Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase | 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 Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase Rebecca M. Murphy, Fang Huang, Matthew E. Loewen, Trent C. Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1975355/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, pulse meal contains anti-nutritional and anti-palatability compounds, including (poly)phenolics (tannic and non-tannic), such that improvements in pulse meal processing are still needed to increase its uptake by the industry. Microbial fermentation is currently used as a strategy to decrease tannin content, but results in the undesirable accumulation of monophenolics. Here we investigate the viability of cell-free biocatalytic reduction of phenolic content in faba bean ( Vicia faba ) meal. A representative catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Its amenability to large scale recombinant production was established, and its ongoing stability in complex environments including resuspension in slurries of faba bean meal demonstrated. Reaction results suggest that BLC23O is effective for biocatalytic phenol reduction in faba bean meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry. pulse meal Vicia faba biocatalysis catechol 2 3 dioxygenase phenol reduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Alternative feed ingredients and innovation of feed technology are progressively being investigated and incorporated into processing pipelines toward enhanced digestibility and efficiency, decreased environmental impact and maximized economic opportunity (Behnke, 1996 ; der Poel et al., 2020 ). Pulses ( Fabaceae family) and their by-products are examples of advantageous sources of animal feed that could be further optimized to increase their value and applicability (Sherasia, Garg, & Bhanderi, 2018 ; Singh, 2017 ). Currently, pulses play an important role in sustainable development based on their ability to fix nitrogen, reducing the need for costly nitrogen fertilizers when used in crop rotations, and sequestering greenhouse gas from the environment (Irisarri et al., 2021 ). However pulse nutrient and protein-rich profiles significantly furthers their potential. For example, pulse meal, a by-product of the industry, is approximately 40–45% protein on a dry mass basis and thus holds great potential to serve as animal feed, contributing to food security and reducing competition with human food (Sherasia et al., 2018 ). Such applications of by-products also reduces costs related to disposal and allows for the conversion of low value products into higher value feed/food (Ominski et al., 2021 ). Tannins and other non-tannic (poly)phenols are intrinsic to pulses, and in the context of animal nutrition, are considered anti-nutritional factors (ANFs) based on observed negative effects on digestion and the bioavailability of nutrients (Kardum & Glibetic, 2018 ; Kumar et al., 2021 ). These effects are due to (poly)phenols forming complexes with storage proteins, interfering with peptide bond proteolysis, and serving as direct inhibitors of digestive enzyme, together decreasing protein and carbohydrate availability (Punia, Siroha, & Kumar, 2021 ). Chelation of metal ions by polyphenolics has also been shown to reduce absorption and decrease uptake of vitamins and minerals (Singh, 2017 ). Finally, the palatability of pulse meal is reduced by tannins in particular, due to undesirable astringency, which lowers food intake and animal performance (Singh, 2017 ). Thus, reducing phenol content (tannic and non-tannic) in pulse products remains an important goal for increased nutritional and economic value. Currently, microbial fermentation is used as a strategy to lower ANFs, including tannins, in feeds (der Poel et al., 2020 ; Olukomaiya, Fernando, Mereddy, Li, & Sultanbawa, 2019 ). For example, Lactobacillus plantarum , a Gram-positive lactic acid bacterium commonly used in the food industry, specifically possesses genes encoding tannin acyl hydrolase (commonly known as tannase) and gallate decarboxylase, yielding the monophenol pyrogallol (Jimenez, Esteban-Torres, Mancheno, de Las Rivas, & Munoz, 2014 ). However, the observed accumulation of such monophenols in L. plantarum fermented feeds is also undesirable (Kardum & Glibetic, 2018 ; Kumar et al., 2021 ). The next step in degradation of monophenols involves breaking the phenolic ring. In nature, this activity relies on a family of microbial catechol dioxygenases that carry out the oxidative cleavage of hydroxylated aromatic rings, to yield linear compounds that feed into the citric acid cycle (Kamimura et al., 2017 ). Phenolic ring cleavage can be intradiol (between two consecutive hydroxyl groups on the ring (positions 1 and 2)), proximal extradiol (next to the hydroxyls; position 2 and 3) or distal extradiol (removed from the hydroxyls; position 4 and 5) (Hou, Patel, & Lillard, 1977 ). A brief review of the current literature and a search of the NCBI database suggests L. plantarum does not encode any members of this family of enzymes, consistent with the high accumulation of phenolics in such fermentations. Thus, as an alternative or complement to microbial fermentation, cell-free biocatalysis has been proposed for reduction of phenolic content. A biocatalytic strategy would decouple tannin and phenolic degradation from cellular physiology, reducing any downregulation imposed by the carbohydrate rich environment and feedback inhibition, as well as allowing absolute control of enzyme types and concentrations applied (Claassens, Burgener, Vogeli, Erb, & Bar-Even, 2019 ). One example of a potential candidate for cell-free phenolic degradation of pulse meal is a Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O; (Adewale et al., 2021 )). B. ligniniphilus L1 is a halotolerant and alkaliphilic bacterium isolated from sediments from the South China Sea, known to use polyphenolic lignin as its sole carbon source. Catechol 2,3-dioxygenases elicit proximal extradiol cleavage. Three catechol 2,3-dioxygenases encoded by B. ligniniphilus L1 have been identified and the shortest one, a protein of 283 amino acids (BLC23O, NCBI accession WP_017726464.1) with a calculated molecular mass of approximately 32 kDa, was recently characterized (Adewale et al., 2021 ). This enzyme was found to have unusually broad substrate specificity and good thermostability, traits proposed to be associated with its atypical monomeric structure. Here the application of BLC23O to cell-free reduction of phenolic content in pulse meal is assessed. Scaled-up recombinant production and purification of the enzyme is described, followed by evaluation of the enzyme’s potential to reduce phenolics in 3 different meal fractions from Vicia faba (faba bean). Results And Discussion Large-scale (4 L fermentative) production and Ni-NTA enrichment of BLC23O Shake flask production of BLC23O from Echerichia coli has been previously reported (Adewale et al., 2021 ). Here production of BLC23O was scaled up to a 4 L fermentative format. Wet weight determination of the obtained cell pellet was 49.8 g, or 12.4 g cell pellet / L of culture. The pellet was re-suspended in lysis buffer and high-pressure homogenization was used to extract proteins. Following centrifugation, the supernatant was collected and BLC23O was further enriched using the fused His-tag. Outcomes from the Ni-NTA affinity chromatography were visualized by SDS-PAGE (Fig. 1 A). Thick bands at the expected Mw of approximately 35 kDa are present in the three elution fractions and the original supernatant fraction. Small amounts of protein were also detected at 35 kDa in the washes and flow-through, suggesting minor losses during the enrichment. No BLC23O was detected in the lysis pellet, indicating that cell lysis was successful. Contaminating proteins were largely eliminated from the final sample. The elution fractions were pooled and protein quantified, yielding 116 mg of enzyme / L of cell culture, or 9.3 mg of BLC23O / g of wet weight cells. Kinetic analysis of BLC23O. A small portion (4%) of the obtained protein from Ni-NTA enrichment was subjected to further purification by size exclusion chromatography. The chromatographic separation curve for BLC23O ( Supplemental Figure S1A ) showed a clear and steep peak at 82 min. Calibration of indicated this is consistent with the expected molecular weight of 35 kDa ( Supplemental Figure S1B ), falling in between ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Collected fractions spanning this peak were visualized by SDS-PAGE, yielding a single significant band around the expected Mw of 35 kDa ( Supplemental Figure