Polyphenol Oxidase Containing Sidestreams as Emulsifiers of Rumen

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Polyphenol oxidase containing sidestreams as emulsifiers of rumen bypass linseed oil emulsions: interfacial characterization and efficacy of protection against in vitro ruminal biohydrogenation Frederik Gadeyne, Nympha De Neve, Bruno Vlaeminck, Erik Claeys, Paul Van Der Meeren, and Veerle Fievez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01022 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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Journal of Agricultural and Food Chemistry

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Polyphenol oxidase containing sidestreams as emulsifiers of rumen bypass linseed

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oil emulsions: interfacial characterization and efficacy of protection against in vitro

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ruminal biohydrogenation

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Frederik Gadeyne1, Nympha De Neve1, Bruno Vlaeminck1, Erik Claeys1, Paul Van der

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Meeren2, Veerle Fievez1*

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Laboratory for Animal Nutrition and Animal Product Quality, Faculty of Bioscience

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Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium

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2

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University, Coupure Links 653, 9000 Ghent, Belgium

Particle and Interfacial Technology Group, Faculty of Bioscience Engineering, Ghent

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* corresponding author ([email protected]; +3292649002)

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Abstract

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The low transfer in ruminants of dietary polyunsaturated fatty acids to the milk or peripheral

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tissues is largely due to ruminal biohydrogenation. Lipids emulsified by a polyphenol oxidase

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(PPO) rich protein extract of red clover were shown before to be protected against this

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breakdown after cross-linking with 4-methylcatechol. Protein extracts of thirteen other vegetal

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resources were tested. Surprisingly, the effectiveness to protect emulsified lipids against in vitro

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ruminal biohydrogenation largely depended on the origin of the extract and its protein

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concentration but was not related to PPO activity. Moreover, PPO isoforms in vegetal sources,

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effectively protecting emulsified lipids, were diverse and their presence at the emulsion interface

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did not seem essential. Potato tuber peels were identified as an interesting biological source of

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emulsifying proteins and PPO, particularly since protein extracts of industrial potato sidestreams

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proved to be suitable for the current application.

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Keywords: Protein sidestream - Polyphenol oxidase - Rumen Bypass - Biohydrogenation -

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Emulsion

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Introduction

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Polyphenol oxidase (PPO) is a widely distributed enzyme known for the characteristic browning

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of damaged plant tissues as a result of polymerization of PPO-created o-quinones with

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nucleophilic compounds1. Browning is generally unwanted in fruits and vegetables because of its

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detrimental effect on product quality, which resulted in numerous browning prevention and

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inhibition techniques2,3. In certain cases, however, polymerization of quinones is of primary

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interest. The common brown color in black tea4,5 and cocoa6, for example, is the result of a PPO-

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induced reaction. Similarly, in the field of ruminant nutrition, it has been suggested that PPO in

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red clover is responsible for increased nitrogen use efficiency7 and the protection of lipids

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against microbial degradation8. Recently, Gadeyne et al.9 successfully used this polymerizing

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reaction to protect emulsified lipids against in vitro ruminal biohydrogenation (BH) and

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oxidation during storage using a PPO-rich protein extract from red clover: due to the PPO-

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induced conversion of the simple diphenol 4-methylcatechol (4-MC) into a quinone,

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polymerization of the interfacial protein layer occurs, resulting in protection of the encapsulated

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lipophilic content from degradation. Indeed, polyunsaturated fatty acids (PUFA) are to a large

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extent subject to microbial BH in the rumen, resulting in the formation of saturated fatty acids

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(FA) and accumulation of cis and trans isomers10. Consequently, protecting PUFA against

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microbial degradation might enable the production of milk and meat of ruminant origin with

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reduced amounts of saturated and trans-FA, which aligns with current public health policies11.

