at a flow rate of 2 ml/min. A 15 ml sample was eluted with 0.05 M of phosphate buffer pH 7 containing NaCl in a concentration of 0.5 M. The chromatographic mobile phase prior to use, and all samples before injection into the column, were filtered through a membrane filter (0.45 um, Millipore). During the chromatographic run, fractions were collected in volumes of 12 ml. Absorbance at 280 nm was applied for protein detection. The column was calibrated with the selected standard proteins included in the low molecular weight (LMW) and high molecular weight (HMW) range calibration kits (Cytivia, USA). The following proteins were used: Aprotinin (6500 Da), Carbonic anhydrase (29 000 Da), Ovalbumin (43 000 Da), Conalbumin (75 000 Da), Aldolase (158 000 Da), Ferritin from Horse spleen (440 000 Da) and Blue Dextran (2 000 000 Da). A calibration curve was prepared by measuring the elution volumes (Ve) of standards, calculating their corresponding partition coefficient (Kav values), and plotting their Kav values vs the logarithm of their molecular weight. The Kav was calculated with the following equation: $${\text{K}}_{av}=\frac{{\text{V}}_{\text{e}}- {\text{V}}_{0}}{{\text{V}}_{\text{t}}- {\text{V}}_{0}}$$ 1 where V e is elution volume for the standard (ml), V 0 is column void volume = elution volume for Blue Dextran 2000 (ml), Vt is total column volume (mL). Calibration data is included in Supplementary Materials. The fractions collected after separation by size exclusion chromatography with a retention volume similar to the lupine ferritin standard were further analyzed (SDS-PAGE, western blot and slot blot). 2.6. SDS-Page separation The fraction separated by FPLC method was subjected to electrophoresis. The protein profile was examined using 14% polyacrylamide gel electrophoresis under denaturing conditions [ 19 ]. Samples obtained from soy chromatography were directly denatured, while lupine samples were first concentrated 10 times through filters with point cut-off 3 kDa (Amicon Ultra, Millipore Ltd., Poland). Gels were dyed with Coomassie Brilliant Blue and documented using CLIQS (TotalLabQuant, UK). Molecular mass of the detected protein was determined by reference to molecular mass marker in range 20–120 kDa (Thermo Scientific,USA). 2.7. Ferritin immunodetection 2.7.1. Slot-blot analysis The collected fractions after FPLC analysis were applied on the PVDF membrane (Immobilon-P 0,45 µm, Merck Millipore Ltd., Poland). The applied volume was 200 µl for lupine fractions and 20 µl for soy fractions. 1% BSA in Tris-buffered saline, pH 7.4 was used as the blocking agent (1-h incubation). Next, the membrane was incubated for 2 hours with the goat sera containing anti-lupine ferritin antibodies, diluted 1:100 (provided for the research by the Institute of Bioorganic Chemistry of the Polish Academy of Sciences in Poznań). As a secondary antibody, rabbit anti-goat IgG polyclonal antibody, marked with horseradish peroxidase was applied for 2 hours in 1:1000 dilution (Invitrogen, USA). Detection of protein-bound antibodies was performed with diaminobenzidine (Sigma-Aldrich, USA) in 20 minutes. 2.7.2. Western blot analysis Protein fractions separated by SDS-PAGE electrophoresis were also transferred by a semi-dry electrotransfer (200 mA current for 30 min. and 120 mA for 90 min.) to a polyvinylidene difluoride membrane (Immobilon-P 0,45 µm, Merck Millipore Ltd., Poland). The same antibodies and method of detection were used as presented above for the slot-blot analysis. The membranes were analysed using the CLIQS program (TotalLab Quant, UK). 2.8. Statistical analysis The necessary statistical analyses were performed using Statistica 13.0 (StatSoft, USA). All numerical data were presented as mean ± SD. The statistical significance of the difference between the control and the treated sample was assessed by one-way ANOVA and post-hoc Tukey’s tests. Results were considered statistically significant at P < 0.05. 3. Results And Discussion 3.1. Protein digestibility The protein content in the tested lupine seeds (43.61 ± 0.22 g/100 g d.m.) and sprouts (47.76 ± 0.40 g/100 g d.m.) is over 4% higher compared to soybean seeds (39.18 ± 0.19 g/100 g d.m.) and sprouts (42.51 ± 0.13 g/100 g d.m.), but it may differ depending on the seed variety and conditions of seed cultivation [ 4 ]. Samples subjected to the digestion process were unified in terms of the amount of total protein in the dry matter (Table 1 ). Only the S.Fe.35 (soybean sprouts enriched in iron, dried continuously in 35 o C) variant differed slightly, but statistically significantly. Table 1 Total protein and soluble protein balance in the test material before and after in vitro digestion (g/d.m.) Sample Content of total protein in the weight of material introduced into digestion [g/100 g d.m.] Content of total protein in sample [g/100 g d.m.] Total digestibility [%] Soluble protein content determined in fluids after digestion by Bradford method [µg/ml] after the 1st stage of digestion after the 2nd stage of digestion Fluid Sediment Fluid Sediment after the 1st stage after the 2nd stage L.0.35 2.63 ± 0.01 a * 1.28 ± 0.00 a 1.35 ± 0.02 b 1.40 ± 0.03 a 1.22 ± 0.03 d 53.23 a 364.17 ± 5.20 b 298.33 ± 1.44 a L.0.80 2.61 ± 0.01 a 1.26 ± 0.01 a 1.35 ± 0.02 b 1.65 ± 0.01 c 0.96 ± 0.02 b 63.22 b 346.67 ± 1.44 a 333.33 ± 3.82 b L.Fe.35 2.65 ± 0.00 a 1.25 ± 0.01 a 1.40 ± 0.01 b 1.84 ± 0.02 d 0.81 ± 0.02 a 69.4 c 350.83 ± 5.00 a 465.00 ± 11.55 d L.Fe.80 2.63 ± 0.02 a 1.37 ± 0.02 b 1.26 ± 0.03 a 1.51 ± 0.02 b 1.12 ± 0.04 c 57.4 d 375.00 ± 2.89 c 379.17 ± 11.46 c S.0.35 2.37 ± 0.01 a 1.16 ± 0.01 c 1.21 ± 0.01 a 1.06 ± 0.01 a 1.31 ± 0.01 b 44.73 a 388.33 ± 13.23 c 462.50 ± 10.10 c S.0.80 2.37 ± 0.01 a 1.07 ± 0.01 b 1.30 ± 0.01 b 1.11 ± 0.01 b 1.26 ± 0.01 a 46.84 b 305.00 ± 6.29 a 370.83 ± 6.29 a S.Fe.35 2.39 ± 0.01 b 0.91 ± 0.01 a 1.48 ± 0.01 c 1.14 ± 0.01 b 1.25 ± 0.01 a 47.70 b 320.83 ± 4.33 b 550.00 ± 7.64 d S.Fe.80 2.36 ± 0.01 a 1.17 ± 0.01 c 1.19 ± 0.01 a 1.11 ± 0.01 b 1.25 ± 0.01 a 47.04 b 411.67 ± 7.64 d 425.00 ± 14.65 b * a, b, c, d – statistically determined (with the Tukey Test) homogeneous groups (separately for results of soybean and lupine in the columns) The availability of nutrients for the human body depends not only on their content in the digested material, but also on the extractivity of the components from digested material at individual stages of digestion. Ingredients remaining in undigested material are excreted with feces. Absorption is only possible for those substances which, after being extracted in the stomach or intestine, are dissolved in digestive fluids. Thus, the nitrogen compounds (recalculated into total protein) released from the digested material were studied. The most of total protein during digestion was released into the gastric fluid from the L.Fe.80 (lupine sprouts enriched in iron and dried in 80 o C for the first 5 hours) and S.Fe.80 (enriched soy sprouts dried in the same conditions for the first 5 hours) variant, and the least from L.Fe.35 and S.Fe.35 (lupine and soy sprouts enriched in iron and dried continuously in 35 o C). It may be explained by the denaturation of proteins during drying at 80°C, which facilitates their hydrolysis, but also extraction in the stomach. On the other hand, as a result of total digestion, the most total protein is secreted into intestinal fluid during digestion of L.Fe.35 and S.Fe.35, while the least is in L.0.35 and S.0.35 (not enriched soy and lupine sprouts dried in 35 o C). Most of protein is extracted to the gastric fluid (respectively ~ 49% for lupine and ~ 