S1C ). A Michaelis-Menten analysis of the obtained purified BLC23O against varying concentrations of the known substrate, 3-methycatechol, yielded a K M of 379 ± 86 µM, a turnover number (kcat) of 0.13 ± 0.01 s − 1 , and a catalytic efficiency (kcat/K M ) of 340 ± 80 M − 1 s − 1 (Fig. 1 B). Comparison of these results with a previous kinetic analysis for BLC23O highlights non-significant differences (previously: K M 418 ± 25 µM, kcat 0.20 ± 0.03 s − 1 and kcat/K M 480 ± 80 M − 1 s − 1 ; (Adewale et al., 2021 )). Measurement of phenolic content in faba bean meal by BLC23O Initially, the phenolic content in three distinct samples of faba bean was meal was assessed, including the original ‘total’ meal, as well as ‘fine’ and ‘course’ fractions of the meal obtained from air-classification. Following reaction of re-suspended meal fractions with the Folin-Ciocalteu (F-C) reagent, the absorbance at 725 nm wavelength was measured and quantified by comparison to a calibration curve made with pure tannic acid (Fig. 2 and Supplemental Figure S2A ). Statistically significant differences in phenolic content were detected, where the fine fraction had the highest total phenolic content, followed by total meal, and then the coarse fraction. This is consistent with the fine fraction being protein enriched and the coarse fraction being carbohydrate enriched (Coda et al., 2015 ). Higher-phenolics in the ‘fine’ protein fraction is consistent with the protein amino acids introducing significant phenolic content, in addition to other (poly)phenolics being fractionated into the sample due to linkages with the protein. Some phenolic content was also observed in the ‘coarse’, carbohydrate rich fraction, likely due to some association between carbohydrates and (poly)phenols (Kardum & Glibetic, 2018 ; Smeriglio, Barreca, Bellocco, & Trombetta, 2017 ). That this assay was able to detect the differences between the meal fractions, demonstrates that the F-C reagent is appropriate for use in phenolic content determination assays in faba bean meal. The kinetics of the F-C reagent were evaluated in more detail at the maximum amount of phenolics detected in meal reactions, using 14 µg/mL tannic acid ( Supplemental Figure S2B ). Under these conditions the reaction was complete within 60–70 min and phenolic content remained constant for an additional 120 min thereafter. These outcomes highlight the minimum amount of time solutions should be left to react before measuring the absorbance, ensuring an efficient and robust assay. Biocatalytic reduction of phenolics in faba bean meal The ability of BLC23O to degrade polyphenols in total faba bean meal and the air classified fractions was evaluated by monitoring phenolic content over time. Following addition of the enzyme to the meal suspensions, samples were taken at 15 min intervals and reacted with the F-C reagent and absorbance obtained at 725 nm. In all cases, BLC23O-treatment led to detectable decreases in phenol content over time, compared to untreated meal samples (Fig. 3 A-C). Importantly, in both the meal-alone and enzyme-alone samples, phenolic content remained constant or even displayed a slight increase over time. That increases in phenol content were observed in the meal fractions (in the absence of any enzyme) is likely representative of the effect of chemical hydrolysis over time. (Poly)phenol content can be inaccessible due to being embedded in large complex structures (Smeriglio et al., 2017 ; Soares et al., 2020 ). Water hydrolysis is known to release phenols from the large complexes and break up interactions with proteins and sugars, increasing phenol available to react with the enzyme and assay reagents. Thus to quantify the effect of the enzymatic treatment on phenolic levels, the ratio between phenolics in the BLC23O-treated samples and phenolics in the meal-only samples were determined. Additionally, background phenolics detected in the enzyme-only sample were subtracted. This analysis revealed a 49.6% decrease in phenolic content in the course meal fraction over 3 h (Fig. 3 F). Phenolic content in the total meal was reduced by 26.7%, while only a 9.0% reduction was observed in the fine fraction (Fig. 3 E & 3 D). The coefficient of determination (R 2 ) values for the linear regressions of each fraction type are low, especially for the assay with the coarse flour, indicating a high amount of variability in the data. To ascertain the stability of the reaction, overnight assays were performed for the total meal fraction in the presence and absence of BLC23O, as well as for the enzyme-alone (background control). Samples were taken at 0 and 24 h, reacted with the F-C reagent for 60 min, and absorbances obtained at 725 nm wavelength. Consistent with previous experiments, a small but significant increase in phenolics was detected in the meal-only sample, while decreases in both the enzyme-only and the BLC23O-treated meal samples were observed (Fig. 4 ). Quantification (as described above) indicates a 68% decrease in phenolics in this meal fraction upon treatment with BLC23O for 24 hrs. This represents a 41% further decrease in phenolic content compared to values obtained after only 3 h, and emphasizes the catalytic stability of BLC23O over time in this complex environment. Conclusions In conclusion, this study demonstrates the viability of cell-free biocatalytic reduction of phenolic content in faba bean meal. The representative catechol dioxygenase, BLC23O, enzyme used in the proof-of concept was selected based on its known stability and broad substrate specificity. Its amenability to high yield fermentative recombinant production has now been established, and its ongoing stability in complex environments including resuspension in a slurry of pulse meal demonstrated. While results suggest that BLC23O is effective for biocatalytic phenolic reduction, the upstream release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) is likely a limiting factor in the absence of other enzymes or microbial fermentation. Analyses of the effect of including other enzymes with BLC23O to improve substrate availability, and the effect of adding BLC23O to fermentative processes is ongoing, toward optimization of the nutritional and economical value of pulse meal in the animal feed industry. Methods And Materials All chemicals were obtained from Sigma Aldrich, except where indicated below. Faba beans were obtained from Faba Canada Ltd, de-hulled at the Canadian International Grains Institute (Winnipeg, Canada), and milled/air classified at the Richardson Center for Food Technology and Research (Winnipeg, Canada), yielding a ‘total’ meal fraction (before air classification), and then air classification-derived ‘fine’ and ‘course’ meal fractions. BLC23O fermentative scale production An optimized version of the BLC23O coding region was cloned into the pET28B + vector such that the expressed fusion protein included an N-terminal 6 x His tag as previously described (Adewale et al., 2021 ). Echerichia coli BL21 cells containing the expression vector encoding the BLC23O gene was used to inoculate an overnight culture containing 50 mL of Terrific Broth (TB) medium (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) with 50 µg/mL kanamycin. The culture was incubated overnight at 37°C in a MaxQ 6000 shaker (Thermo Scientific) at 150 rpm. The overnight culture was used to inoculate a BioFlo/CelliGen 115 benchtop fermentor & bioreactor (New Brunswick Scientific) containing 4 L of TB medium, 50 mg/L kanamycin, and 0.5 mL of antifoam. The bioreactor had an agitation of 250 rpm, its water jacket temperature was set to 37°C, and the dissolved oxygen setpoint was 30%. The culture was grown to an OD600 between 0.6 and 0.7 at which time protein expression was induced with the addition of 0.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was left growing in the bioreactor overnight at 18°C. The media was centrifuged in a Sorvall Lynx 4000 centrifuge (Thermo scientific) at 3250 x g for 30 min at 4°C. Purification of recombinantly produced BLC23O The obtained pellet was resuspended in 500 mL of 1x lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 3U/mL benzonase, 1 mg/mL lysozyme, pH 8.0). Proteins were extracted by high pressure homogenization using an EmulsiFlex-C5 (Avestin) according to the manufacturer’s protocol. The lysate was centrifuged (Thermo scientific) at 12,000 rpm for 15 min at 4°C. The supernatant was collected and applied to nickel-nitriloacetic acid (Ni-NTA) resin (Qiagen) with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) by a batch method, where resin was mixed with the cleared lysate in a MaxQ 6000 shaker (Thermo Scientific) set to 200 rpm at 4°C for 2 hrs. The sample was centrifuged in a Sorvall Legend X1R centrifuge at 1000 x g for 3 min at 4°C (Thermo scientific) and the supernatant discarded. The remaining beads were washed three times using this same process with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and then BLC23O was eluted using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Obtained elution supernatants containing the BLC23O enzymes were pooled and concentrated using an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentration of the obtained BLC23O sample was determined using a Quick Start Bradford protein assay (Bio-Rad) according to the manufacturer’s protocol. Proteins samples were visualized by 12% SDS-PAGE. A small portion (approx. 