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However, the seasonal availability and the low-fat content of red clover limits the potential

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industrial applicability of the current technology. Nevertheless, PPO or tyrosinase activity has

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been reported in almost any organism, both in plants, fungi, bacteria and animals12. Although

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only PPO extracted from red clover has been used to create protection against ruminal BH,

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extrapolation to other resources could be hypothesized. In this perspective, industrial sidestreams

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of plant resources are widely available, some of which are now used in animal feeds or 3 ACS Paragon Plus Environment


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discarded, although they still might contain valuable molecules and characteristics. Therefore,

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the aim of this paper is to assess the possibility to protect PUFA against ruminal BH using 4-MC

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as diphenolic cross-linking mediator after emulsification using protein extracts from different

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plant sources or possible sidestreams as emulsifiers. In addition, protein extracts and emulsions

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were further characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE), PPO zymography, conventional PPO activity assays and protein load tests to get

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further insight in factors affecting the protection efficiency (PE) in the rumen.

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Materials and methods

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Materials

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Different vegetal sources were used for a screening test depending on their availability, formerly

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reported PPO activity in literature13-23 or potential as upgradable industrial sidestream24-26: apple

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peels (Malus domestica Mill. cv. Jonagold), artichoke flower leaves (Cynara scolymus L.),

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broccoli stems (Brassica oleracea L. convar. botrytis var. cymosa), carrot peels (Daucus carota

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subsp sativus (Hoffm.) Schübl & G. Martens), cauliflower florets, stems and leaves (Brassica

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oleracea L. convar. botrytis var. botrytis), eggplant pulp (Solanum melongena L.), pineapple

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peels (Ananas comosus (L.) Merr.), potato tuber peels (Solanum tuberosum L.), red clover stems

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& leaves (Trifolium pratens L. cv. Lemmon), spinach leaves (Spinacia olereacea L.) and tomato

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stems & leaves (Solanum lycopersicum L.). Plant sources were either bought in a local grocery

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store or kindly provided by the Institute for Agricultural and Fisheries Research (ILVO,

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Belgium). Immediately upon arrival in the laboratory, plant material was cut into fine pieces,

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snap frozen in liquid nitrogen and stored at -80 °C until further use.

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In addition to these sources, sidestreams of the production process of two potato processing

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companies were collected including solid (cutter scraps and slivers) as well as liquid sidestreams


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after steam peeling, but were taken before any blanching step was performed. Samples were

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stored at -80 °C until further use.

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Crude linseed oil (40, 194, 169 and 586 mg per g total FA of C18:0, C18:1n-9, C18:2n-6 and

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C18:3n-3, respectively) was a gift from Dumoulin (Kortrijk, Belgium). 4-MC was purchased

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from Sigma-Aldrich (Bornem, Belgium). All other chemicals were of analytical grade and were

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purchased from Sigma-Aldrich (Bornem, Belgium), Merck (Darmstadt, Germany), Carl-Roth

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(Karlsruhe, Germany) or VWR (Heverlee, Belgium), unless stated otherwise. Any centrifugal

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step was performed using a refrigerated Beckman J2-HS centrifuge (Beckman Coulter, Brea,

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California, USA).

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Preparation of protected PUFA emulsions and experimental setup

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Emulsions were prepared as described by Gadeyne et al.9 In brief, a three-step process was

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performed: first, proteins were isolated from frozen plant material by extraction in 0.1 M sodium

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phosphate buffer (SPB) with ascorbic acid (pH=7), protein precipitation with acetone (4:1

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volumetric acetone to SPB ratio) and recovery of soluble protein in 0.01 M SPB (pH=7); second,

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this protein extract was used to emulsify oil with a high speed Ultra-Turrax (T25 Basic, Ika

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Werke, Staufen, Germany) for pre-emulsification and passed 5 times at 25 MPa through a

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microfluidizer (M110S, Microfluidics Corporation, Newton, Massachusetts, USA); third,

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emulsions were treated with 4-MC and shaken for 24 h to induce protein-phenol complexing. All

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emulsions were prepared using 20 mg linseed oil per ml of extract and stored at 4 C.