45% for soybean sprouts) and average increase in the total content of protein in the intestinal fluid (i.e. total digestibility) is up to ~ 61% for lupine and only up to ~ 46% for soybean sprouts. These results suggest that proteins of lupine sprouts were more digestible, and it may result from the decreased content of trypsin inhibitors compared to soy. Lupine is usually indicated as a legume with trace content of trypsin inhibitor activity (M. [ 14 ]. However, both the thermal treatment and the sprouting processes reduce the activity of these inhibitors in the material [ 20 , 21 ]. Total protein content informs us about extractivity of nitrogen compounds from the digested material. Intestinal enterocytes absorb mainly free amino acids or very short peptides. Only few protein are absorbed via endocytosis, but in the group ferritin is located [ 8 , 9 ]. Thus, in order to distinguish the amount of proteins that are released during digestion from amino acids, short peptides and nucleotides, determination of the protein extracted from the tested material was done by the Bradford method. The method allows to determine peptides/proteins that exceed 3–5 kDa [ 22 ], i.e. peptides composed of at least ~ 27 amino acids. The highest increase in the soluble protein content in intestine fluid compared to stomach fluid was observed in samples fortified in iron and dried in 35 o C (L.Fe.35 - ~35%, S.Fe.35 - ~70%). Thus, the thermal denaturation of protein in the studied material could increase their digestibility. This may confirm the thesis that not all proteins contained in the test material are susceptible to the action of digestive enzymes, which may affect their further absorption. Ferritin is a protein resistant to high temperatures, i.e. 85° C, low pH and a number of proteolytic enzymes, with confirmed extractivity in pH close to 8.0 [ 10 , 23 ]. And in vivo studies confirm its resistance to digestion [ 9 ]. Thus, the presented results suggest possible ferritin extraction among other proteins in the intestine. 3.2. Iron release As a next step in the presented experiments, the release of iron during digestion of the studied material was checked. At this stage of experiment L.Fe.35 and S.Fe.35 samples were analysed. Significant differences were observed even in the color of the liquids obtained after digestion of lupine (Fig. 1 a) and soybean (Fig. 1 b) sprouts. The color observation indicates the release of iron from the tested material in the intestine, and a different form of iron released in the two studied steps of digestion. For the bioavailability of iron from the studied material not only compound resistance to the digestive enzymes may be important, but also their susceptibility to extraction from the food matrix. The observed ‘red’ color of the intestine fluid obtained after lupine sprouts digestion suggests the ‘red’ iron presence, which can be the result of both the presence of ferric ions (Fe 3+ ) and a complexed form of iron (such as ferritin). Thus, iron speciation in the obtained digestive fluids was performed. During the first step of digestion from the studied material most of released iron was in ionic form, as ferrous iron (85%±3% in lupine and 95%±2% in soybean) (Fig. 2 ). Moreover, when the same model of digestion was performed without the use of enzymes, solely acid action caused the release of ~ 80% of these iron amounts. Intestine digestion allowed to almost double the amount of iron released from lupine sprouts (from ~ 21% up to 38% of total iron), while in soybean it was not such a significant increase (from ~ 16% up to ~ 23%). Ferric iron (which is less available) constituted ~ 25% of total iron in intestine after lupine digestion and ~ 18% after soybean digestion. It suggests that administration of vitamin C together with the prepared sprouts should increase the iron absorption in the intestine. The increased content of complexed iron (Fig. 2 ) was detected and it constituted ~ 16,5% of total iron for lupine and ~ 12% for soybean sprouts. However, this result still does not prove or exclude the stability of ferritin during digestion. Iron may be complexed here also by polyphenolic compounds, synthetized in response to stress conditions in time of sprouting [ 24 , 25 ]. 3.3. Isolation of ferritin from the digestive fluids Thus, as a next step, chromatographic isolation of ferritin was attempted from the fluids obtained after digestion to confirm or deny the possibility of extraction and stability of ferritin during digestion of the studied samples. Chromatogram presented in Fig. 3 A and 3 B suggests that ferritin (collected in the fraction between 125–155 mL) is not present in the fluid after gastric digestion. This may result mainly from the inability to extract ferritin under such conditions. The best extraction conditions to obtain ferritin fractions with good yield correspond to pH approx. 8.0 [ 18 ]. Another essential factor that could have influenced the ferritin level after gastric digestion is the low pH of the environment in which this process takes place. As Bejjani, Pullakhandam, Punjal and Madhavan [ 26 ] demonstrated, pea ferritin dissociates when exposed to a low pH, releasing iron into the digestive fluid simultaneously. Under these conditions, the proportion between the number of α-helices and β-sheet structures changes, wherein the number of the latter increases significantly. It is worth noting that in the case of the presented results, correction of stomach fluid pH after the end of this step of digestion up to 7.4 did not modify the results. It excludes the possibility of reassembling ferritin degraded in the stomach after modification of pH, as it was observed in other studies [ 27 , 28 ]. Solely intestine digestion of sprouts, both lupine and soybean, resulted in extraction ferritin from the material. Moreover, this extraction was many times higher in case of soybean. Simultaneously, after application of two-step digestion of the material, the amount of extracted ferritin decreased, especially for soybean. This suggests the advisability of administering ferritin preparations after encapsulation, limiting its contact with the gastric fluid (e.g. in eudragit), which is not consistent with the observations of Theil [ 9 ]. Intestine condition seems to be more convenient to ferritin extraction, but also safe to maintain its structure and, consequently, to ensure the possibility of its absorption by endocytosis [ 8 ]. However, this extraction is still limited – the pH 7.4 is far from pH 8.0 suggested for ferritin extraction. 3.4. The detection of ferritin in the digestive fluids The samples were subjected to the SDS-PAGE analysis. Analysis of the obtained gels (Fig. 4 ) suggest the presence in the studied fraction of peptides with molecular weight close to the molecular weight of ferritin subunits. Molecular weight suggested for ferritin is close to ~ 450 kDa. It is multimeric protein composed from 16 subunits with molecular weight ~ 28 kDa, although for lupine ferritin the presence of the second subunit, the product of deletion of amino acids from the C-end, is also confirmed [ 23 , 29 , 30 ]. However, results of the electrophoretical separation of such not homogenous material (even partially purified ferritin isolate – line F, Fig. 5 ) could be very misleading. Proteins separated by gel electrophoresis were transferred to the membrane and detected by polyclonal anti-ferritin antibodies. Western blot analysis (Fig. 5 A and B) confirmed reaction of the goat serum with the protein present in the fraction separated by FPLC from fluids obtained after digestion of soy sprouts and lupine sprouts. The antibodies recognised even the ferritin subunits in fluids after gastric digestion, which suggests that some ferritin was extracted and denatured during gastric digestion. The same result was observed in slot-blot analysis (Fig. 5 C). It must be remembered that samples from gastric digestion had to be concentrated before analysis and the consistency of soy samples from intestine (both solely I, as well as SI digestion) significantly impeded the migration of the sample through the membrane. 