4% of the total sample) of the obtained enriched enzyme was further subjected to Fast Performance Liquid Chromatography (FPLC; Amersham Pharmacia Biotech) size-exclusion chromatography (SEC) with a HiLoad 16/600 Superdex 200 (Cytvia) column. Size exclusion buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) was degassed and the FPLC flowrate was set to 1 mL/min. Elution fractions were collected in increments of 1.4 mL. Fractions containing BLC23O were combined and concentrated with an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentrations of BLC23O were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. Proteins samples were visualized by 12% SDS-PAGE. BLC23O kinetic analysis Reaction solutions with total volumes of 250 µL were prepared in triplicate in a 96-well microplate (Greiner Bio-One) with 50 mM phosphate buffer pH 7.5, 18.7 µg/mL of BLC23O purified by Ni-NTA and SEC, 10 mM Mn 2+ , and substrate (3-methylcatechol) concentrations varying from 0.05 mM to 3 mM. Plates were pre-incubated for 5 min at 32.5°C and reactions were initiated with the addition of substrate and samples left at 32.5°C. The absorbance was read at 388 nm for 60 min in 30 sec intervals using a SpectraMax M5e spectrophotometer (Molecular Devices). The initial reaction rate, in µM of product formed per second, was plotted and the data fitted to the Michaelis-Menten equation using Microsoft Excel’s Solver add-in. Determination of phenolic content by F-C reagent. Based on previous reports (Blainski, Lopes, & de Mello, 2013 ; Everette et al., 2010 ), a calibration curve for the quantification of phenolic content was created where reaction solutions with a volume of 1 mL were prepared with 0.125 N F-C reagent, 0.125 g/mL of sodium carbonate, and tannic acid concentrations ranging from 0 to 30 µg/mL. The solutions were left to react for 45 min and then 250 µL of each was transferred into a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using a SpectraMax M5e spectrophotometer (Molecular Devices). This was repeated in triplicate. Tannic acid reaction kinetics were assessed in a similar manner, but with reaction solution volumes of 250 µL and tannic acid concentrations ranging from 0 to 14 µg/mL. In this case, as soon as the tannic acid was added, the absorbance at 725 nm was monitored for 180 min in 15 min intervals using an Epoch 2 microplate spectrophotometer (Biotek). Initial phenolic content in the three faba bean meal fractions was determined using the same procedure as described above, but with 0.1, 0.2, and 0.3 mg/mL of each type of faba bean sample instead of tannic acid, and incubation with the F-C reagent for 60 min. Results were compared to the tannic acid calibration curve for quantification, based on the assumption that a µg of commercial tannic acid is equivalent to a µg of phenolic content in the meal. BLC23O-mediated phenolic reduction in faba bean flour BLC23O reaction solutions with volumes of 4 mL were prepared for each of the faba bean meal fractions with 1.2 mg/mL of each fraction type and reactions initiated with the addition of 0.06 mg/mL of BLC23O enzyme. A blank solution with no meal or enzyme, as well negative and background controls of meal only and enzyme only were also prepared. Samples from the reaction mixtures were taken at 15 min intervals for 3 hrs, vortexed into 0.125 N F-C reagent and 0.125 g/mL of sodium carbonate and then left to react for 60 min at room temperature. Subsequently, 250 µL from each mixture was transferred to a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using Epoch 2 microplate spectrophotometer (Biotek). This was performed in triplicate. The % phenolic reduction was calculated using the following equation: (phenolic content of meal in reaction mixture (%) = (R_abs (t) - E_abs (t)) / (F_abs (t)) * 100%, where Rabs(t) is the absorbance of the reaction mixture at time t, Eabs(t) is the absorbance of the enzyme sample at time t, and Fabs(t) is the absorbance of the specific flour (fine, meal or coarse) at time t. Additional assays were left to react for 24 h before being added to the F-C and sodium carbonate. Statistical analysis and graphing Statistical analyses and graphing of data were performed using Microsoft Excel. Unless otherwise stated, statistical analyses were done using a t-test and a p-value of 0.05 or lower was considered significant. Abbreviations Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O); anti-nutritional factors (ANFs); Folin-Ciocalteu (F-C); Declarations Ethics approval and consent to participate – Not Applicable Consent for publication – Not Applicable Availability of data and materials - All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests - The authors declare that they have no competing interests Funding - This work was funded by the Natural Sciences and Engineering Research Council - Alliance Program [Project # ALLRP 550057-20] to M.E.L and M.C.L. This funding body had no input into the design of the study or collection, analysis, or interpretation of data or in writing the manuscript. Authors' contributions - RM designed, conducted and interpreted all experiments, wrote first draft of manuscript. FH trained RM, designed experiments, interpreted results. MEL conceived of some ideas, conducted faba bean meal de-hulling, grinding and air-classification. TCY conceived of some ideas, interpreted the data. MCL conceived of some ideas, supervised FH and RM, designed experiments, interpreted the data and finalized the manuscript. All authors read and approved the manuscript. Acknowledgements - This manuscript represents National Research Council of Canada Communication NRCC# 58344. References Adewale P, Lang A, Huang F, Zhu D, Sun J, Ngadi M, Yang TC (2021) A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement. Sci Rep 11(1):23982. doi: 10.1038/s41598-021-03144-8 Behnke KC (1996) Feed manufacturing technology: current issues and challenges. Anim Feed Sci Technol 62:49–57 Blainski A, Lopes GC, de Mello JC (2013) Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from Limonium brasiliense L. Molecules 18(6):6852–6865. doi: 10.3390/molecules18066852 Claassens NJ, Burgener S, Vogeli B, Erb TJ, Bar-Even A (2019) A critical comparison of cellular and cell-free bioproduction systems. Curr Opin Biotechnol 60:221–229. doi: 10.1016/j.copbio.2019.05.003 Coda R, Melama L, Rizzello CG, Curiel JA, Sibakov J, Holopainen U, Sozer N (2015) Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties. Int J Food Microbiol 193:34–42. doi: 10.1016/j.ijfoodmicro.2014.10.012 der Poel AFB, v., Abdollahi MR, Cheng H, Colovic R, den Hartog LA, Miladinovic D, Hendriks WH (2020) Future directions of animal feed technology research to meet the challenges of a changing world. Anim Feed Sci Technol 270. doi: 10.1016/j.anifeedsci.2020.114692 Everette JD, Bryant QM, Green AM, Abbey YA, Wangila GW, Walker RB (2010) Thorough study of reactivity of various compound classes toward the F-C reagent. J Agric Food Chem 58(14):8139–8144. doi: 10.1021/jf1005935 Hou T, Patel R, Lillard MO (1977) Extradiol cleavage of 3-methylcatechol by catechol 1,2-dioxygenase from various microorganisms. Appl Environ Microbiol 33(3):725–727 Irisarri P, Imperial J, Lattanzi FA, Monza J, Palacios J, Sanjuan J, Grossman J (2021) Editorial: Maximizing Nitrogen Fixation in Legumes as a Tool for Sustainable Agriculture Intensification. Front Agron 3:796717. doi: 10.3389/fagro.2021.796717 Jimenez N, Esteban-Torres M, Mancheno JM, de Las Rivas B, Munoz R (2014) Tannin degradation by a novel tannase enzyme present in some Lactobacillus plantarum strains. Appl Environ Microbiol 80(10):2991–2997. doi: 10.1128/AEM.00324-14 Kamimura N, Takahashi K, Mori K, Araki T, Fujita M, Higuchi Y, Masai E (2017) Bacterial catabolism of lignin-derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism. Environ Microbiol Rep 9(6):679–705. doi: 10.1111/1758-2229.12597 Kardum N, Glibetic M (2018) Polyphenols and Their Interactions With Other