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Three experiments were performed to assess the potential of different sources as emulsifier to

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protect against ruminal degradation. Emulsions in experiment 1 were created using protein

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extracts from the thirteen selected vegetal sources mentioned in the Materials sections.

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Emulsions (before addition of diphenol) were prepared in triplicate, starting from three different

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protein extracts. Afterwards, 4-MC was added to obtain a final concentration of 0, 10, 20 or 40

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mM 4-MC. Experiment 2 attempted to perform a first screening using industrial sidestreams of 5 ACS Paragon Plus Environment


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potato processing facilities. Samples were taken from two companies at similar places in the

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production process and kept as replicates throughout the experiment. Extraction procedures of

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solid and liquid sidestreams aimed at obtaining protein concentrations of at least 1 mg per ml

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solution. Therefore, extraction of solid sidestreams was as described before9. Volumes of liquid

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byproducts needed for extraction in 0.1 M SPB with ascorbic acid were determined based on

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preliminary analysis of the protein content. Final 4-MC concentrations of emulsions were 0 or 20

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mM 4-MC. Finally, in experiment 3 four vegetal sources were selected from the first experiment

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for further characterization of proteins of the continuous phase and present at the interface: carrot

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peels, cauliflower florets, eggplant pulp and potato tuber peels. Protein solutions were obtained

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as described before, but were diluted with 0.01 M SPB to obtain similar protein concentrations

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across treatments. Emulsions were prepared in triplicate starting from the same protein solution

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and contained a final concentration of 0 or 40 mM 4-MC.

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Protein extract characterization

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Spectrophotometric analysis

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Protein solutions were characterized by measuring the PPO activity and the concentration of total

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protein and protein-bound phenol (PBP). The adaptations described by Winters and Minchin27

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were used to perform a modified Lowry assay, in order to determine total protein and PBP

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concentration simultaneously. Protein content and PBP were expressed as mg of protein per ml

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extract and mg tyrosine-equivalents per mg protein, respectively, assuming 1 g of bovine serum

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albumin, used for the standard solution, contains 0.043 g tyrosine-equivalents. Further, PPO

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activity was determined by measuring the absorbance at 400 nm each 5 seconds for 1 min. For

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this, 0.75 ml 0.04 M 4-MC (containing 5 mM HCl to prevent autoxidation) was added to a

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solution of 2.2 ml 0.01 M SPB (pH=7) and 50 µl of protein extract. The increase in absorbance

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of the initial linear part of the curve was retained as a measure of activity. PPO activity was

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finally expressed in nkatal by recalculating the increase in absorbance per time unit to amounts 6 ACS Paragon Plus Environment

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of formed quinones using a standard series of 4-methylbenzoquinone, obtained by treating 3 ml

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of 4-MC with 3 ml 0.045 M sodium periodate and measuring the absorbance at 400 nm after 10

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min. Specific PPO activity was expressed as µkatal per mg protein. All spectrophotometric

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analyses were done in triplicate (analytical replicates).

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Gel electrophoretic analysis

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Next to spectrophotometric analyses of protein and PPO activity, gel electrophoresis was

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performed to visualize different protein patterns in the extracts using a vertical slab gel system

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(SE600, Hoefer Scientific Instruments, Holliston, Massachusetts, USA). Gels were prepared as

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described by Greaser et al.28 Briefly, separating gels contained 10 or 12 % of total acrylamide

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and N,N′-methylene-bis-acrylamide. In experiment 1, wells were loaded with the protein

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solution, to which SDS (2 %, w/v), approximately 15 % sucrose (w/v) and 0.3 mg/ml

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bromophenol blue were added. Twenty µl of this mixture was loaded per well, while volumes

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loaded in experiment 3 varied from 10 to 50 µl per well, depending on the protein concentration

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of the samples, in order to load equal amounts of protein. Separation was carried out at constant

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current. After the bromophenol front reached the bottom of the gel, proteins were fixed in a 20 %

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methanol / 10 % acetic acid solution and visualized by staining with Coomassie brilliant blue