4. Conclusion Presented results confirmed previous observations of protein shell dissociation induced by acid in gastric conditions [ 6 , 26 ]. Simultaneously, these conditions are considered not to be suitable for the extraction of ferritin from complex matrices (plant materials or food). Thus, it may be expected that ferritin did not ‘partially escape’ from stomach digestion [ 31 ], but just that it was not extracted (or only partially extracted) in stomach. These explain also differences between the results of in vitro studies on pure, isolated ferritin and in vivo studies, when ferritin was administered as a food ingredient. In order to use ferritin effectively as a unique source of iron absorbed in the intestines, the matrix / food should not only be processed to keep the ferritin undenatured. It is also important to release this protein from the matrix uniquely during digestion at the intestinal stage. This will allow the use of ferritin both as a source of iron (released from ferritin by its denaturation and transported by DMT1), and as a protein absorbed by endocytosis, and thus, by a unique system mechanism in intestinal enterocytes [ 8 ]. The presented experiment confirmed the protective effect of food ingredients on the stability of ferritin during digestion. The results are unique because they do not present studies carried out on isolated ferritin, but on a material containing the protein that can be a separate dietary supplement or added as a food ingredient. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: All authors agreed to publish the results in Plant Foods for Human Nutrition Competing Interests The authors declare that they have no known competing financial interests Author contributions: Magdalena Zielińska-Dawidziak – conceptualization, methodology, validation, investigation, resources, writing original draft; Wojciech Białas - conceptualization, methodology, validation, investigation, resources, writing original draft; Dorota Piasecka-Kwiatkowska – investigation (total protein content supervision); Halina Staniek - investigation (total iron content); Przemysław Niedzielski - investigation (iron speciation) Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. It was financed by our Universities. 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World J Gastroenterol 13:2083–2088. https://doi.org/10.3748/wjg.v13.i14.2083 Stühn L, Auernhammer J, Dietz C (2019) pH-depended protein shell dis- and reassembly of ferritin nanoparticles revealed by atomic force microscopy. Sci Rep 9:1–9 Zhang C, Zhang X, Zhao G (2020) Ferritin nanocage: A versatile nanocarrier utilized in the field of food, nutrition, and medicine. Nanomaterials 10:1–25. https://doi.org/10.3390/nano10091894 Smól J, Astriab M, Dudzińska-Madej B, Twardowski T (2001) Stress conditions applied to the interpretation of translation machinery. Acta Biol Hung 52:161–170. https://doi.org/10.1556/ABiol.52.2001.1.15 Strozycki PM, Szczurek A, Lotocka B et al (2007) Ferritins and nodulation in Lupinus luteus: Iron management in indeterminate type nodules. J Exp Bot 58:3145–3153. https://doi.org/10.1093/jxb/erm152 Kalgaonkar S, Lönnerdal B (2008) Effects of dietary factors on iron uptake from ferritin in Caco-2 cells. J Nutr Biochem 19:33–39. https://doi.org/10.1016/j.jnutbio.2007.02.001 Additional Declarations No competing interests reported. Supplementary Files Superdexcalibration.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 28 Oct, 2022 Reviewers agreed at journal 03 Oct, 2022 Reviewers agreed at journal 02 Oct, 2022 Reviews received at journal 02 Oct, 2022 Reviewers agreed at journal 01 Oct, 2022 Reviewers invited by journal 01 Oct, 2022 Editor assigned by journal 23 Sep, 2022 Submission checks completed at journal 23 Sep, 2022 First submitted to journal 20 Sep, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2084755","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":138924263,"identity":"4e530504-3ce1-4577-97d3-dc47264d049c","order_by":0,"name":"Magdalena Zielińska-Dawidziak","email":"data:image/png;base64,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","orcid":"","institution":"Poznań University of Life Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Zielińska-Dawidziak","suffix":""},{"id":138924264,"identity":"2e7a017a-59b3-4198-b069-41faddd96290","order_by":1,"name":"Wojciech Białas","email":"","orcid":"","institution":"Poznań University of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Białas","suffix":""},{"id":138924266,"identity":"19a220f0-be47-4318-9dee-6afcb2f05670","order_by":2,"name":"Dorota Piasecka-Kwiatkowska","email":"","orcid":"","institution":"Poznań University of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dorota","middleName":"","lastName":"Piasecka-Kwiatkowska","suffix":""},{"id":138924267,"identity":"0c58acfc-1625-4c2b-baf2-6cea80e2f50f","order_by":3,"name":"Halina Staniek","email":"","orcid":"","institution":"Poznań University of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Halina","middleName":"","lastName":"Staniek","suffix":""},{"id":138924269,"identity":"a8ea200c-feeb-44d8-a263-956bebee303d","order_by":4,"name":"Przemysław Niedzielski","email":"","orcid":"","institution":"Adam Mickiewicz University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Przemysław","middleName":"","lastName":"Niedzielski","suffix":""}],"badges":[],"createdAt":"2022-09-20 11:59:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2084755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2084755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":27042581,"identity":"90429658-2a45-47fa-9c76-2d17f7c858e0","added_by":"auto","created_at":"2022-09-27 17:05:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":390395,"visible":true,"origin":"","legend":"\u003cp\u003ePhotography presenting liquids obtained after \u003cem\u003ein vitro\u003c/em\u003e digestion of A\\ lupine B \\soybean sprouts. I – liquids taken after the first stage of digestion (stomach) II - liquids taken after the second stage of digestion (after stomach+small intestine digestion); 0 – control sample, Fe – samples enriched in iron; 35oC – sprouts dried in the temperature 35oC, 80oC – sprouts dried in the temperature 80oC\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/caed6e9c3442523cd0d77723.png"},{"id":27042576,"identity":"03f6077b-f29d-423b-8b5a-ee6cf8a38eb2","added_by":"auto","created_at":"2022-09-27 17:05:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58034,"visible":true,"origin":"","legend":"\u003cp\u003eIron released during digestion of A) lupine B) soybean sprouts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/ccf9b08f8ceff5653097b86a.png"},{"id":27042577,"identity":"7ba278fc-3cc7-4999-b3b2-736de791ed53","added_by":"auto","created_at":"2022-09-27 17:05:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63079,"visible":true,"origin":"","legend":"\u003cp\u003eSize exclusion chromatographic analyses of the fluids obtained after: \u003cstrong\u003e―\u003c/strong\u003e - stomach (S); \u003cstrong\u003e----\u003c/strong\u003e - solely intestine digestion (I); \u003cstrong\u003e….