Dietary Compounds: Implications for Human Health. Adv Food Nutr Res 84:103–144. doi: 10.1016/bs.afnr.2017.12.001 Kumar Y, Basu S, Goswami D, Devi M, Shivhare US, Vishwakarma RK (2021) Anti-nutritional compounds in pulses: Implications and alleviation methods. Legume Sci 4(2). doi: 10.1002/leg3.111 Olukomaiya O, Fernando C, Mereddy R, Li X, Sultanbawa Y (2019) Solid-state fermented plant protein sources in the diets of broiler chickens: A review. Anim Nutr 5(4):319–330. doi: 10.1016/j.aninu.2019.05.005 Ominski K, McAllister T, Stanford K, Mengistu G, Kebebe EG, Omonijo F, Wittenberg K (2021) Utilization of by-products and food waste in livestock production systems: a Canadian perspective. Anim Front 11(2):55–63. doi: 10.1093/af/vfab004 Punia S, Siroha AK, Kumar M (2021) Handbook of Cereals, Pulses, Roots, and Tubers: Functionality, Health Benefits, and Applications (1st ed.) . Boca Raton: CRC Press Sherasia PL, Garg MR, Bhanderi BM (2018) Pulses and their By-Products as Animal Feed. United Nations Singh N (2017) Pulses: an overview. J Food Sci Technol 54(4):853–857. doi: 10.1007/s13197-017-2537-4 Smeriglio A, Barreca D, Bellocco E, Trombetta D (2017) Proanthocyanidins and hydrolysable tannins: occurrence, dietary intake and pharmacological effects. Br J Pharmacol 174(11):1244–1262. doi: 10.1111/bph.13630 Soares S, Brandao E, Guerreiro C, Soares S, Mateus N, de Freitas V (2020) Tannins in Food: Insights into the Molecular Perception of Astringency and Bitter Taste. Molecules 25(11). doi: 10.3390/molecules25112590 Supplementary Files C23OFabaBeanFiguresSUPPLEMENTAL220818SUBMITTED.pdf FababeanGraphicalAbstract.tiff Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 Sep, 2022 Reviewers invited by journal 06 Sep, 2022 Editor assigned by journal 02 Sep, 2022 First submitted to journal 29 Aug, 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1975355","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":134410498,"identity":"a5068adc-acfc-44f4-9b1b-1f14c00b166b","order_by":0,"name":"Rebecca M. Murphy","email":"","orcid":"","institution":"University of Ottawa Department of Chemistry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"M.","lastName":"Murphy","suffix":""},{"id":134410499,"identity":"645d5f8a-41fa-4b97-871c-a1a08e6a2105","order_by":1,"name":"Fang Huang","email":"","orcid":"","institution":"National Research Council Canada","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Huang","suffix":""},{"id":134410500,"identity":"60e91def-6b03-4253-8a1d-2ed28fcabf68","order_by":2,"name":"Matthew E. Loewen","email":"","orcid":"","institution":"University of Saskatchewan Western College of Veterinary Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"E.","lastName":"Loewen","suffix":""},{"id":134410501,"identity":"61635050-80c8-4c6c-9a2c-9926ed0f12a8","order_by":3,"name":"Trent C. Yang","email":"","orcid":"","institution":"National Research Council Canada","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Trent","middleName":"C.","lastName":"Yang","suffix":""},{"id":134410502,"identity":"81cdde2b-c5df-4843-827b-4967893b2960","order_by":4,"name":"Michele C. Loewen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACZgY2BoYCBgY+CNeGWC0GDCASBNKIsgdFy2HC6nXbmZ89+GBgw8DG3nxM4uOO83nm7QcYP/zAo8XsMJu54QyDNAY2nmNpkjPP3C6WOZPALNmDVwsPmzSPwWEGNokcs9u8bbcTZ0gAHclDSMsfg/9ALfnfbv9tOwfWwviHkBYGgwMgW9huM7YdAGthxm8Lm5lkj0EyD9Av5j9725ITZ/AkNkvL4NNy/vAziR8VdnL87M2PDX622SXOYD988OMbPFpgANkljA1EaBgFo2AUjIJRgA8AAPmdQd5fAnTrAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5053-9512","institution":"National Research Council Canada","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Michele","middleName":"C.","lastName":"Loewen","suffix":""}],"badges":[],"createdAt":"2022-08-18 15:15:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1975355/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1975355/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26280323,"identity":"b75c104a-550d-4ba0-ba3b-6382404ae1d8","added_by":"auto","created_at":"2022-09-09 19:35:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":894052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurification and Characterization of Recombinant BLC23O\u003c/strong\u003e. \u003cstrong\u003eA) \u003c/strong\u003eAffinity purification of recombinant BLC23O. SDS–PAGE showing the obtained pellet (P) and supernatant (S) fractions from an \u003cem\u003eE. coli \u003c/em\u003elysate. Subsequent flow through (FT), washes (W) and elutions (E) are included. \u0026nbsp;The elution fractions were pooled and used in all subsequent experiments. A small portion of the pooled elution was further purified by SEC (\u003cstrong\u003eSupplemental Figure S1\u003c/strong\u003e). \u003cstrong\u003eB)\u003c/strong\u003e Kinetic analysis of Ni-NTA+SEC purified BLC23O. Following SEC purification, a Michaelis-Menten kinetic analysis of BLC23O catalytic activity was conducted. BLC23O (18.7 µg/mL) was combined with10 mM of the co-factor Mn2+ and varying concentrations of the substrate 3-methylcatechol. The data was fitted to the Michaelis-Menten equation, using Microsoft Excel’s Solver.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/04f1aa4cb1b96eaf8e318432.png"},{"id":26280324,"identity":"683ad15a-8331-43b2-9477-2f707f56e096","added_by":"auto","created_at":"2022-09-09 19:35:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal phenolic content of three types of faba bean flour. \u003c/strong\u003eFine, total, and coarse faba bean meal fractions were reacted with the Folin-Ciocalteu reagent for 45 min. The absorbance at 725 nm was obtained and compared to a calibration curve for tannic acid (\u003cstrong\u003eSupplemental Figure S2\u003c/strong\u003e). Total phenolic content is represented as µg of phenolics per mg (dry weight) meal. Data is presented as the mean and standard deviation of the results (n=3). \u003cem\u003e**p\u0026lt;0.01, ***p\u0026lt;0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/befed9d95299f649b8691a32.png"},{"id":26280325,"identity":"617c01b1-e451-41cb-95d8-fe93d140d45b","added_by":"auto","created_at":"2022-09-09 19:35:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":437131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiocatalytic reduction of phenols in faba bean meal using BLC23O. \u003c/strong\u003ePhenolic content in faba bean total, fine and course meal fractions was evaluated in the presence (black) and absence (blue) of BLC23O over time. An enzyme only control sample was also assessed (grey). Reactions between each meal type and BLC23O were initiated with the addition of 0.06 mg/mL of enzyme. Samples were taken in 15 min intervals and reacted with the Folin-Ciocalteu reagent. Time zero was set to 100 % phenol and changes in absorbance at 725nm plotted on a percentage change basis with\u003cstrong\u003e A)\u003c/strong\u003e showing fine meal, \u003cstrong\u003eB)\u003c/strong\u003e showing total meal and \u003cstrong\u003eC)\u003c/strong\u003e showing coarse meal fractions. \u0026nbsp;The observed changes in phenol content of BLC23O treated \u003cstrong\u003eD)\u003c/strong\u003e fine, \u003cstrong\u003eE)\u003c/strong\u003e total and \u003cstrong\u003eF)\u003c/strong\u003e course meal fractions were quantified relative to untreated fractions. The data represents the mean and standard deviation (n=3). Linear fits of the data and the corresponding equations and coefficients of determination are shown.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/1ca539daef522b8c80bda747.png"},{"id":26280430,"identity":"7dcb494e-e2bd-4f25-ab3c-2ca0347b0c36","added_by":"auto","created_at":"2022-09-09 19:40:23","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of extended BLC23O reaction time on phenol content of faba bean meals. \u003c/strong\u003eChange in total meal phenol content after 24 h reaction with BLC23O. 1.2 mg/mL of meal flour and 0.06 mg/mL of BLC23O were left to react at room temperature for 24 h and then reacted with the Folin-Ciocalteu reagent. Data is presented as the mean and standard deviation of the % absorbance at 725 nm (n=3). \u003cem\u003e***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/37969c2093914f50bbf5f310.jpeg"},{"id":26280431,"identity":"f7dbe1e7-d10b-4f14-842c-0c994e4a80d4","added_by":"auto","created_at":"2022-09-09 19:40:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1024197,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/deab59ba-a9f8-4af7-9908-0d384b47de7d.pdf"},{"id":26280322,"identity":"a2f19094-3061-4b83-bf8c-57ed1a94d763","added_by":"auto","created_at":"2022-09-09 19:35:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":416330,"visible":true,"origin":"","legend":"","description":"","filename":"C23OFabaBeanFiguresSUPPLEMENTAL220818SUBMITTED.