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R250 according to Claeys et al.29, followed by scanning with a Bio-Rad computing densitometer

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(model CDS-100; Bio-Rad, Temse, Belgium). Further, similar gels were used to visualize PPO

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activity, based on the method described by Rescigno et al.30 For this, 10 % gels were prepared

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similarly as for the protein visualization. The presence of SDS is of particular interest as it is

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known to activate PPO isoforms from its latent state, in contrast to many enzymes which are

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inactivated by SDS.31 After proteins were separated, gels were gently washed with demineralized

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water and soaked in 0.1 M SPB (pH=7) for 3 min. Next, the gels were transferred to a solution

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containing 80 mM 4-MC (dissolved in 0.1 M SPB, pH=7). After 5 min, gels were washed in the

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original SPB solution and immediately transferred to a 16 mM 4-amino-N,N-diethylaniline

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sulfate solution, which reacts with 4-methylbenzoquinone, resulting in the visualization of PPO 7 ACS Paragon Plus Environment


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isoforms as purple blue bands. Zymograms were scanned at least 10 minutes after addition of 4-

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amino-N,N-diethylaniline sulfate.

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Emulsion characterization

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The particle size distribution of emulsions was assessed immediately after preparation with a

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Mastersizer S (Malvern Instruments, Malvern, UK) as described before9. Droplet sizes were

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characterized in terms of volume-weighted mean diameter (D43), surface-weighted mean

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diameter (D32), median volume-weighted distribution value D[v,0.5] or 90 % percentile of the

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volume-weighted distribution D[v,0.9]. Specific surface areas (in m²/g oil) were calculated as SSA

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= 6 / [D32 x ρ], assuming a linseed oil density ρ of 930 kg/m³.

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Further, the protein load of emulsions from experiment 3 was determined. To separate proteins

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present in the continuous phase from proteins at the interface after emulsification, 25 ml of

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emulsion was centrifuged for at least 30 min (30000 g; 4° C). The protein fraction of the

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continuous phase was obtained by removing 5 ml of the subnatant with a fine needle and was

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filtered through a 0.20 µm cellulose syringe filter (Chromafil, Macherey-Nagel, Düren,

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Germany). These unadsorbed proteins were further characterized using the modified Lowry and

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gel electrophoretic procedures (vide supra). As demonstrated before32, the aqueous phases

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obtained after filtration only contained negligible volumes of oil (data not shown). Accordingly,

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adsorbed protein in the continuous phase can be assumed negligible as well. Protein loads were

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expressed as mg protein per m² interfacial area or relatively to the initial protein concentration in

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the aqueous extract (g per 100 g protein). Next, to recover the interfacial proteins for gel

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electrophoresis, an extra 15 ml was removed from the centrifuged sample and the remaining

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fraction was washed twice by redispersing in fresh 0.01 M SPB (pH=7), thorough vortexing and

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centrifuging for at least 30 min (30000 g; 4° C). Then, 1 % SDS solution was added to the

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remaining fraction, vortexed and gently shaken for 1 h to allow interfacial proteins to be replaced

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by SDS, before centrifuging for at least 30 min (30000 g; 4° C). Finally, 5 ml of the subnatant 8 ACS Paragon Plus Environment

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was removed and passed through a 0.20 µm cellulose syringe filter to obtain the adsorbed protein

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fraction for gel electrophoretic analysis.

