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Impact of particle size reduction and carbohydrate hydrolyzing enzyme treatment on protein recovery from rapeseed (Brassica rapa L.) press cake Katariina Rommi,* Ulla Holopainen, Sari Pohjola, Terhi K. Hakala, Raija Lantto, Kaisa Poutanen, Emilia Nordlund VTT Technical Research Centre of Finland Ltd. P.O. Box 1000, FI-02044 VTT, Finland *Corresponding author; phone: +358 20 722 4514; fax: +358 20 722 7001; email: [email protected]

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Abstract

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The aim was to assess how particle size reduction and carbohydrate hydrolyzing

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enzyme treatment influence protein recovery from rapeseed cold-press cake, and to

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determine the effect of these pretreatments in protein extraction procedures varying in

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ionic strength, pH and total solid content. Defatted press cake (median particle size 600

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µm) was milled to 21-164 µm and 7 µm median particle sizes by pin disc milling and

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air-flow milling, respectively. The milled press cake samples were treated with a

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carbohydrate hydrolyzing enzyme preparation, after which proteins were extracted in

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saline (pH 6) or alkaline (pH 12) buffer at 5% solid content, or in water at 20% solid

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content. Particle size reduction of the press cake did not influence enzyme action or

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protein yield, suggesting that protein release from the press cake is not physically

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limited by cell walls or internal cell structures. As an exception, protein release from the

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aleuronic cells appeared to be hindered by intact cell walls. Enzyme treatment improved

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protein recovery, more substantially when the extraction was carried out in water at

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20% solid content than in saline or alkaline conditions at 5% solid content. The enzyme

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mediated its positive effect most probably by reducing the water holding capacity of the

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press cake, thereby facilitating solid-liquid separation, and releasing anionic compounds

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which improved protein solubility through electrostatic stabilization. The results suggest

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that carbohydrate hydrolyzing enzymes are beneficial for rapeseed protein extraction at

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reduced water content or when no salt or alkali is added to increase protein solubility.

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Keywords: milling, pectinase, microscopy, differential scanning calorimetry

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Introduction

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Rapeseed cold-press cake, the coproduct of food oil production, has gained growing

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interest in the past decade as a potential plant-based protein source. In addition to the

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current use as animal feed, production of food protein ingredients from the press cake

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could generate added value for the manufacturing industry. Rapeseed protein has

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favorable features for human use, including well-balanced amino acid composition and

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suitable technological properties (Bell and Keith 1991; Salleh et al. 2002). The direct

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use of rapeseed press cake as food is, however, hindered by its high carbohydrate

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content and presence of secondary plant metabolites such as phytates and phenolics

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having negative effects on the digestibility and sensory properties. For partial removal

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of the interfering compounds, proteins are commonly extracted from the press cake

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using alkali or salt solutions (Bérot et al. 2005; Ghodsvali et al. 2005) and precipitated

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by decreasing the pH or ionic strength of the extracts. Furthermore, the use of proteases

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and/or cell wall degrading enzymes has been recognized to facilitate the extraction

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processes (Niu et al. 2012; Rommi et al. 2014; Sari et al. 2013; Tang 2010).

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Consideration of the technological and economic feasibility of the production processes

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is a prerequisite for successful commercialization of rapeseed protein production.

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Although 70-90% protein yields have been obtained by water-intensive alkaline

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extraction procedures (Ghodsvali et al. 2005; Zhou et al. 1990), water-lean processes

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are suggested to be favorable for sustainable protein production (Von Der Haar et al.

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2014). New possibilities for more feasible protein extraction processes could be opened

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by increased understanding of the influence of cell wall structures and protein-

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carbohydrate interactions on protein release from the press cake. Most of rapeseed

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protein is stored in the embryo (i.e. cotyledons and embryonic axis) cells which are

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surrounded by cell walls comprising of a cellulose, hemicellulose and pectin network

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(Pustjens et al. 2013). Since cold pressing only partially disrupts the embryonic cell

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walls, integrity of the tissue and cell wall structures in the press cake is expected to

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influence protein release (Rommi et al. 2014).