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/c64970bf85f092cb0e59e6a3.pdf"},{"id":26280327,"identity":"360a62a7-a61b-4e4f-a490-6957ed6bbd43","added_by":"auto","created_at":"2022-09-09 19:35:24","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5395878,"visible":true,"origin":"","legend":"","description":"","filename":"FababeanGraphicalAbstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-1975355/v1/823b2add58a3b2ae39f2116b.tiff"}],"financialInterests":"","formattedTitle":"Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlternative feed ingredients and innovation of feed technology are progressively being investigated and incorporated into processing pipelines toward enhanced digestibility and efficiency, decreased environmental impact and maximized economic opportunity (Behnke, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; der Poel et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePulses (\u003cem\u003eFabaceae\u003c/em\u003e family) and their by-products are examples of advantageous sources of animal feed that could be further optimized to increase their value and applicability (Sherasia, Garg, \u0026amp; Bhanderi, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Singh, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Currently, pulses play an important role in sustainable development based on their ability to fix nitrogen, reducing the need for costly nitrogen fertilizers when used in crop rotations, and sequestering greenhouse gas from the environment (Irisarri et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However pulse nutrient and protein-rich profiles significantly furthers their potential. For example, pulse meal, a by-product of the industry, is approximately 40\u0026ndash;45% protein on a dry mass basis and thus holds great potential to serve as animal feed, contributing to food security and reducing competition with human food (Sherasia et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Such applications of by-products also reduces costs related to disposal and allows for the conversion of low value products into higher value feed/food (Ominski et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTannins and other non-tannic (poly)phenols are intrinsic to pulses, and in the context of animal nutrition, are considered anti-nutritional factors (ANFs) based on observed negative effects on digestion and the bioavailability of nutrients (Kardum \u0026amp; Glibetic, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These effects are due to (poly)phenols forming complexes with storage proteins, interfering with peptide bond proteolysis, and serving as direct inhibitors of digestive enzyme, together decreasing protein and carbohydrate availability (Punia, Siroha, \u0026amp; Kumar, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Chelation of metal ions by polyphenolics has also been shown to reduce absorption and decrease uptake of vitamins and minerals (Singh, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Finally, the palatability of pulse meal is reduced by tannins in particular, due to undesirable astringency, which lowers food intake and animal performance (Singh, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, reducing phenol content (tannic and non-tannic) in pulse products remains an important goal for increased nutritional and economic value.\u003c/p\u003e \u003cp\u003eCurrently, microbial fermentation is used as a strategy to lower ANFs, including tannins, in feeds (der Poel et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Olukomaiya, Fernando, Mereddy, Li, \u0026amp; Sultanbawa, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, a Gram-positive lactic acid bacterium commonly used in the food industry, specifically possesses genes encoding tannin acyl hydrolase (commonly known as tannase) and gallate decarboxylase, yielding the monophenol pyrogallol (Jimenez, Esteban-Torres, Mancheno, de Las Rivas, \u0026amp; Munoz, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the observed accumulation of such monophenols in \u003cem\u003eL. plantarum\u003c/em\u003e fermented feeds is also undesirable (Kardum \u0026amp; Glibetic, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The next step in degradation of monophenols involves breaking the phenolic ring. In nature, this activity relies on a family of microbial catechol dioxygenases that carry out the oxidative cleavage of hydroxylated aromatic rings, to yield linear compounds that feed into the citric acid cycle (Kamimura et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Phenolic ring cleavage can be intradiol (between two consecutive hydroxyl groups on the ring (positions 1 and 2)), proximal extradiol (next to the hydroxyls; position 2 and 3) or distal extradiol (removed from the hydroxyls; position 4 and 5) (Hou, Patel, \u0026amp; Lillard, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). A brief review of the current literature and a search of the NCBI database suggests \u003cem\u003eL. plantarum\u003c/em\u003e does not encode any members of this family of enzymes, consistent with the high accumulation of phenolics in such fermentations.\u003c/p\u003e \u003cp\u003eThus, as an alternative or complement to microbial fermentation, cell-free biocatalysis has been proposed for reduction of phenolic content. A biocatalytic strategy would decouple tannin and phenolic degradation from cellular physiology, reducing any downregulation imposed by the carbohydrate rich environment and feedback inhibition, as well as allowing absolute control of enzyme types and concentrations applied (Claassens, Burgener, Vogeli, Erb, \u0026amp; Bar-Even, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne example of a potential candidate for cell-free phenolic degradation of pulse meal is a \u003cem\u003eBacillus ligniniphilus\u003c/em\u003e L1 catechol 2,3-dioxygenase (BLC23O; (Adewale et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)). \u003cem\u003eB. ligniniphilus\u003c/em\u003e L1 is a halotolerant and alkaliphilic bacterium isolated from sediments from the South China Sea, known to use polyphenolic lignin as its sole carbon source. Catechol 2,3-dioxygenases elicit proximal extradiol cleavage. Three catechol 2,3-dioxygenases encoded by \u003cem\u003eB. ligniniphilus\u003c/em\u003e L1 have been identified and the shortest one, a protein of 283 amino acids (BLC23O, NCBI accession WP_017726464.1) with a calculated molecular mass of approximately 32 kDa, was recently characterized (Adewale et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This enzyme was found to have unusually broad substrate specificity and good thermostability, traits proposed to be associated with its atypical monomeric structure.\u003c/p\u003e \u003cp\u003eHere the application of BLC23O to cell-free reduction of phenolic content in pulse meal is assessed. Scaled-up recombinant production and purification of the enzyme is described, followed by evaluation of the enzyme\u0026rsquo;s potential to reduce phenolics in 3 different meal fractions from \u003cem\u003eVicia faba\u003c/em\u003e (faba bean).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLarge-scale (4 L fermentative) production and Ni-NTA enrichment of BLC23O\u003c/h2\u003e \u003cp\u003eShake flask production of BLC23O from \u003cem\u003eEcherichia coli\u003c/em\u003e has been previously reported (Adewale et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Here production of BLC23O was scaled up to a 4 L fermentative format. Wet weight determination of the obtained cell pellet was 49.8 g, or 12.4 g cell pellet / L of culture. The pellet was re-suspended in lysis buffer and high-pressure homogenization was used to extract proteins. Following centrifugation, the supernatant was collected and BLC23O was further enriched using the fused His-tag. Outcomes from the Ni-NTA affinity chromatography were visualized by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Thick bands at the expected Mw of approximately 35 kDa are present in the three elution fractions and the original supernatant fraction. Small amounts of protein were also detected at 35 kDa in the washes and flow-through, suggesting minor losses during the enrichment. No BLC23O was detected in the lysis pellet, indicating that cell lysis was successful. Contaminating proteins were largely eliminated from the final sample. The elution fractions were pooled and protein quantified, yielding 116 mg of enzyme / L of cell culture, or 9.3 mg of BLC23O / g of wet weight cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKinetic analysis of BLC23O.