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Assessment of protection against ruminal biohydrogenation

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Batch in vitro incubations were performed to assess protection of PUFA in emulsions against

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ruminal BH as described before9. In brief, incubation flasks contained 1 ml emulsion, 250 mg

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hay and 24 ml buffer/rumen fluid solution in a 4:1 ratio, which has been shown before10 to be

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appropriate to estimate BH of C18:2n-6 or C18:3n-3. Rumen contents were collected before the

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morning feeding from three rumen fistulated sheep, which were fed grass hay ad libitum and a

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grain based concentrate (200 g/day), combined and filtered before incubation. Fistulation was

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approved by the ethical commission (file number 114, 2009) of the Institute for Agricultural and

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Fisheries Research (ILVO, Belgium). Flasks were flushed with CO2 and incubated under

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intermittent shaking at 39 °C for 24 h in a batch culture incubator (Edmund Bühler GmbH,

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Hechingen, Germany). All emulsions were incubated in duplicate (analytical replicates). To

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monitor the quality of the incubations, gas composition33, pH (Hanna Instruments, Temse,

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Belgium) and volatile FA34 was assessed. Gas composition was converted to absolute gas

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production rates based on the pressure accumulation in the flask (Infield 7C handheld read-out

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device equipped with a T1 Stitch-Tensiometer; UMS GmbH, München, Germany). Incubation

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characteristics are not reported as no major differences were observed between treatments,

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indicating changes in the extent of BH were not due to changes in microbial activity.

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To calculate BH, 5 ml of incubation fluid was taken before and after incubation, freeze-dried and

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analyzed for long chain FA after direct transesterification using gas chromatograph, according to

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Gadeyne et al.9 Peaks were identified based on their retention times, compared to external

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standards (GLC463, Nu-Check Prep Inc., Elysian, Minnesota, USA). Quantification of FA

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methyl esters was based on the area of the internal standard (C21:0 for experiment 1 and C13:0

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for experiments 2 and 3) and on the conversion of peak areas to the weight of FA by a theoretical 9 ACS Paragon Plus Environment


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response factor for each FA35,36. Finally, BH (%), which is the disappearance of C18:3n-3 after

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incubation, was calculated as [(proportion of C18:3n-3 in total C18 FA)0h – (proportion of

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C18:3n-3 in total C18 FA)24h] × 100 / (proportion of C18:3n-3 in total C18 FA)0h. PE of

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C18:3n-3 (%) was calculated as [(BH of C18:3n-3)non-protected (0 mM) – (BH of C18:3n-3)protected (10,

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20 or 40 mM)]

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C18 FA BH, was calculated as [(proportion of C18:0 in total C18 FA)24h – (proportion of C18:0

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in total C18 FA)0h] × 100 / [(proportion of C18:3n-3 and C18:2n-6 in total C18 FA)0h –

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(proportion of C18:3n-3 and C18:2n-6 in total C18 FA)24h].

× 100 / (BH of C18:3n-3)non-protected (0 mM). The formation of C18:0, the end product of

224 225

Statistics

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Results were analyzed with the MIXED procedure of SAS (SAS Enterprise Guide 7, SAS

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Institute Inc., Cary, North Carolina, USA). Analytical replicates were averaged prior to statistical

228

analysis. Linear regression analyses and two-sample T-tests were performed using the REG and

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TTEST procedure of SAS, respectively.

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For experiment 1, the following model was used: Yij = µ + Pi + Dj + Pi × Dj + εij, with Yij the

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variable of interest, µ the overall mean, Pi the fixed effect of protein source (i = apple peel,

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artichoke flower leaf, broccoli stem, etc.), Dj the fixed effect of diphenol concentration (j = 0, 10,

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20 or 40 mM 4-MC) and εij the residual error. Hierarchical cluster analysis was performed on

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C18:3n-3 BH using 10, 20 and 40 mM 4-MC to distinguish between protein source using the

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CLUSTER and TREE procedures of SAS with average linkage.

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In experiment 2 and 3, the same model as for experiment 1 was used, with Pi the fixed effect of

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protein source (i = solid or liquid sidestreams or i = carrot peel, cauliflower floret, eggplant pulp

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or potato tuber peel for experiment 2 and 3, respectively).

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Principal component analysis was performed to determine components which account for most

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of the variation in PE. The PRINCOMP procedure of SAS was applied using data from the 40


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mM 4-MC emulsions of experiment 1 and 3, whereby PE of C18:3n-3, protein concentration,

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PPO activity and D32 were included as variables.

