Functional properties and chemical composition of fractionated brown and yellow linseed meal (Linum usitatissimum L.)

Journal of Food Engineering 98 (2010) 453–460

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Functional properties and chemical composition of fractionated brown and yellow linseed meal (Linum usitatissimum L.) Klaus Mueller a,*, Peter Eisner a, Yumiko Yoshie-Stark b, Reiko Nakada b, Eva Kirchhoff a a b

Department of Process Engineering, Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Tokyo, Japan

a r t i c l e

i n f o

Article history: Received 5 November 2009 Received in revised form 13 January 2010 Accepted 23 January 2010 Available online 28 January 2010 Keywords: Linum usitatissimum L. Linola Fractionation Functional properties Carbohydrate composition Lignans Secoisolariciresinol diglucoside Ferulic acid glucoside Coumaric acid glucoside

a b s t r a c t Considering its high content of protein and dietary fiber, linseed meal is a remarkable source for food ingredient and food additive production. In this study, brown and yellow linseed meal (Linum usitatissimum L.) were fractionated via pH control, to obtain five linseed meal fractions rich in protein and fiber. The fractions were characterized by measuring functional properties, proximate and carbohydrate composition, and lignan contents. Acid soluble protein fractions were characterized by lower emulsification capacities and foaming activities in comparison to a commercial soy protein isolate. Alkaline soluble protein fractions showed emulsification activities comparable to whole egg and relatively high contents of secoisolariciresinol diglucoside (SDG) of 110 mg/g DM and 56.2 mg/g DM, respectively. The good emulsification and foaming activities, as well as the enriched concentration of SDG and therefore high nutritional value, make especially the alkaline soluble protein fraction highly interesting for food ingredient production. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Linseed is one of the most important cultivated plants concerning its linen and oil (FAO, 2008). A protein-rich linseed cake results as a by-product of linseed oil production, which is currently mainly used as animal feed or fertilizer. Linseed meal is reported to have a high nutritional potential, not only based on its high protein content (Oomah and Mazza, 1993a), but also because of its water-soluble fiber fraction (Warrand et al., 2005) and lignan content (Hyvärinen et al., 2006a). Lignans especially secoisolariciresinol diglucoside (SDG), are claimed to be effective in reducing the risk of cardiovascular diseases and might be efficient in inhibiting the development of diabetes (Westcott and Muir, 2003). The health benefits of these compounds are suggested to be due to their antioxidant activity and estrogenic and antiestrogenic properties (Hall et al., 2006). Ideally, the protein and/or fiber containing fraction that results from the fractionation of linseed meal, could be applied as a food additive or a food ingredient in processed foods. Proteins are expected to contribute to emulsification and foaming activities in

food processing, and soluble fibers could additionally stabilize emulsions. Dev and Quensel (1986) reported that functional properties of alkaline-extracted linseed protein fractions showed functional properties comparable to those of soy protein isolates. Oomah and Mazza (1998) and Chung et al. (2005) found that dehulling treatment can provide an effective fractionation for gum and proteins. However, de-hulling procedure costs are relatively high and therefore the linseed meal resulting from the usual industrial oil production includes the hulls. The objective of this study was to fractionate industrial brown linseed meal and evaluate the resulting fractions by measuring functional properties, chemical composition and lignan contents for applicability as food ingredients or food additives. Because a dark color of the final food products is not desirable using the resulting fractions as food ingredients, also a yellow (light color) linseed variety was investigated in this study.

2. Materials and methods 2.1. Materials

* Corresponding author. Address: Department of Process Engineering, Fraunhofer Institute for Process Engineering and Packaging, Giggenhauser Str. 35, 85354, Freising, Germany. Tel.: +49 (0)8161 491405; fax: +49 (0)8161 491444. E-mail address: [email protected] (K. Mueller). 0260-8774/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.01.028

Brown and yellow (Linola) linseed (Linum usitatissimum L.) were obtained from C. Thywissen GmbH, Germany. The brown linseed has a predominant market share in comparison to Linola seed.