\u003c/strong\u003e – two-step digestion (stomach and intestine) (SI)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/864be90af1717840c709ac8f.png"},{"id":27043662,"identity":"74a93d1a-980f-4d8b-bdba-d09167fd2045","added_by":"auto","created_at":"2022-09-27 17:10:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136761,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoregram obtained after separation of fluid after A\\ lupine B\\soybean\u003c/p\u003e\n\u003cp\u003eS - stomach digestion, I - intestine digestion, SI - two-step digestion (stomach and intestine); F - partially purified lupine ferritin isolate; MW – molecular weight marker (\u003cem\u003e1\u003c/em\u003e- 120 kDa, \u003cem\u003e2\u003c/em\u003e- 85 kDa, \u003cem\u003e3\u003c/em\u003e – 50 kDa, \u003cem\u003e4\u003c/em\u003e – 35 kDa, \u003cem\u003e5\u003c/em\u003e- 25 kDa, - 20 kDa). *- peptide subunits with moleculare weight comparable to ferritin subunits\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/4a658449036e8427417ce6a4.png"},{"id":27043663,"identity":"6ea918c5-28dd-439f-9f78-daaaad9fa710","added_by":"auto","created_at":"2022-09-27 17:10:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182621,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot (A-B) and slot blot analysis (C). Samples of A\\ lupine B\\soybean after S- stomach digestion, I - intestine digestion, SI - two-step digestion (stomach and intestine); F - partially purified lupine ferritin isolate; MW – molecular weight marker (\u003cem\u003e1\u003c/em\u003e- 120 kDa, \u003cem\u003e2\u003c/em\u003e- 85 kDa, \u003cem\u003e3\u003c/em\u003e – 50 kDa, \u003cem\u003e4\u003c/em\u003e – 35 kDa, \u003cem\u003e5 \u003c/em\u003e– 25 kDa, \u003cem\u003e6 \u003c/em\u003e– 20 kDa)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/e84583dd443d47512dcb68a5.png"},{"id":27043664,"identity":"bec0a4dd-b1e0-453e-aadc-d80e1dff7818","added_by":"auto","created_at":"2022-09-27 17:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1278327,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/0425ec72-8344-4c0b-b842-4438121ccdbc.pdf"},{"id":27042579,"identity":"bd354f17-b7b8-4e60-84df-e699b7743d37","added_by":"auto","created_at":"2022-09-27 17:05:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17781,"visible":true,"origin":"","legend":"","description":"","filename":"Superdexcalibration.docx","url":"https://assets-eu.researchsquare.com/files/rs-2084755/v1/0e8f2500d8590472d73f8e49.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Digestibility of protein and iron bioavailability from enriched sprouts","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIron deficiency anemia (IDA) is the most common type of anemia (about 50% incidence) affecting\u0026thinsp;~\u0026thinsp;1.48\u0026nbsp;billion people in 2015 and more than 1.2\u0026nbsp;billion in 2016, particularly infants, children, adolescents, pregnant women, the elderly or people suffering from eating disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To treat IDA, diet modification and supplements intake are recommended. However, iron supplementation (both as pills application as well as enriched food consumption) may bring numerous side effects, such as constipation, diarrhea, abdominal pain and nausea [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. An alternative to this type of strategy may be the use of food biofortified in iron, such as legumes sprouts enriched in ferritin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ferritin is a protein able to contain up to 4,500 Fe ions, and it also protects iron from chelating agents present in the diet. However, studies on the bioavailability of ferritin iron are contradictory [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. I\u003cem\u003en vivo\u003c/em\u003e results suggest higher stability of ferritin than \u003cem\u003ein vitro\u003c/em\u003e studies on the ferritin iron bioavailability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This may be due to the fact that pure, isolated ferritin is usually used in \u003cem\u003ein vitro\u003c/em\u003e studies, whereas food rich in ferritin is often part of the human diet in \u003cem\u003ein vivo\u003c/em\u003e studies. Clinical studies, despite the large development of \u003cem\u003ein vitro\u003c/em\u003e methods, are still considered a \"gold standard\" of nutritional research [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, \u003cem\u003ein vitro\u003c/em\u003e methods are characterized by a significantly shorter time, the ease of sampling a large number of samples, lower costs and labor consumption, a guarantee of repeatability and no ethical restrictions. That is why still \u003cem\u003ein vitro\u003c/em\u003e methods simulating digestive processes are widely used in research on the behavior of food and pharmaceutical products in the digestive tract [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The aim of the presented research was to study the bioavailability of iron and proteins from the sprouts enriched in iron at individual stages of digestion in a simplified \u003cem\u003ein vitro\u003c/em\u003e digestion model. It is hypothesised that food constituents may have a protective effect on ferritin during digestion and increase its potential as a source of bioavailable iron.\u003c/p\u003e"},{"header":"2. Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sprouts preparation\u003c/h2\u003e \u003cp\u003eSoy (\u003cem\u003eGlycine max\u003c/em\u003e, Augusta variety) and lupine seeds (\u003cem\u003eLupinus luteus\u003c/em\u003e, Lord var.) from the Poznan University of Life Sciences cultivations were used for the preparation of iron-enriched sprouts.\u003c/p\u003e \u003cp\u003eThe sprouting process was carried out according to the procedure presented previously [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The seeds were cultured for seven days under controlled conditions and watered with 25 mM FeSO\u003csub\u003e4\u003c/sub\u003e from the third day of cultivation. Then the sprouts were dried, until their moisture was decreased to below 14%, and milled. Two manners of material drying were applied: continuous drying in 35\u003csup\u003eo\u003c/sup\u003eC, or two-stage drying: 5 hours in 80\u003csup\u003eo\u003c/sup\u003eC, followed by drying in 35\u003csup\u003eo\u003c/sup\u003eC. As a control sample the sprouts not fortified in iron (watered with tap water only), prepared in the same conditions, were applied. The sprouts were cultivated and dried in 7-fold repetition, mixed, and stored in an airtight glass container prior to further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003eIn vitro\u003c/em\u003e digestion experiment\u003c/h2\u003e \u003cp\u003eThe digestion of the prepared sprouts was done by an \u003cem\u003ein vitro\u003c/em\u003e method, simulating two-stage multi-enzymatic (gastric and intestinal) digestion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The digestion in the oral cavity was not considered, because it has no effect on their digestibility (the studied material is rich in protein, poor in starch). The large intestine stage was also omitted, as not important in iron bioavailability studies.