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Differences were assigned at the 0.05 significance level and differences among least square

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means evaluated using Tukey’s multiple comparison test.

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Results

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Experiment 1: screening of different plant sources

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In the first experiment, a screening test was performed to assess whether PPO containing protein

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extracts from different sources could be used to protect PUFA against ruminal BH. Therefore,

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thirteen possible vegetal sources were selected, proteins extracted and used as emulsifiers.

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Protein concentrations of the extracts largely differed (Table 1), ranging from 0.204 to 2.73 mg

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of protein per ml extract for apple peel and potato tuber peel, respectively. Negligible

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concentrations of PBP were observed in the extracts (Table 1), except for artichoke flower leaf

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(10.5 mg tyrosine equivalents per mg protein), which was also reflected in a dark discoloration

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during the extraction procedure prior to acetone precipitation. The protein extracts from the latter

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also contained the highest specific PPO activity (5.23 µkatal per mg protein). Substantial

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amounts of quinones were formed in most extracts after addition of 4-MC, ranging from 1.53

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nkatal for pineapple peel to 116 nkatal for artichoke flower leaf (Table 1). Very low activities

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were found for broccoli, carrot and cauliflower.

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Gel electrophoretic analysis was performed to separate proteins according to their molecular

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weights. Large variation in molecular weight and intensity of protein bands within and between

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vegetal extracts was observed (Figure 1A). Also, reactivity towards 4-MC of these proteins,

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attributed to the activity of PPO (Figure 1B), varied to a large extent among the protein sources.

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Most sources showed one or more bands indicating PPO activity, except for carrot peel.

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Remarkably, protein sources were quite diverse in PPO isoforms as indicated by the lack of 11 ACS Paragon Plus Environment


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uniformity in PPO bands, showing proteins with varying molecular weight being responsible for

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PPO activity. No relation between the spectrophotometric PPO activity (Table 1) and intensity of

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bands after electrophoretic analysis was observed: for example, no (detectable) or very low PPO

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activity was measured spectrophotometrically for protein extracts of cauliflower, broccoli,

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spinach and pineapple, whereas intense bands were detected on gels. Furthermore, intensities of

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bands with high PPO activity (Figure 1B) were not related to intensities of protein as such

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(Figure 1A), meaning for some sources PPO represented only a minor proportion of the proteins.

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Emulsion droplet sizes and specific surface areas (Table 1) illustrate the protein concentrations

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of the extracts were sufficient to obtain stable emulsions with a D32 ranging from 0.67 to 3.91

276

µm for cauliflower floret and carrot peel, respectively, associated with the highest (9.69 m²/g oil)

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and lowest (1.67 m²/g oil) specific surface area. Artichoke, broccoli, cauliflower and spinach

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protein extracts resulted in emulsions with the smallest droplet sizes, with 90 % of the droplet

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population showing a diameter below 10 µm. Regression analysis revealed no linear link

280

between D32 of the emulsions and the protein concentration of the different extracts (p=0.927).

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In order to create protection, 4-MC was added to the emulsions to obtain final concentrations of

282

0, 10, 20 or 40 mM. Results for BH of C18:3n-3 are depicted in Figure 2A, while the associated

283

PE for 40 mM 4-MC emulsions are presented in Table 1. Numeric averages and standard

284

deviations for BH of C18:3n-3 and C18:2n-6 as well as the PE of C18:3n-3 and the formation of

285

C18:0 for all treatments are reported in Supplement 1. On average across all protein sources,

286

96.3 % of C18:3n-3 in emulsions without 4-MC was hydrogenated after 24 h in vitro incubation.

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Upon addition of 4-MC, a decrease in BH of C18:3n-3 was observed with the extent of the

288

decrease depending on the source of protein used as emulsifier. C18:3n-3 BH of emulsions

289

containing 10 mM 4-MC, prepared using potato tuber peel, was significantly lower compared

290

with other protein sources (p

Polyphenol Oxidase Containing Sidestreams as Emulsifiers of Rumen

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