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Whereas brown linseed is normally processed by a combination of cold and hot pressing, yellow linseed is usually treated by hot pressing to recover the oil. In this study, the brown linseed was cold pressed (C. Thywissen GmbH, Germany) in order to obtain protein fractions with as unmodified protein structures as possible. Yellow linseed meal was only available obtained pretreated by hot pressing to remove oil (C. Thywissen GmbH, Germany). After pressing, linseed meal was de-oiled using iso-hexane and followed by milling using a Retsch ZM-100 mill (Duesseldorf, Germany) obtaining a powder (d50 < 400 lm). This de-oiled linseed meal powders were used for the fractionation of protein and fibers. All reagents used for the experiments were of analytical grade. 2.2. Proximate analysis The chemical composition (dry matter, nitrogen content and ash content) of the linseeds, linseed meal, de-oiled linseed meal, and the processed fractions, rich in protein or rich in fiber, were analyzed in accordance with the German Food Act (1980). Fat content was analyzed according to Pendl et al. (1998). 2.3. Protein solubility of linseed meal and de-oiled linseed meal The protein solubility of linseed meal and de-oiled linseed meal was analyzed following the method of Morr et al. (1985) at pH 3–8. The nitrogen solubility index (NSI) value was determined in accordance with the official AOCS (1998) or AACC (2000a) method. The protein solubility curve was obtained by mixing protein samples (1 g) in 50 mL 100 mM sodium chloride solution at a set pH and at room temperature for 60 min. The non-dissolved fraction was separated by centrifugation at 20,000g for 15 min. The protein remaining in solution was determined by nitrogen analysis. The protein solubility is given as the percentage of dissolved nitrogen compared to the nitrogen content of the starting sample. 2.4. Fractionation of de-oiled linseed meal Following the result of pH study of protein solubility from linseed meal, the fractionation method was started with acidic extraction. The detailed procedure is shown in Fig. 2. The ratio of solid/solvent, temperature and pH were optimized by a preliminary experiment (Mueller and Eisner, 2007). De-oiled linseed meal was extracted under acidic conditions, then, acid soluble protein fraction and acid soluble carbohydrate fraction were obtained. From acid insoluble residue, alkaline soluble protein fraction, alkaline soluble carbohydrate (soluble dietary fiber) fraction, and insoluble dietary fiber fraction were prepared. Adjustment of pH was carried out using 1 M NaOH solution and 1 M HCl solution, respectively. 2.5. Functional properties The functional properties of the linseed meal fractions were determined using standardized methods. Protein solubility was determined at pH 7 according to the method of Morr et al. (1985), whereas the NSI value was determined in accordance with the official AOCS method (1998) or AACC official method (2000a). Water binding capacity analysis was conducted according to the AACC official method (2000b). Oil-binding capacity was determined by dispersing the sample in oil and subsequent centrifugation following a method described by Ludwig et al. (1989). In order to determine the (oil/water) emulsification capacity, a 1 L laboratory reactor (IKA) with a stirrer unit and an emulsifying system (Ultra-Turrax, IKA-Werke GmbH & Co. KG, Staufen, Germany) was used. The protein solution (1 g/100 g, w/w) was agi-

tated in the reactor, which was held at a constant temperature by a cooling or heating system. The oil was automatically added by a titration system (Metrohm GmbH & Co. KG, Herisau, Switzerland). The conductivity was continuously measured and used for determining the inversion point of the emulsion. The amount of oil which was added up to the inversion point of the emulsion was used to calculate the emulsification capacity (mL oil/g protein). Foams were generated using a whipping machine (Hobart N 50, Hobart GmbH, Offenburg, Germany). The foaming activities of 5 g/ 100 g sample solutions were obtained by comparing the foam volume after 8 min of whipping with the volume of starting sample solution. The foaming activity was calculated according to:

%FA ¼ foam vol after whipping=vol of sample solution  100 2.6. Viscosity measurements Rheological determinations were performed at steady share stress with a Bohlin rheometer CVO 50 (Bohlin Instruments, Pforzheim, Germany). Linseed fractions were suspended at 20 g/L in deionized water with stirring until suspended well. Under constant pH value (pH 7.0) and shear rate (300 s1) the influence of temperature on the viscosity was evaluated from 10 to 90 °C. 2.7. Further chemical analysis of linseed fractions The molecular weight of linseed meal fractions was analyzed by gel permeation chromatography. The HPLC was performed on Shodex Asahipak GS-510HQ (300 mm  7.6 mm i.d., Showa Denko Co., Tokyo, Japan) by elution with Milli-Q water at flow rate of 1.0 mL/ min. Column temperature was set at 40 °C, and RI detector was used. Pullulan (P-82, Showa Denko Co., Tokyo, Japan) standard was used to evaluate the molecular weight. Total sugar content of linseed meal fractions (except insoluble dietary fiber fraction) was determined by the phenol–sulfuric acid method using glucose as a standard (Dubois et al., 1956; Southgate, 1991). Total uronic acid content was determined by a colorimetric method of Blumenkrantz and Asboe-Hansen (1973) using galacturonic acid as a standard. Acid soluble carbohydrate and soluble dietary fiber fractions were hydrolyzed with 2 M trifluoroacetic acid for 1 h at 120 °C. Monosaccharide composition of them was analyzed by HPLC fitted with Intertsil NH2 column (250  4.6 mm i.d., 5 lm, GL Science Co., Tokyo, Japan) operating at 40 °C with a flow rate of 0.7 mL/ min. Elution was effected using an isocratic elution of acetonitrile/water (75/25, v/v) as a solvent. Components were detected by RI detector and identified by comparison of their retention times with those of authentic standards under analysis conditions and quantified by external standard method. Phenolic acid glucosides (secoisolariciresinol diglucoside and hydroxycinnamic acid glucosides) in linseed fractions were extracted following the method of Eliasson et al. (2003) and analyzed by HPLC. Linseed fractions (200 mg), vortexed together with 1.0 mL internal standard (o-coumaric acid, 0.8 mg/mL methanol), was continuously mixed with 4 mL distilled water and 5 mL 2 M aqueous sodium hydroxide for 1 h at 20 °C by constant rotation. The hydrolysate was acidified to pH 3 using 2 M sulfuric acid and centrifuged (1700g, 10 min). The supernatant was recentrifuged in microcentrifuge tubes (11,000g, 5 min) to a clear liquid phase. The liquid phase (0.6 mL) was mixed with 95% aqueous ethanol (0.9 mL) in microcentrifuge tubes, left at room temperature for at least 10 min and centrifuged (11,000g, 5 min) to precipitate and remove water-soluble polysaccharides and proteins. Before HPLC analysis, the sample was filtered through 0.45 lm

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nents were detected by diode array detector and identified by comparison of their retention times and spectra with those of authentic standards under analysis conditions. Recovery was calculated by internal standard (o-coumaric acid) and quantified by external standard method.

cellulose acetate filter. The sample solution was further subjected to PLC fitted with TSK-GEL column ODS-100V (5 lm, 150  4.6 mm, Tosoh, Japan) operating at 40 °C with a flow rate of 1.0 mL/min. Elution was performed using an isocratic elution of acetonitrile/0.1% phosphate (75/25, v/v) as a solvent. Compo-

100 brown linseed press cake (12.7 % fat) brown linseed meal (1.7 % fat) yellow linseed meal (1.5 % fat)

90

Protein solubility [%]

80 70 60 50 40 30 20 10 0 2

3

4

5

6

7

8

9

10

pH value Fig. 1. Protein solubility as a function of pH for linseed press cake.

Linseed

Supernatant Heat treatment at 90°C 10 min

Cold oil pressing

Centrifugation Linseed press cake Residue, neutralization

Supernatant, neutralization

Hexane de-oiling Spray drying

Spray drying

Acid soluble protein

Acid soluble carbohydrates

De-oiled linseed meal Acid extraction at pH 4.0, 15o C 45 min

Supernatant

Centrifugation Residue, neutralization Residue

Precipitation at pH 4.0, 35o C 45 min

Drying by convection oven

Alkaline extraction at pH 8.0, 35oC, 45min

Insoluble dietary fiber

Centrifugation Residue, neutralization

Supernatant, neutralization

Centrifugation Spray drying

Spray drying

Alkaline soluble protein

Soluble dietary fiber

Fig. 2. General procedure for the fractionation of linseed meal from oil production.