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA small portion (4%) of the obtained protein from Ni-NTA enrichment was subjected to further purification by size exclusion chromatography. The chromatographic separation curve for BLC23O (\u003cb\u003eSupplemental Figure S1A\u003c/b\u003e) showed a clear and steep peak at 82 min. Calibration of indicated this is consistent with the expected molecular weight of 35 kDa (\u003cb\u003eSupplemental Figure S1B\u003c/b\u003e), falling in between ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Collected fractions spanning this peak were visualized by SDS-PAGE, yielding a single significant band around the expected Mw of 35 kDa (\u003cb\u003eSupplemental Figure S1C\u003c/b\u003e). A Michaelis-Menten analysis of the obtained purified BLC23O against varying concentrations of the known substrate, 3-methycatechol, yielded a K\u003csub\u003eM\u003c/sub\u003e of 379\u0026thinsp;\u0026plusmn;\u0026thinsp;86 \u0026micro;M, a turnover number (kcat) of 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and a catalytic efficiency (kcat/K\u003csub\u003eM\u003c/sub\u003e) of 340\u0026thinsp;\u0026plusmn;\u0026thinsp;80 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Comparison of these results with a previous kinetic analysis for BLC23O highlights non-significant differences (previously: K\u003csub\u003eM\u003c/sub\u003e 418\u0026thinsp;\u0026plusmn;\u0026thinsp;25 \u0026micro;M, kcat 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eand kcat/K\u003csub\u003eM\u003c/sub\u003e 480\u0026thinsp;\u0026plusmn;\u0026thinsp;80 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; (Adewale et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of phenolic content in faba bean meal by BLC23O\u003c/h2\u003e \u003cp\u003eInitially, the phenolic content in three distinct samples of faba bean was meal was assessed, including the original \u0026lsquo;total\u0026rsquo; meal, as well as \u0026lsquo;fine\u0026rsquo; and \u0026lsquo;course\u0026rsquo; fractions of the meal obtained from air-classification. Following reaction of re-suspended meal fractions with the Folin-Ciocalteu (F-C) reagent, the absorbance at 725 nm wavelength was measured and quantified by comparison to a calibration curve made with pure tannic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Supplemental Figure S2A\u003c/b\u003e). Statistically significant differences in phenolic content were detected, where the fine fraction had the highest total phenolic content, followed by total meal, and then the coarse fraction. This is consistent with the fine fraction being protein enriched and the coarse fraction being carbohydrate enriched (Coda et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Higher-phenolics in the \u0026lsquo;fine\u0026rsquo; protein fraction is consistent with the protein amino acids introducing significant phenolic content, in addition to other (poly)phenolics being fractionated into the sample due to linkages with the protein. Some phenolic content was also observed in the \u0026lsquo;coarse\u0026rsquo;, carbohydrate rich fraction, likely due to some association between carbohydrates and (poly)phenols (Kardum \u0026amp; Glibetic, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Smeriglio, Barreca, Bellocco, \u0026amp; Trombetta, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). That this assay was able to detect the differences between the meal fractions, demonstrates that the F-C reagent is appropriate for use in phenolic content determination assays in faba bean meal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe kinetics of the F-C reagent were evaluated in more detail at the maximum amount of phenolics detected in meal reactions, using 14 \u0026micro;g/mL tannic acid (\u003cb\u003eSupplemental Figure S2B\u003c/b\u003e). Under these conditions the reaction was complete within 60\u0026ndash;70 min and phenolic content remained constant for an additional 120 min thereafter. These outcomes highlight the minimum amount of time solutions should be left to react before measuring the absorbance, ensuring an efficient and robust assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiocatalytic reduction of phenolics in faba bean meal\u003c/h2\u003e \u003cp\u003eThe ability of BLC23O to degrade polyphenols in total faba bean meal and the air classified fractions was evaluated by monitoring phenolic content over time. Following addition of the enzyme to the meal suspensions, samples were taken at 15 min intervals and reacted with the F-C reagent and absorbance obtained at 725 nm. In all cases, BLC23O-treatment led to detectable decreases in phenol content over time, compared to untreated meal samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Importantly, in both the meal-alone and enzyme-alone samples, phenolic content remained constant or even displayed a slight increase over time. That increases in phenol content were observed in the meal fractions (in the absence of any enzyme) is likely representative of the effect of chemical hydrolysis over time. (Poly)phenol content can be inaccessible due to being embedded in large complex structures (Smeriglio et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Soares et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Water hydrolysis is known to release phenols from the large complexes and break up interactions with proteins and sugars, increasing phenol available to react with the enzyme and assay reagents. Thus to quantify the effect of the enzymatic treatment on phenolic levels, the ratio between phenolics in the BLC23O-treated samples and phenolics in the meal-only samples were determined. Additionally, background phenolics detected in the enzyme-only sample were subtracted. This analysis revealed a 49.6% decrease in phenolic content in the course meal fraction over 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Phenolic content in the total meal was reduced by 26.7%, while only a 9.0% reduction was observed in the fine fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) values for the linear regressions of each fraction type are low, especially for the assay with the coarse flour, indicating a high amount of variability in the data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo ascertain the stability of the reaction, overnight assays were performed for the total meal fraction in the presence and absence of BLC23O, as well as for the enzyme-alone (background control). Samples were taken at 0 and 24 h, reacted with the F-C reagent for 60 min, and absorbances obtained at 725 nm wavelength. Consistent with previous experiments, a small but significant increase in phenolics was detected in the meal-only sample, while decreases in both the enzyme-only and the BLC23O-treated meal samples were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Quantification (as described above) indicates a 68% decrease in phenolics in this meal fraction upon treatment with BLC23O for 24 hrs. This represents a 41% further decrease in phenolic content compared to values obtained after only 3 h, and emphasizes the catalytic stability of BLC23O over time in this complex environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study demonstrates the viability of cell-free biocatalytic reduction of phenolic content in faba bean meal. The representative catechol dioxygenase, BLC23O, enzyme used in the proof-of concept was selected based on its known stability and broad substrate specificity. Its amenability to high yield fermentative recombinant production has now been established, and its ongoing stability in complex environments including resuspension in a slurry of pulse meal demonstrated. While results suggest that BLC23O is effective for biocatalytic phenolic reduction, the upstream release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) is likely a limiting factor in the absence of other enzymes or microbial fermentation. Analyses of the effect of including other enzymes with BLC23O to improve substrate availability, and the effect of adding BLC23O to fermentative processes is ongoing, toward optimization of the nutritional and economical value of pulse meal in the animal feed industry.