\u003c/p\u003e \u003cp\u003eA studied sample was introduced into distilled water containing pepsin (60,000 U) (Sigma) and pH of the mixture was lowered to 2.0 with 1 M HCl. Gastric digestion was carried out for 2 hours, at 37\u003csup\u003eo\u003c/sup\u003eC. Then, the pH of the solution was adjusted to 7.4 and a solution containing pancreatic-intestine extract (0.005 g, Sigma) and bile salts (0.03 g, Sigma) in 5 ml 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e was added. The digestion was again performed at 37\u0026deg;C for 2 hours. The solution was centrifuged and the remaining extracted and not digested proteins were precipitated with trichloroacetic acid. In the sample prepared in this way, protein nitrogen was determined using the Kjeldahl [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] method and was related in percentage to the amount of protein nitrogen introduced with the sample into the digestion test. Additionally, the concentration of protein released from the tested material into digestive fluids at individual stages of digestion (i.e. stomach and intestine) before precipitation by TCA was determined with the Bradford method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Iron determination\u003c/h2\u003e \u003cp\u003eThe iron released during digestion from the sprouted seeds was determined as total iron and iron in ionic form. The total iron determination was conducted with atomic absorption spectrometry with air-acetylene flame atomization, while the ionic iron was determined as Fe(II) and Fe(III) forms by the colorimetric procedure. Fe(II) ions content was determined in reaction with 2,2\u0026prime;-bipirydyl in the environment of acetate buffer (pH 4.5), while Fe(III) content was determined in reaction with thiocyanate in the environment of hydrochloric acid (pH\u0026thinsp;\u0026lt;\u0026thinsp;2) using photometry (wavelength 470 nm). For each sample, a blank sample was prepared and the obtained value was used to correct the interference of the sample color [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe complexed iron content (in the studied preparation considered as iron bound mainly in ferritin form) was calculated from the difference between the total iron content obtained with the atomic absorption spectrometry and ionic iron, which was the sum of Fe(II) and Fe(III) contents obtained during the photometric analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Lupine ferritin standard preparation\u003c/h2\u003e \u003cp\u003ePurification of lupine ferritin was made using Korcz and Twardowski method [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The procedure included two-step salting out of protein from the homogenized sprouts (in the buffer containing 50 mM Tris-HCI, pH 8,0, and 30 mM NaCl). Next, the ferritin from the isolated protein was purified by fourfold ultracentrifugation (100000 x g/120 min).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. FPLC separation\u003c/h2\u003e \u003cp\u003eThe chromatographic studies were performed with an AKTA Explorer 100 Air System (Amersham Pharmacia Biotech, Uppsala, Sweden). A HiLoad 26/60 Superdex 200 column from Amersham Pharmacia Biotech (Uppsala, Sweden) was used. Assays on digested samples were performed at room temperature, at a flow rate of 2 ml/min. A 15 ml sample was eluted with 0.05 M of phosphate buffer pH 7 containing NaCl in a concentration of 0.5 M. The chromatographic mobile phase prior to use, and all samples before injection into the column, were filtered through a membrane filter (0.45 um, Millipore). During the chromatographic run, fractions were collected in volumes of 12 ml. Absorbance at 280 nm was applied for protein detection.\u003c/p\u003e \u003cp\u003eThe column was calibrated with the selected standard proteins included in the low molecular weight (LMW) and high molecular weight (HMW) range calibration kits (Cytivia, USA). The following proteins were used: Aprotinin (6500 Da), Carbonic anhydrase (29 000 Da), Ovalbumin (43 000 Da), Conalbumin (75 000 Da), Aldolase (158 000 Da), Ferritin from Horse spleen (440 000 Da) and Blue Dextran (2 000 000 Da). A calibration curve was prepared by measuring the elution volumes (Ve) of standards, calculating their corresponding partition coefficient (Kav values), and plotting their Kav values vs the logarithm of their molecular weight. The Kav was calculated with the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${\\text{K}}_{av}=\\frac{{\\text{V}}_{\\text{e}}- {\\text{V}}_{0}}{{\\text{V}}_{\\text{t}}- {\\text{V}}_{0}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere V\u003csub\u003ee\u003c/sub\u003e is elution volume for the standard (ml), V\u003csub\u003e0\u003c/sub\u003e is column void volume\u0026thinsp;=\u0026thinsp;elution volume for Blue Dextran 2000 (ml), Vt is total column volume (mL). Calibration data is included in Supplementary Materials.\u003c/p\u003e \u003cp\u003eThe fractions collected after separation by size exclusion chromatography with a retention volume similar to the lupine ferritin standard were further analyzed (SDS-PAGE, western blot and slot blot).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. SDS-Page separation\u003c/h2\u003e \u003cp\u003eThe fraction separated by FPLC method was subjected to electrophoresis. The protein profile was examined using 14% polyacrylamide gel electrophoresis under denaturing conditions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Samples obtained from soy chromatography were directly denatured, while lupine samples were first concentrated 10 times through filters with point cut-off 3 kDa (Amicon Ultra, Millipore Ltd., Poland). Gels were dyed with Coomassie Brilliant Blue and documented using CLIQS (TotalLabQuant, UK). Molecular mass of the detected protein was determined by reference to molecular mass marker in range 20\u0026ndash;120 kDa (Thermo Scientific,USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Ferritin immunodetection\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Slot-blot analysis\u003c/h2\u003e \u003cp\u003eThe collected fractions after FPLC analysis were applied on the PVDF membrane (Immobilon-P 0,45 \u0026micro;m, Merck Millipore Ltd., Poland). The applied volume was 200 \u0026micro;l for lupine fractions and 20 \u0026micro;l for soy fractions. 1% BSA in Tris-buffered saline, pH 7.4 was used as the blocking agent (1-h incubation). Next, the membrane was incubated for 2 hours with the goat sera containing anti-lupine ferritin antibodies, diluted 1:100 (provided for the research by the Institute of Bioorganic Chemistry of the Polish Academy of Sciences in Poznań). As a secondary antibody, rabbit anti-goat IgG polyclonal antibody, marked with horseradish peroxidase was applied for 2 hours in 1:1000 dilution (Invitrogen, USA). Detection of protein-bound antibodies was performed with diaminobenzidine (Sigma-Aldrich, USA) in 20 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein fractions separated by SDS-PAGE electrophoresis were also transferred by a semi-dry electrotransfer (200 mA current for 30 min. and 120 mA for 90 min.) to a polyvinylidene difluoride membrane (Immobilon-P 0,45 \u0026micro;m, Merck Millipore Ltd., Poland). The same antibodies and method of detection were used as presented above for the slot-blot analysis. The membranes were analysed using the CLIQS program (TotalLab Quant, UK).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe necessary statistical analyses were performed using Statistica 13.0 (StatSoft, USA). All numerical data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical significance of the difference between the control and the treated sample was assessed by one-way ANOVA and post-hoc Tukey\u0026rsquo;s tests. Results were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Protein digestibility\u003c/h2\u003e \u003cp\u003eThe protein content in the tested lupine seeds (43.