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2.8. Statistical analysis The results are presented as mean values ± SD (n = 3–5). ANOVA was used to determine significant differences. 3. Results and discussion

The acid soluble carbohydrate and soluble dietary fiber fractions, respectively, contained 14.9 g/100 g and 15.6 g/100 g protein, 75.27 g/100 g and 72.3 g/100 g carbohydrate and 0.50 g/100 g fat each. The insoluble dietary fiber fractions of brown and yellow linseed contained, respectively, 46.1 g/100 g and 39.0 g/100 g protein, 47.3 g/100 g and 55.6 g/100 g carbohydrate and 2.0 g/100 g fat each.

3.1. Protein solubility of linseed meal 3.3. Yield of each fraction The effect of pH on protein solubility of linseed meal is shown in Fig. 1. Egg white ovalbumin is known to have a minimum solubility at pH 4.5 (iso-electric point), similar to egg, percent soluble protein of both, brown and yellow linseed meal was the lowest between pH 3 and 5. Oomah and Mazza (1997) determined the protein solubility of de-hulled linseed with 87–92% at pH 7. Comparison to de-hulled linseed, brown linseed meal including hull showed in this study less than 60% of protein solubility at pH 7. After optimization of the fractionation process by a preliminary experiment, first step of the fractionation was performed at pH 4, and, also considering the report of Dev and Quensel (1986), following alkaline extraction was applied at pH 8.0 (cf. Fig. 2).

The dry matter yield and protein yield of each fraction are shown in Table 2. From fractionation process, acid and alkaline soluble protein fractions of brown and yellow linseed had in summary a protein yield of 31.0% and 18.5%, respectively. Concerning the yields of the soluble dietary fiber fractions, 21.0% of dry matter was recovered from brown linseed meal whilst only 6.80% of dry matter was recovered from yellow linseed. The protein and carbohydrate recovery of brown linseed were significant higher than that of yellow linseed, indicating the brown variety being more effectively and easily fractionated than yellow variety. 3.4. Functional properties

3.2. Chemical composition The chemical compositions of raw linseed, linseed meals, deoiled linseed meals, acid soluble protein fractions, alkaline soluble protein fractions, acid soluble carbohydrate fractions, and soluble dietary fiber fractions are summarized in Table 1. Brown linseed contains 23.4 g/100 g protein and 45.2 g/100 g fat, while yellow linseed contains 23.3 g/100 g protein and 44.0 g/100 g fat. After pressing of seed, brown and yellow linseed press cake showed a fat content of 12.4 g/100 g and 7.55 g/100 g, respectively. After de-oiling by hexane, the content of fat of brown and yellow linseed meal was 1.67 g/100 g, and 1.13 g/100 g, respectively. After fractionation process of brown linseed meal, the acid soluble protein and alkaline soluble protein fractions, respectively, contained 66.5 g/100 g and 71.1 g/100 g protein, 23.0 g/100 g and 21.5 g/100 g carbohydrate, and 1.80 g/100 g and 2.10 g/100 g fat. Acid soluble carbohydrate and soluble dietary fiber fractions, respectively, contained 33.3 g/100 g and 32.3 g/100 g protein, 55.6 g/100 g and 57.3 g/100 g carbohydrate and 0.30 g/100 g and 0.60 g/100 g fat. After fractionation process of yellow linseed meal, the acid soluble protein and alkaline soluble protein fractions, respectively, contained 23.1 g/100 g and 63.4 g/100 g protein, 71.5 g/100 g and 29.2 g/100 g carbohydrate and 1.90 g/100 g and 2.00 g/100 g fat.