\u003c/p\u003e"},{"header":"Methods And Materials","content":"\u003cp\u003eAll chemicals were obtained from Sigma Aldrich, except where indicated below. Faba beans were obtained from Faba Canada Ltd, de-hulled at the Canadian International Grains Institute (Winnipeg, Canada), and milled/air classified at the Richardson Center for Food Technology and Research (Winnipeg, Canada), yielding a \u0026lsquo;total\u0026rsquo; meal fraction (before air classification), and then air classification-derived \u0026lsquo;fine\u0026rsquo; and \u0026lsquo;course\u0026rsquo; meal fractions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBLC23O fermentative scale production\u003c/h2\u003e \u003cp\u003eAn optimized version of the BLC23O coding region was cloned into the pET28B\u0026thinsp;+\u0026thinsp;vector such that the expressed fusion protein included an N-terminal 6 x His tag as previously described (Adewale et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eEcherichia coli\u003c/em\u003e BL21 cells containing the expression vector encoding the BLC23O gene was used to inoculate an overnight culture containing 50 mL of Terrific Broth (TB) medium (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) with 50 \u0026micro;g/mL kanamycin. The culture was incubated overnight at 37\u0026deg;C in a MaxQ 6000 shaker (Thermo Scientific) at 150 rpm. The overnight culture was used to inoculate a BioFlo/CelliGen 115 benchtop fermentor \u0026amp; bioreactor (New Brunswick Scientific) containing 4 L of TB medium, 50 mg/L kanamycin, and 0.5 mL of antifoam. The bioreactor had an agitation of 250 rpm, its water jacket temperature was set to 37\u0026deg;C, and the dissolved oxygen setpoint was 30%. The culture was grown to an OD600 between 0.6 and 0.7 at which time protein expression was induced with the addition of 0.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The culture was left growing in the bioreactor overnight at 18\u0026deg;C. The media was centrifuged in a Sorvall Lynx 4000 centrifuge (Thermo scientific) at 3250 x g for 30 min at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePurification of recombinantly produced BLC23O\u003c/h2\u003e \u003cp\u003eThe obtained pellet was resuspended in 500 mL of 1x lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 3U/mL benzonase, 1 mg/mL lysozyme, pH 8.0). Proteins were extracted by high pressure homogenization using an EmulsiFlex-C5 (Avestin) according to the manufacturer\u0026rsquo;s protocol. The lysate was centrifuged (Thermo scientific) at 12,000 rpm for 15 min at 4\u0026deg;C. The supernatant was collected and applied to nickel-nitriloacetic acid (Ni-NTA) resin (Qiagen) with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) by a batch method, where resin was mixed with the cleared lysate in a MaxQ 6000 shaker (Thermo Scientific) set to 200 rpm at 4\u0026deg;C for 2 hrs. The sample was centrifuged in a Sorvall Legend X1R centrifuge at 1000 x g for 3 min at 4\u0026deg;C (Thermo scientific) and the supernatant discarded. The remaining beads were washed three times using this same process with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and then BLC23O was eluted using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Obtained elution supernatants containing the BLC23O enzymes were pooled and concentrated using an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentration of the obtained BLC23O sample was determined using a Quick Start Bradford protein assay (Bio-Rad) according to the manufacturer\u0026rsquo;s protocol. Proteins samples were visualized by 12% SDS-PAGE.\u003c/p\u003e \u003cp\u003eA small portion (approx. 4% of the total sample) of the obtained enriched enzyme was further subjected to Fast Performance Liquid Chromatography (FPLC; Amersham Pharmacia Biotech) size-exclusion chromatography (SEC) with a HiLoad 16/600 Superdex 200 (Cytvia) column. Size exclusion buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) was degassed and the FPLC flowrate was set to 1 mL/min. Elution fractions were collected in increments of 1.4 mL. Fractions containing BLC23O were combined and concentrated with an Amicon Ultra-15 centrifugal filter with a 10 kDa cutoff (MilliporeSigma). The final concentrations of BLC23O were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer\u0026rsquo;s protocol. Proteins samples were visualized by 12% SDS-PAGE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBLC23O kinetic analysis\u003c/h2\u003e \u003cp\u003eReaction solutions with total volumes of 250 \u0026micro;L were prepared in triplicate in a 96-well microplate (Greiner Bio-One) with 50 mM phosphate buffer pH 7.5, 18.7 \u0026micro;g/mL of BLC23O purified by Ni-NTA and SEC, 10 mM Mn\u003csup\u003e2+\u003c/sup\u003e, and substrate (3-methylcatechol) concentrations varying from 0.05 mM to 3 mM. Plates were pre-incubated for 5 min at 32.5\u0026deg;C and reactions were initiated with the addition of substrate and samples left at 32.5\u0026deg;C. The absorbance was read at 388 nm for 60 min in 30 sec intervals using a SpectraMax M5e spectrophotometer (Molecular Devices). The initial reaction rate, in \u0026micro;M of product formed per second, was plotted and the data fitted to the Michaelis-Menten equation using Microsoft Excel\u0026rsquo;s Solver add-in.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of phenolic content by F-C reagent.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on previous reports (Blainski, Lopes, \u0026amp; de Mello, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Everette et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), a calibration curve for the quantification of phenolic content was created where reaction solutions with a volume of 1 mL were prepared with 0.125 N F-C reagent, 0.125 g/mL of sodium carbonate, and tannic acid concentrations ranging from 0 to 30 \u0026micro;g/mL. The solutions were left to react for 45 min and then 250 \u0026micro;L of each was transferred into a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using a SpectraMax M5e spectrophotometer (Molecular Devices). This was repeated in triplicate. Tannic acid reaction kinetics were assessed in a similar manner, but with reaction solution volumes of 250 \u0026micro;L and tannic acid concentrations ranging from 0 to 14 \u0026micro;g/mL. In this case, as soon as the tannic acid was added, the absorbance at 725 nm was monitored for 180 min in 15 min intervals using an Epoch 2 microplate spectrophotometer (Biotek).\u003c/p\u003e \u003cp\u003eInitial phenolic content in the three faba bean meal fractions was determined using the same procedure as described above, but with 0.1, 0.2, and 0.3 mg/mL of each type of faba bean sample instead of tannic acid, and incubation with the F-C reagent for 60 min. Results were compared to the tannic acid calibration curve for quantification, based on the assumption that a \u0026micro;g of commercial tannic acid is equivalent to a \u0026micro;g of phenolic content in the meal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBLC23O-mediated phenolic reduction in faba bean flour\u003c/h2\u003e \u003cp\u003eBLC23O reaction solutions with volumes of 4 mL were prepared for each of the faba bean meal fractions with 1.2 mg/mL of each fraction type and reactions initiated with the addition of 0.06 mg/mL of BLC23O enzyme. A blank solution with no meal or enzyme, as well negative and background controls of meal only and enzyme only were also prepared. Samples from the reaction mixtures were taken at 15 min intervals for 3 hrs, vortexed into 0.125 N F-C reagent and 0.125 g/mL of sodium carbonate and then left to react for 60 min at room temperature. Subsequently, 250 \u0026micro;L from each mixture was transferred to a 96-well microplate (Greiner Bio-One) and the absorbance was read at 725 nm using Epoch 2 microplate spectrophotometer (Biotek). This was performed in triplicate. The % phenolic reduction was calculated using the following equation: (phenolic content of meal in reaction mixture (%) = (R_abs (t) - E_abs (t)) / (F_abs (t)) * 100%, where Rabs(t) is the absorbance of the reaction mixture at time t, Eabs(t) is the absorbance of the enzyme sample at time t, and Fabs(t) is the absorbance of the specific flour (fine, meal or coarse) at time t. Additional assays were left to react for 24 h before being added to the F-C and sodium carbonate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis and graphing\u003c/h2\u003e \u003cp\u003eStatistical analyses and graphing of data were performed using Microsoft Excel. Unless otherwise stated, statistical analyses were done using a t-test and a p-value of 0.05 or lower was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eBacillus ligniniphilus\u003c/em\u003e L1 catechol 2,3-dioxygenase (BLC23O); anti-nutritional factors (ANFs); Folin-Ciocalteu (F-C);\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u0026ndash; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u0026ndash; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e- All data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e- The authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e- This work was funded by the Natural Sciences and Engineering Research Council - Alliance Program [Project # ALLRP 550057-20] to M.E.L and M.C.L. This funding body had no input into the design of the study or collection, analysis, or interpretation of data or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e- RM designed, conducted and interpreted all experiments, wrote first draft of manuscript. FH trained RM, designed experiments, interpreted results. MEL conceived of some ideas, conducted faba bean meal de-hulling, grinding and air-classification. TCY conceived of some ideas, interpreted the data. MCL conceived of some ideas, supervised FH and RM, designed experiments, interpreted the data and finalized the manuscript.