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Most of the published rapeseed protein extraction studies have focused on improving

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protein solubility as a function of pH and ionic strength, whereas the physical barriers of

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protein release have received little attention. Particle size reduction by milling has been

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reported to clearly enhance protein extraction from soybean flour (Rosenthal et al. 1998,

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2001; Russin et al. 2007), but to our knowledge, respective studies on rapeseed press

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cake have not been published. Milling is also known to benefit enzyme-aided

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fractionation processes by increasing the accessible surface area of the substrate to the

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enzyme and thus improving the hydrolysis efficiency, as has been demonstrated for

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several lignocellulosic feedstocks such as brewer’s spent grain (Niemi et al. 2012), corn

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grain (Blasel et al. 2006) and wheat straw (Silva et al. 2012). Furthermore, particle size

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reduction has been reported to decrease the water holding capacity and viscosity of

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biomass slurries, with positive impacts on processing especially at reduced water

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content (Santala et al. 2013; Viamajala et al. 2009).

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The aim of the present study was to obtain insight on the factors affecting protein

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recovery from rapeseed press cake, with focus on determining the effect of particle size

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reduction and carbohydrate hydrolyzing enzyme treatment on aqueous protein

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extraction. These factors where evaluated in water-intensive saline or alkaline

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conditions where the protein solubility and extract recovery was expected to be high, as

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well as during water extraction at 20% solid content where the protein solubility and

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extract separation efficiency was expected to be limited. 4

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Materials and Methods

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Materials and enzymes

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Rapeseed press cake was obtained from Kankaisten Öljykasvit Oy (Turenki, Finland).

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Oil was cold-pressed from turnip rape (Brassica rapa L.) seeds at 50–60°C and the

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press cake was pelletized. A commercial carbohydrate hydrolase preparation, Pectinex

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Ultra SP-L, was obtained from Novozymes A/S (Bagsvaerd, Denmark). The preparation

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contained 2876 nkat of polygalacturonase, 135 nkat of endo- -glucanase, 22 nkat of

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endoglucanase and 7 nkat endo-1,4- -xylanase activity per total protein (61 mg/mL) in

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the enzyme preparation (Rommi et al. 2014).

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Defatting and particle size reduction

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The pelletized rapeseed press cake was ground at 2000 rpm using an SM 300 cutting

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mill (Retsch GmbH, Düsseldorf, Germany) equipped with a 8 mm square hole bottom

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sieve, and defatted by supercritical carbon dioxide (SC-CO2) extraction at 42°C

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according to (Rommi et al. 2015). The defatted press cake (with 35.5% protein content

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on dry matter basis) was further milled in a pin disc mill (100 UPZ-II Fine Impact Mill,

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Hosokawa Alpine Ag, Ausburg, Germany) or in an air-flow mill (MKCL8-15J DAU,

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Masuko Sangyo, Kawaguchi, Japan) which was set to 3 min residence time. Particle

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size distribution in the press cake samples after grinding and milling was determined by

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laser light (750 nm) diffraction using an LS320 particle size analyzer (Beckman Coulter

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Inc., Indianapolis, IN, USA) and Beckman Coulter LS software, version 3.2. The

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ground sample was sieved in a shaker through 2 mm holes to exclude particles over 2

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mm (representing 2% of the total sample) which exceeded the maximum detection limit

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of the analyzer. All samples were dispersed and run in ethanol, and before analysis

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treated with ultrasound for 5 min to prevent aggregate formation. Duplicate 5

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measurements were performed for each sample. The median particle size of the ground,

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SC-CO2-defatted press cake was 600 µm, whereas further pin disc milling at 8900 rpm

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produced coarse flour with a median (D50) particle size of 164 µm. Fine flour with a

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median particle size of 21 µm was obtained by passing the press cake twice through the

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pin disc mill at 17800 rpm. Ultrafine flour with a median particle size of 7 µm was

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produced by dry milling at 7600 rpm in an air-flow mill (Table 1).