Protein solubility at pH 7, water binding, oil binding, emulsification capacity, and foaming activity of linseed fractions are shown in Table 3. The protein solubility of the alkaline soluble protein fraction from brown linseed was to a degree of 48.0% higher that of the corresponding fraction of yellow linseed and comparable to that of soy

Table 2 Mass and protein yield of the fractionation process. Dry matter yield (%) Protein yield (%) Brown linseed meal (hexane de-oiled) Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

100 14.0 10.0 21.0 11.0 44.0

100 16.0 15.0 14.0 10.0 45.0

Yellow linseed meal (hexane deoiled) Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

100

100

8.9 4.6 6.8 12.3 68.2

15.4 3.1 3.7 5.3 72.5

Table 1 Chemical composition of linseed, linseed meal, de-oiled linseed meal, and processed fractions of linseed meal (mean ± SD, n = 3). Dry matter (g/100 g)

Protein (g/100 g DM)

Ash (g/100 g DM)

Fat (g/100 g DM)

Carbohydrate (g/100 g DM)

Brown linseed (raw) Brown linseed press cake Brown linseed meal (hexane de-oiled) Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

92.6 ± 0.41 87.4 ± 0.28 90.3 ± 2.21 94.2 ± 0.05 96.4 ± 0.96 93.1 ± 0.06 93.8 ± 0.03 96.0 ± 0.23

23.4 ± 0.06 40.9 ± 2.54 43.3 ± 1.13 71.1 ± 0.15 66.5 ± 0.03 32.3 ± 0.01 33.3 ± 0.11 46.1 ± 0.21

3.50 ± 0.10 6.30 ± 0.85 6.40 ± 0.03 5.3 ± 0.16 8.7 ± 0.26 9.7 ± 0.29 10.8 ± 0.32 4.5 ± 0.14

45.2 ± 1.12 12.4 ± 1.97 1.67 ± 0.04 2.1 ± 0.19 1.8 ± 0.19 0.6 ± 0.08 0.3 ± 0.01 2.0 ± 0.04

27.8 ± 1.08 45.3 ± 1.48 48.7 ± 1.14 21.5 ± 1.98 23.0 ± 1.80 57.3 ± 1.95 55.6 ± 2.08 47.3 ± 2.21

Yellow linseed (raw) Yellow linseed press cake Yellow linseed meal (hexane de-oiled) Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

92.7 ± 1.36 91.4 ± 0.64 89.2 ± 0.73 93.9 ± 0.05 88.7 ± 0.03 92.5 ± 0.19 89.5 ± 0.02 94.2 ± 0.04

23.3 ± 0.88 36.9 ± 2.58 39.8 ± 1.41 63.4 ± 0.12 23.1 ± 0.02 15.6 ± 0.04 14.9 ± 0.01 39.0 ± 0.03

3.38 ± 0.03 6.19 ± 1.01 6.34 ± 0.35 5.4 ± 0.09 3.5 ± 0.07 11.6 ± 0.55 9.3 ± 0.02 3.4 ± 0.29

44.0 ± 1.97 7.55 ± 1.91 1.13 ± 0.20 2.0 ± 0.06 1.9 ± 0.06 0.5 ± 0.01 0.5 ± 0.01 2.0 ± 0.06

29.4 ± 1.06 44.5 ± 3.14 52.7 ± 1.96 29.2 ± 1.06 71.5 ± 1.92 72.3 ± 2.04 75.2 ± 2.34 55.6 ± 2.01

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K. Mueller et al. / Journal of Food Engineering 98 (2010) 453–460 Table 3 Functional properties of processed fractions of linseed meal (mean ± SD, n = 3). Protein solubility at pH 7 (%)

Water binding (mL/g)

Oil binding (mL/g)

Emulsification capacity (mL/g)

Foaming activity (%)

Brown linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

48.0 ± 0.28 <20 N.A. N.A. N.A.

0.8 ± 0.05 5.0 ± 0.34 0 0 6.7 ±

2.5 ± 0.11 1.8 ± 0.13 1.6 ± 0.07 1.6 ± 0.07 3.7 ± 0.17

535 ± 24.0 250 ± 11.2 145 ± 6.5 165 ± 7.4 N.A.

1651 ± 78.9 563 ± 26.9 1512 ± 72.3 1673 ± 80.0 N.A.