\u0026nbsp;All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements -\u0026nbsp;\u003c/strong\u003eThis manuscript represents National Research Council of Canada Communication NRCC# 58344.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAdewale P, Lang A, Huang F, Zhu D, Sun J, Ngadi M, Yang TC (2021) A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement. Sci Rep 11(1):23982. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-021-03144-8\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBehnke KC (1996) Feed manufacturing technology: current issues and challenges. Anim Feed Sci Technol 62:49\u0026ndash;57\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBlainski A, Lopes GC, de Mello JC (2013) Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from Limonium brasiliense L. Molecules 18(6):6852\u0026ndash;6865. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules18066852\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eClaassens NJ, Burgener S, Vogeli B, Erb TJ, Bar-Even A (2019) A critical comparison of cellular and cell-free bioproduction systems. Curr Opin Biotechnol 60:221\u0026ndash;229. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.copbio.2019.05.003\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCoda R, Melama L, Rizzello CG, Curiel JA, Sibakov J, Holopainen U, Sozer N (2015) Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties. Int J Food Microbiol 193:34\u0026ndash;42. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijfoodmicro.2014.10.012\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eder Poel AFB, v., Abdollahi MR, Cheng H, Colovic R, den Hartog LA, Miladinovic D, Hendriks WH (2020) Future directions of animal feed technology research to meet the challenges of a changing world. Anim Feed Sci Technol 270. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.anifeedsci.2020.114692\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEverette JD, Bryant QM, Green AM, Abbey YA, Wangila GW, Walker RB (2010) Thorough study of reactivity of various compound classes toward the F-C reagent. J Agric Food Chem 58(14):8139\u0026ndash;8144. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jf1005935\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHou T, Patel R, Lillard MO (1977) Extradiol cleavage of 3-methylcatechol by catechol 1,2-dioxygenase from various microorganisms. Appl Environ Microbiol 33(3):725\u0026ndash;727\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eIrisarri P, Imperial J, Lattanzi FA, Monza J, Palacios J, Sanjuan J, Grossman J (2021) Editorial: Maximizing Nitrogen Fixation in Legumes as a Tool for Sustainable Agriculture Intensification. Front Agron 3:796717. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fagro.2021.796717\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJimenez N, Esteban-Torres M, Mancheno JM, de Las Rivas B, Munoz R (2014) Tannin degradation by a novel tannase enzyme present in some Lactobacillus plantarum strains. Appl Environ Microbiol 80(10):2991\u0026ndash;2997. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.00324-14\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKamimura N, Takahashi K, Mori K, Araki T, Fujita M, Higuchi Y, Masai E (2017) Bacterial catabolism of lignin-derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism. Environ Microbiol Rep 9(6):679\u0026ndash;705. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/1758-2229.12597\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKardum N, Glibetic M (2018) Polyphenols and Their Interactions With Other Dietary Compounds: Implications for Human Health. Adv Food Nutr Res 84:103\u0026ndash;144. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/bs.afnr.2017.12.001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKumar Y, Basu S, Goswami D, Devi M, Shivhare US, Vishwakarma RK (2021) Anti-nutritional compounds in pulses: Implications and alleviation methods. Legume Sci 4(2). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/leg3.111\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOlukomaiya O, Fernando C, Mereddy R, Li X, Sultanbawa Y (2019) Solid-state fermented plant protein sources in the diets of broiler chickens: A review. Anim Nutr 5(4):319\u0026ndash;330. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.aninu.2019.05.005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOminski K, McAllister T, Stanford K, Mengistu G, Kebebe EG, Omonijo F, Wittenberg K (2021) Utilization of by-products and food waste in livestock production systems: a Canadian perspective. Anim Front 11(2):55\u0026ndash;63. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/af/vfab004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePunia S, Siroha AK, Kumar M (2021) \u003cem\u003eHandbook of Cereals, Pulses, Roots, and Tubers: Functionality, Health Benefits, and Applications (1st ed.)\u003c/em\u003e. Boca Raton: CRC Press\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSherasia PL, Garg MR, Bhanderi BM (2018) Pulses and their By-Products as Animal Feed. United Nations\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSingh N (2017) Pulses: an overview. J Food Sci Technol 54(4):853\u0026ndash;857. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13197-017-2537-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSmeriglio A, Barreca D, Bellocco E, Trombetta D (2017) Proanthocyanidins and hydrolysable tannins: occurrence, dietary intake and pharmacological effects. Br J Pharmacol 174(11):1244\u0026ndash;1262. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/bph.13630\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSoares S, Brandao E, Guerreiro C, Soares S, Mateus N, de Freitas V (2020) Tannins in Food: Insights into the Molecular Perception of Astringency and Bitter Taste. Molecules 25(11). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules25112590\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pulse meal, Vicia faba, biocatalysis, catechol 2,3 dioxygenase, phenol reduction","lastPublishedDoi":"10.21203/rs.3.rs-1975355/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1975355/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, pulse meal contains anti-nutritional and anti-palatability compounds, including (poly)phenolics (tannic and non-tannic), such that improvements in pulse meal processing are still needed to increase its uptake by the industry. Microbial fermentation is currently used as a strategy to decrease tannin content, but results in the undesirable accumulation of monophenolics. Here we investigate the viability of cell-free biocatalytic reduction of phenolic content in faba bean (\u003cem\u003eVicia faba\u003c/em\u003e) meal. A representative catechol dioxygenase, \u003cem\u003eBacillus ligniniphilus \u003c/em\u003eL1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Its amenability to large scale recombinant production was established, and its ongoing stability in complex environments including resuspension in slurries of faba bean meal demonstrated. Reaction results suggest that BLC23O is effective for biocatalytic phenol reduction in faba bean meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (tannins, proteins, carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.\u003c/p\u003e","manuscriptTitle":"Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-09 19:35:22","doi":"10.21203/rs.3.rs-1975355/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-09-06T04:48:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-09-06T04:18:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-09-02T17:45:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2022-08-29T18:49:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bf27009-c7a0-45e7-b595-ed78c1e187d3","owner":[],"postedDate":"September 9th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-01-20T09:26:45+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-09 19:35:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1975355","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1975355","identity":"rs-1975355","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}