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Enzymatic hydrolysis

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The ground (600 µm) and milled (7-164 µm) press cake samples (Table 1) were treated

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with Pectinex Ultra SP-L using the procedure described by Rommi et al. 2015 with

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justified modifications. Pectinex was dosed at 10 mg total protein / g dry press cake and

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the treatment was continued for 4 h. The enzyme dose and treatment time were adjusted

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based on the study of Rommi et al. (2015) in which hydrolysis was found to be feasible

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at 40% solid content, but the selected enzyme dose (5 mg/g) and hydrolysis time (1 h)

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were suggested to be insufficient. The samples were mixed with MQ water and enzyme

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to 40% (w/w) total solid content and placed in a closed 800 mL stirred tank reactor

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(Protoshop, Espoo, Finland) and incubated at 40°C with 40 rpm mixing. After

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hydrolysis the slurries were immediately frozen, freeze-dried and gently ground in a

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mortar. All enzyme treatments were performed in duplicate.

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Protein extraction

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Proteins were recovered from the enzyme-treated and non-enzymatically treated press

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cake samples by a two-step saline extraction into 0.2 M sodium phosphate buffer, pH 6.

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The samples were mixed with buffer to 5% (w/w) total solid content and agitated

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horizontally in a shaking incubator at 230 rpm, 25 °C for 30 min. Extracts were

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separated by centrifugation. The extraction was repeated for the residual solids and the 6

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first and second extracts were combined and weighed. From the fine (21 µm) and

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ultrafine samples (7 µm) after enzyme treatment or without enzyme treatment, protein

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was additionally recovered by a two-step alkaline extraction into 0.2 M sodium

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phosphate buffer, pH 12. In addition, the ground (600 µm) and fine (21 µm) press cake

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samples were subjected to a two-step water extraction at 20% total solid content. The

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extraction pH was defined by the natural pH of the substrate after enzyme treatment (pH

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5.5) or without enzyme treatment (pH 6). The extraction was performed twice at 25°C

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in MQ water for 30 min, and extracts were obtained by centrifugation. All extractions

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were performed in duplicate for each of the two parallel enzyme-treated samples (N =

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4) and in triplicate or quadruple for each non-enzymatically treated sample (N = 3). The

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yield of solubilized protein was determined by multiplying the protein concentration of

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extracts by the total liquid volume during extraction and dividing by the amount of total

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protein. Respectively, the yield of recovered protein was determined by multiplying the

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protein concentration of extracts by the recovered extract volume and dividing by the

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amount of total protein. Protein yields from the water extraction at 20% solid content

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were differentiated for the first extraction step (1-step extraction) and for the first and

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second extraction steps (2-step extraction).

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Analyses

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Total nitrogen content of the defatted press cake and protein extracts was analyzed by

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Kjeldahl according to the method by (Kane 1986). Protein concentration was calculated

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from the total nitrogen content using a conversion factor of 6.25. The press cake

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samples were prepared for microscopy according to (Holopainen-Mantila et al. 2013),

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and the sections were stained with Acid Fuchsin and Calcofluor White as described by

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(Dornez et al. 2011). The samples were examined and micrographs were obtained

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according to (Holopainen-Mantila et al. 2013). Images taken from replicate sample

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blocks were examined and representative images were selected for publication.

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Denaturation enthalpy of proteins in the press cake samples was measured by

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differential scanning calorimetry (DSC) (Mettler Toledo DSC820, Greifensee,

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Switzerland).

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hydrated with water to 75% moisture content, sealed and equilibrated for 1.5 h before

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heating from 10°C to 150°C at a temperature increase rate of 10°C/min. The

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measurements were performed in duplicate.

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Statistical analysis

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Statistical analysis of the protein solubilization and recovery yields from 3-4 replicate

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experiments was carried out by general linear model multivariate analysis using SPSS

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Statistics software (version 22, IBM, Armonk, NY). Level of significance was set at p