Yellow linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber Soy protein isolate

30.0 ± 0.18 25.0 ± 0.15 N.A. N.A. N.A. 45.0 ± 0.27

6.9 ± 0.46 N.A. 1.6 ± 0.11 1.0 ± 0.07 6.8 ± 0.46 1.9 ± 0.13

0.9 ± 0.04 0.7 ± 0.03 2.0 ± 0.09 1.0 ± 0.04 4.3 ± 0.19 1.8 ± 0.08

540 ± 24.2 216 ± 9.7 315 ± 14.1 400 ± 17.9 N.A. 605 ± 27.2

1059 ± 50.6 812 ± 38.8 640 ± 30.6 424 ± 20.3 N.A. 900 ± 43.0

N.A. = not analyzed.

protein isolate. However, it was significantly lower than that of whole egg (65.0%). Protein solubility of the acid soluble protein fraction of brown linseed was below the determination limit at pH 7. However, the corresponding fraction of yellow linseed showed 25.0% protein solubility. Water binding capacity of both insoluble dietary fiber fractions were the strongest, with values of 6.70 mL/g brown linseed and 6.80 mL/g of yellow linseed, respectively. Fedeniuk and Biliaderis (1994) reported, that water binding capacity of linseed mucilage was found with 16–30 g H2O/g sample, comparable to commercial guar gum (22 g/g guar gum). Oil-binding capacity of the alkaline soluble protein fraction of brown linseed was found with a significantly higher value of 2.5 mL/g in comparison to commercial soy protein isolate (1.80 mL/g). This value confirms the results of preliminary studies of Dev and Quensel (1988) and Krause et al. (2002), respectively. They found relatively high oil-binding capacities for linseed protein isolates obtained by alkaline extraction. The oil-binding capacity values were higher with lower pentosan concentration in the specific fraction (Krause et al., 2002). The oil-binding capacity of the alkaline soluble fraction of yellow linseed was found with a relatively low value of 0.9 mL/g, presumably due to the heat influence during pressing. Madhusudhan and Singh (1985) found lower oil-binding capacities of boiled linseed meal in comparison with the raw linseed meal material. Brown and yellow linseed alkaline soluble protein fractions were characterized by emulsification capacity values of 535 mL oil/g protein and 540 mL oil/g protein, respectively. Determining the emulsification capacity of whole egg and egg white for comparison purposes, the capacity of alkaline soluble protein fractions from both linseed varieties were found to be higher than for whole egg (495 mL/g) and lower than for egg white (800 mL/g). The alkaline soluble protein fractions from brown and yellow linseeds showed foaming activity values of 1650% and 1060%, respectively. Commercially available soy protein isolate, usable as a food ingredient adding foaming properties to a food, shows a value of 900%. Oomah and Mazza (1993b) mentioned hexane deoiled commercial flaxseed meal had foaming capacity 59.2%, Wanasundara and Shahidi (1997) reported that linseed protein isolate showed a foam expansion of 112% whipping 1% solutions for 30 s. In comparison, all obtained linseed protein containing fractions were found to be characterized by significant higher foaming activities while whipping 5% solutions for 8 min. 3.5. Viscosity of linseed fractions Viscosities of the linseed fractions in dependence of the temperature are shown in Figs. 3 and 4. Viscosity of 20 g/L (2%) brown and yellow linseed fraction solutions was found to be 4.0–26 and 3.8– 10 mPa/s at a temperature of 20 °C.