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 g/100 g d.m.) and sprouts (47.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 g/100 g d.m.) is over 4% higher compared to soybean seeds (39.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 g/100 g d.m.) and sprouts (42.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 g/100 g d.m.), but it may differ depending on the seed variety and conditions of seed cultivation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Samples subjected to the digestion process were unified in terms of the amount of total protein in the dry matter (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only the S.Fe.35 (soybean sprouts enriched in iron, dried continuously in 35\u003csup\u003eo\u003c/sup\u003eC) variant differed slightly, but statistically significantly.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal protein and soluble protein balance in the test material before and after \u003cem\u003ein vitro\u003c/em\u003e digestion (g/d.m.)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eContent of total protein in the weight of material introduced into digestion [g/100 g d.m.]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eContent of total protein in sample\u003c/p\u003e \u003cp\u003e[g/100 g d.m.]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal digestibility [%]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c9\" namest=\"c8\" rowspan=\"2\"\u003e \u003cp\u003eSoluble protein content determined in fluids after digestion by Bradford method\u003c/p\u003e \u003cp\u003e[\u0026micro;g/ml]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eafter the 1st stage of digestion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eafter the 2nd stage of digestion\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSediment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFluid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSediment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eafter the 1st stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eafter the 2nd stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL.0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea *\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e53.23 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e364.17\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e298.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL.0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.22 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e 346.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e333.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL.Fe.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e69.4 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e350.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e465.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.55 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL.Fe.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57.4 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e375.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e379.17\u0026thinsp;\u0026plusmn;\u0026thinsp;11.46 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44.73 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e388.33\u0026thinsp;\u0026plusmn;\u0026thinsp;13.23 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e462.50\u0026thinsp;\u0026plusmn;\u0026thinsp;10.10 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.84 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e305.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.29 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e370.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.29 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.Fe.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47.70 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e320.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.33 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e550.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.64 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.Fe.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e47.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e411.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.64 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e425.00\u0026thinsp;\u0026plusmn;\u0026thinsp;14.65 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e* a, b, c, d \u0026ndash; statistically determined (with the Tukey Test) homogeneous groups (separately for results of soybean and lupine in the columns)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe availability of nutrients for the human body depends not only on their content in the digested material, but also on the extractivity of the components from digested material at individual stages of digestion. Ingredients remaining in undigested material are excreted with feces. Absorption is only possible for those substances which, after being extracted in the stomach or intestine, are dissolved in digestive fluids. Thus, the nitrogen compounds (recalculated into total protein) released from the digested material were studied.\u003c/p\u003e \u003cp\u003eThe most of total protein during digestion was released into the gastric fluid from the L.Fe.80 (lupine sprouts enriched in iron and dried in 80\u003csup\u003eo\u003c/sup\u003eC for the first 5 hours) and S.Fe.80 (enriched soy sprouts dried in the same conditions for the first 5 hours) variant, and the least from L.Fe.35 and S.Fe.35 (lupine and soy sprouts enriched in iron and dried continuously in 35\u003csup\u003eo\u003c/sup\u003eC). It may be explained by the denaturation of proteins during drying at 80\u0026deg;C, which facilitates their hydrolysis, but also extraction in the stomach.\u003c/p\u003e \u003cp\u003eOn the other hand, as a result of total digestion, the most total protein is secreted into intestinal fluid during digestion of L.Fe.35 and S.Fe.35, while the least is in L.0.35 and S.0.35 (not enriched soy and lupine sprouts dried in 35\u003csup\u003eo\u003c/sup\u003eC). Most of protein is extracted to the gastric fluid (respectively\u0026thinsp;~\u0026thinsp;49% for lupine and ~\u0026thinsp;45% for soybean sprouts) and average increase in the total content of protein in the intestinal fluid (i.e. total digestibility) is up to ~\u0026thinsp;61% for lupine and only up to ~\u0026thinsp;46% for soybean sprouts. These results suggest that proteins of lupine sprouts were more digestible, and it may result from the decreased content of trypsin inhibitors compared to soy. Lupine is usually indicated as a legume with trace content of trypsin inhibitor activity (M. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, both the thermal treatment and the sprouting processes reduce the activity of these inhibitors in the material [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTotal protein content informs us about extractivity of nitrogen compounds from the digested material. Intestinal enterocytes absorb mainly free amino acids or very short peptides. Only few protein are absorbed via endocytosis, but in the group ferritin is located [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, in order to distinguish the amount of proteins that are released during digestion from amino acids, short peptides and nucleotides, determination of the protein extracted from the tested material was done by the Bradford method. The method allows to determine peptides/proteins that exceed 3\u0026ndash;5 kDa [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], i.e. peptides composed of at least\u0026thinsp;~\u0026thinsp;27 amino acids. The highest increase in the soluble protein content in intestine fluid compared to stomach fluid was observed in samples fortified in iron and dried in 35\u003csup\u003eo\u003c/sup\u003eC (L.Fe.35 - ~35%, S.Fe.35 - ~70%). Thus, the thermal denaturation of protein in the studied material could increase their digestibility. This may confirm the thesis that not all proteins contained in the test material are susceptible to the action of digestive enzymes, which may affect their further absorption. Ferritin is a protein resistant to high temperatures, i.e. 85\u0026deg; C, low pH and a number of proteolytic enzymes, with confirmed extractivity in pH close to 8.0 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. And \u003cem\u003ein vivo\u003c/em\u003e studies confirm its resistance to digestion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, the presented results suggest possible ferritin extraction among other proteins in the intestine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Iron release\u003c/h2\u003e \u003cp\u003eAs a next step in the presented experiments, the release of iron during digestion of the studied material was checked. At this stage of experiment L.Fe.35 and S.Fe.35 samples were analysed.\u003c/p\u003e \u003cp\u003eSignificant differences were observed even in the color of the liquids obtained after digestion of lupine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and soybean (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) sprouts. The color observation indicates the release of iron from the tested material in the intestine, and a different form of iron released in the two studied steps of digestion. For the bioavailability of iron from the studied material not only compound resistance to the digestive enzymes may be important, but also their susceptibility to extraction from the food matrix. The observed \u0026lsquo;red\u0026rsquo; color of the intestine fluid obtained after lupine sprouts digestion suggests the \u0026lsquo;red\u0026rsquo; iron presence, which can be the result of both the presence of ferric ions (Fe\u003csup\u003e3+\u003c/sup\u003e) and a complexed form of iron (such as ferritin). Thus, iron speciation in the obtained digestive fluids was performed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the first step of digestion from the studied material most of released iron was in ionic form, as ferrous iron (85%\u0026plusmn;3% in lupine and 95%\u0026plusmn;2% in soybean) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, when the same model of digestion was performed without the use of enzymes, solely acid action caused the release of ~\u0026thinsp;80% of these iron amounts. Intestine digestion allowed to almost double the amount of iron released from lupine sprouts (from ~\u0026thinsp;21% up to 38% of total iron), while in soybean it was not such a significant increase (from ~\u0026thinsp;16% up to ~\u0026thinsp;23%). Ferric iron (which is less available) constituted\u0026thinsp;~\u0026thinsp;25% of total iron in intestine after lupine digestion and ~\u0026thinsp;18% after soybean digestion. It suggests that administration of vitamin C together with the prepared sprouts should increase the iron absorption in the intestine. The increased content of complexed iron (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was detected and it constituted\u0026thinsp;~\u0026thinsp;16,5% of total iron for lupine and ~\u0026thinsp;12% for soybean sprouts. However, this result still does not prove or exclude the stability of ferritin during digestion. Iron may be complexed here also by polyphenolic compounds, synthetized in response to stress conditions in time of sprouting [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Isolation of ferritin from the digestive fluids\u003c/h2\u003e \u003cp\u003eThus, as a next step, chromatographic isolation of ferritin was attempted from the fluids obtained after digestion to confirm or deny the possibility of extraction and stability of ferritin during digestion of the studied samples. Chromatogram presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB suggests that ferritin (collected in the fraction between 125\u0026ndash;155 mL) is not present in the fluid after gastric digestion. This may result mainly from the inability to extract ferritin under such conditions. The best extraction conditions to obtain ferritin fractions with good yield correspond to pH approx. 8.0 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Another essential factor that could have influenced the ferritin level after gastric digestion is the low pH of the environment in which this process takes place. As Bejjani, Pullakhandam, Punjal and Madhavan [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] demonstrated, pea ferritin dissociates when exposed to a low pH, releasing iron into the digestive fluid simultaneously. Under these conditions, the proportion between the number of α-helices and β-sheet structures changes, wherein the number of the latter increases significantly. It is worth noting that in the case of the presented results, correction of stomach fluid pH after the end of this step of digestion up to 7.4 did not modify the results. It excludes the possibility of reassembling ferritin degraded in the stomach after modification of pH, as it was observed in other studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Solely intestine digestion of sprouts, both lupine and soybean, resulted in extraction ferritin from the material. Moreover, this extraction was many times higher in case of soybean. Simultaneously, after application of two-step digestion of the material, the amount of extracted ferritin decreased, especially for soybean. This suggests the advisability of administering ferritin preparations after encapsulation, limiting its contact with the gastric fluid (e.g. in eudragit), which is not consistent with the observations of Theil [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Intestine condition seems to be more convenient to ferritin extraction, but also safe to maintain its structure and, consequently, to ensure the possibility of its absorption by endocytosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, this extraction is still limited \u0026ndash; the pH 7.4 is far from pH 8.0 suggested for ferritin extraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The detection of ferritin in the digestive fluids\u003c/h2\u003e \u003cp\u003eThe samples were subjected to the SDS-PAGE analysis. Analysis of the obtained gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) suggest the presence in the studied fraction of peptides with molecular weight close to the molecular weight of ferritin subunits. Molecular weight suggested for ferritin is close to ~\u0026thinsp;450 kDa. It is multimeric protein composed from 16 subunits with molecular weight\u0026thinsp;~\u0026thinsp;28 kDa, although for lupine ferritin the presence of the second subunit, the product of deletion of amino acids from the C-end, is also confirmed [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, results of the electrophoretical separation of such not homogenous material (even partially purified ferritin isolate \u0026ndash; line F, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) could be very misleading.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteins separated by gel electrophoresis were transferred to the membrane and detected by polyclonal anti-ferritin antibodies. Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B) confirmed reaction of the goat serum with the protein present in the fraction separated by FPLC from fluids obtained after digestion of soy sprouts and lupine sprouts. The antibodies recognised even the ferritin subunits in fluids after gastric digestion, which suggests that some ferritin was extracted and denatured during gastric digestion. The same result was observed in slot-blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). It must be remembered that samples from gastric digestion had to be concentrated before analysis and the consistency of soy samples from intestine (both solely I, as well as SI digestion) significantly impeded the migration of the sample through the membrane.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003ePresented results confirmed previous observations of protein shell dissociation induced by acid in gastric conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Simultaneously, these conditions are considered not to be suitable for the extraction of ferritin from complex matrices (plant materials or food). Thus, it may be expected that ferritin did not \u0026lsquo;partially escape\u0026rsquo; from stomach digestion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], but just that it was not extracted (or only partially extracted) in stomach. These explain also differences between the results of \u003cem\u003ein vitro\u003c/em\u003e studies on pure, isolated ferritin and \u003cem\u003ein vivo\u003c/em\u003e studies, when ferritin was administered as a food ingredient.\u003c/p\u003e \u003cp\u003eIn order to use ferritin effectively as a unique source of iron absorbed in the intestines, the matrix / food should not only be processed to keep the ferritin undenatured. It is also important to release this protein from the matrix uniquely during digestion at the intestinal stage. This will allow the use of ferritin both as a source of iron (released from ferritin by its denaturation and transported by DMT1), and as a protein absorbed by endocytosis, and thus, by a unique system mechanism in intestinal enterocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presented experiment confirmed the protective effect of food ingredients on the stability of ferritin during digestion. The results are unique because they do not present studies carried out on isolated ferritin, but on a material containing the protein that can be a separate dietary supplement or added as a food ingredient.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors agreed to publish the results in Plant Foods for Human Nutrition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare that they have no known competing financial interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003cu\u003eMagdalena Zielińska-Dawidziak\u003c/u\u003e \u0026ndash; conceptualization, methodology, validation, investigation, resources, writing original draft; \u003cu\u003eWojciech Białas\u003c/u\u003e - conceptualization, methodology, validation, investigation, resources, writing original draft; Dorota Piasecka-Kwiatkowska \u0026ndash; investigation (total protein content supervision); Halina Staniek - investigation (total iron content); \u003cu\u003ePrzemysław Niedzielski\u003c/u\u003e - investigation (iron speciation)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. It was financed by our Universities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards -\u0026nbsp;\u003c/strong\u003eThe presented manuscripts represents honest research work of authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVos T, Abajobir AA, Abbafati C et al (2017) Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990\u0026ndash;2016: A systematic analysis for the Global Burden of Disease Study 2016. 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J Nutr Biochem 19:33\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jnutbio.2007.02.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jnutbio.2007.02.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-foods-for-human-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Plant Foods for Human Nutrition](https://www.springer.com/journal/11130)","snPcode":"11130","submissionUrl":"https://submission.nature.com/new-submission/11130/3","title":"Plant Foods for Human Nutrition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ferritin, digestibility, soybean, lupine, fortified sprouts","lastPublishedDoi":"10.21203/rs.3.rs-2084755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2084755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant ferritin is suggested as a good source of iron for human. Usually present in trace amounts, it was induced in legumes seeds by their sprouting in FeSO\u003csub\u003e4\u003c/sub\u003e solution. Fortified sprouts were digested in the \u003cem\u003ein vitro\u003c/em\u003e model of the human gastrointestinal tract. ~49% of lupine and ~\u0026thinsp;45% of soy proteins were extracted into gastric fluid and next\u0026thinsp;~\u0026thinsp;12% and only\u0026thinsp;~\u0026thinsp;1% into intestine fluid from lupine and soybean, respectively. Gastric digestion released mainly ferrous iron (~\u0026thinsp;85% from lupine and ~\u0026thinsp;95% in soybean sprouts). Complexed iron constituted\u0026thinsp;~\u0026thinsp;43% of total iron in intestine after lupine digestion and ~\u0026thinsp;55% after soybean digestion. Intestine digestion doubled the total iron released from lupine sprouts (from ~\u0026thinsp;21% up to 38%), while in soybean it increase from ~\u0026thinsp;16% up to ~\u0026thinsp;23%. Ferritin presence was confirmed by the specific antibodies in digestive fluids, but it is only partially extracted from sprouts during \u003cem\u003ein vitro\u003c/em\u003e digestion.\u003c/p\u003e","manuscriptTitle":"Digestibility of protein and iron bioavailability from enriched sprouts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-27 17:05:17","doi":"10.21203/rs.3.rs-2084755/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-28T22:06:57+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"edb6711a-88ec-4b46-a4dc-2b015da12b66","date":"2022-10-03T06:05:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"abfbd6a0-6291-49fc-8f09-f25b858c44a7","date":"2022-10-02T12:03:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-02T04:20:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17a82cbe-68b4-49cb-956e-5970b6da101f","date":"2022-10-01T23:26:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-01T17:29:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-09-23T04:13:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-09-23T04:13:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Foods for Human Nutrition","date":"2022-09-20T11:52:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-foods-for-human-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Plant Foods for Human Nutrition](https://www.springer.com/journal/11130)","snPcode":"11130","submissionUrl":"https://submission.nature.com/new-submission/11130/3","title":"Plant Foods for Human Nutrition","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bab9b57f-07d8-49f6-b6c6-90f2f6b71b52","owner":[],"postedDate":"September 27th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-01-16T02:59:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-27 17:05:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2084755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2084755","identity":"rs-2084755","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}