As shown in Fig. 3, among four of the brown linseed fractions, acid soluble carbohydrate fraction showed the highest viscosity and as temperature increased, the viscosity decreased dramatically. In comparison to the other fractions, alkaline soluble protein fraction of brown linseed was not affected by temperature. As shown in Fig. 4, the acid soluble carbohydrate fraction of yellow linseed showed the highest viscosity. The temperature dependent change of viscosity of acid soluble carbohydrate fraction of yellow linseed was similar to that of brown linseed. However, the viscosities of acid soluble carbohydrate fractions from brown and yellow linseed were different, with values of 30 and 10 mPa/s at 20 °C, 7.0 and 4.1 mPa/s at 90 °C, respectively. Oomah and Mazza (1993b) found an apparent viscosity of 112.5 mPa/s for hexane de-oiled commercial flaxseed meal. In comparison, acid soluble carbohydrate fraction from brown linseed with the highest viscosity, which is significantly lower, indicating the influence of the fractionation process on the rheological characteristics of the products. Fedeniuk and Biliaderis (1994) found that viscosity at 20 g/L solution of linseed mucilage fraction was affected by pH, especially between pH 2 and 6, and higher than pH 10. In this study, ambient pH was applied, referring to a neutral pH in the final product. In future studies, not only temperature influence, but also pH dependence of viscosity should be investigated, to evaluate the possibilities for food application of the different fractions. Wang et al. (2008) and Kalloufi et al. (2008) investigated the rheological changes by addition of linseed gums to maize starch and whey protein emulsions. Both of them mentioned a significant increase in viscosities of a solution and a dispersion, respectively, by addition of linseed gum at 0.1–0.3%. The use of linseed fractions containing protein and soluble fiber may affect the rheological properties of emulsions and foams occurring during dough and sausage processing. In food development, it has to be considered that these soluble fiber fractions may influence the gel building properties of a food system. 3.6. Further chemical analysis of linseed fractions Molecular weight, total sugar and uronic acid contents of linseed fractions are summarized in Table 4. Monosaccharide composition of soluble dietary fiber and acid soluble carbohydrate fractions are shown in Table 5. Main molecular weights of linseed fractions were in the range from 100,000 to 550,000. Madhusudhan and Singh (1985) reported a molecular weight of the major protein of linseed in the range of 250,000–300,000. Deviant to the investigations of Madhusudhan and Singh (1985), all fractions contained certain percentages of polysaccharides and therefore showed a higher value for the highest main molecular weight. Focusing on an effective and reasonable separation of protein and fiber for food application, not the purification of the linseed protein, the molecular weight distribution was quite wide. Total sugar

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35 Brown linssed Concentration: 2% Shear stress: 300 1/s

30

Viscosity [mPas]

25 Soluble dietary fibre Acid soluble carbohydrates

20

Alkaline soluble protein Acid soluble protein

15

10

5

0 0

10

20

30

40

50

60

70

80

90

100

Temperature [°C] Fig. 3. Steady shear rheological curves of fractions of meal from brown linseed.

12 Yellow linssed Concentration: 2% Shear stress: 300 1/s

Viscosity [mPas]

10

8

6

4 Soluble dietary fibre Acid soluble carbohydrates

2

Alkaline soluble protein Acid soluble protein

0 0

10

20

30

40

50

60

70

80

90

100

Temperature [°C] Fig. 4. Steady shear rheological curves of fractions of meal from yellow linseed.

content of alkaline soluble protein fractions of brown and yellow linseed, calculated as glucose, were 13.5 and 17.2 g/100 g, respectively, and acid soluble protein fractions showed values of 20.3 and 42.1 g/100 g. Uronic acid contents of the brown and yellow linseed fractions did not show significant differences. Acid soluble carbohydrates from both linseed varieties showed similar molecular weight distribution, total sugar and uronic acid contents. However, monosaccharide composition of acid soluble carbohydrates and soluble dietary fiber fractions of brown and yellow linseed showed significant differences (Table 5). The difference in sugar composition may be one reason for the different viscosities of yellow and brown linseed fractions (Figs. 3 and 4). During monosaccharide investigation, fucose and galacturonic acid were also

analyzed. However, in these studies, both monosaccharides could not be detected. In contrast to the brown linseed, both yellow linseed fractions, soluble dietary fiber and acid soluble carbohydrates, did not contain galactose. As shown in Table 5, brown linseed soluble dietary fiber fraction mainly consisted of glucose followed by galactose, and xylose. Brown linseed acid soluble carbohydrates mainly consisted of glucose followed by arabinose and xylose. Oomah et al. (1995) investigated the monosaccharide composition of the water-soluble polysaccharides of 109 brown and yellow linseed varieties originating from different regions of the world. Most of the monosaccharide contents measured in the fractions of this study correspond to the results of Oomah et al. (1995). Differing, we found higher concentrations of arabinose in each of the

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K. Mueller et al. / Journal of Food Engineering 98 (2010) 453–460 Table 4 Molecular weight, total sugar content, and uronic acid content of processed fractions of linseed meal (mean ± SD, n = 3). Main molecular weight (103)

Total sugar (g/100 g)

Uronic acid (g/100 g)

Brown linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

550, 540, 530, 520, N.A.

130 370, 150 190, 130 370, 200

13.5 ± 1.09a 20.3 ± 3.44bc 50.2 ± 3.62e 53.6 ± 4.67e N.A.

5.15 ± 1.11a 5.05 ± 0.96a 7.39 ± 1.59bc 9.30 ± 2.40c N.A.

Yellow linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates Insoluble dietary fiber

520, 500, 550, 500, N.A.

400 200 300 330, 95

17.2 ± 4.09b 42.1 ± 3.98d 29.3 ± 6.81c 45.4 ± 3.98de N.A.

4.44 ± 2.38ab 3.42 ± 2.00a 7.84 ± 2.06bc 9.19 ± 1.38c N.A.

N.A. = not analyzed. Different letters within the same column indicate significant differences (p < 0.05).

Table 5 Relative monosaccharide composition of carbohydrate rich fractions of linseed meal (mean ± SD, n = 3). Sugar composition (molar ratio, %) Rhamnose

Xylose

Arabinose

Glucose

Galactose

Brown linseed Soluble dietary fiber Acid soluble carbohydrates

14.3 ± 1.61a 18.0 ± 8.79ab

17.2 ± 1.68a 18.8 ± 5.91ab

14.8 ± 1.77a 19.6 ± 0.95b

36.5 ± 4.08b 36.4 ± 5.36b

17.3 ± 1.00c 7.10 ± 1.24b

Yellow linseed Soluble dietary fiber Acid soluble carbohydrates

30.3 ± 1.27c 18.9 ± 0.41b

18.5 ± 5.58ab 14.6 ± 0.85a

25.5 ± 5.48c 23.5 ± 0.05c

26.2 ± 3.59a 42.9 ± 1.31b

0 ± 0a 0 ± 0a

Different letters within the same column indicate significant differences (p < 0.05).

fractions, and a relatively high rhamnose content in the soluble dietary fiber fraction of the yellow linseed. Deviant to the studies of Oomah et al. (1995), we could not detect galactose in the yellow linseed meal fractions. Warrand et al. (2005) performed the fractionation of linseed meal in a different way and they reported the monosaccharides D-xylose and D-glucose in one fraction and D-galactose and D-arabinose in the other fraction, respectively, as main components. The high glucose contents in the fractions of this study were confirmed by Warrand et al. (2005). Fedeniuk and Biliaderis (1994) and Alix et al. (2008) investigated the influence of extraction temperature on monosaccharide composition. The results of these studies indicate a strong influence of extraction temperature on monosaccharide compositions of the carbohydrate rich fractions. The contents of phenolic acid compounds in linseed fractions are shown in Table 6. In all four linseed fractions, SDG, p-coumaric acid, and ferulic acid were detected. Only trace amounts of these lignans could be found in the insoluble dietary fiber fractions.

The acid soluble protein fraction of brown linseed contained the smallest content of phenolic acid glucosides among all fractions. Alkaline soluble protein fraction of Linola contained the highest amount of lignans. The yield of the total extracted amount of SDG, p-coumaric acid glucoside, and ferulic acid glucoside of the de-oiled brown or yellow linseed were calculated using the values shown in Table 2. Calculated 21.5 mg/g SDG, 0.22 mg/g p-coumaric acid glucoside, and 1.09 mg/g ferulic acid glucoside were extracted from de-oiled brown linseed, while 13.3 mg/g SDG, 0.11 mg/g pcoumaric acid glucoside, and 0.39 mg/g ferulic acid glucoside were extracted from de-oiled yellow linseed. Li et al. (2008) reported a SDG content of de-oiled brown linseed of 15.4 mg/g. Eliasson et al. (2003) and Strandås et al. (2008a) reported lignan concentrations in de-oiled brown linseed of 12–26 mg/g of SDG, 1.2–8.5 mg/g of p-coumaric acid glucoside and 1.6–5.0 mg/g of ferulic acid glucoside. The lignan contents were depending on the samples origin. In comparison to these reports, all fractions were found to contain 21 mg of SDG in sum. This indicates, that fractionation

Table 6 Content of phenolic compounds in linseed meal fractions (mean ± SD, n = 3). Phenolic compounds (mg/g DM) SDG

p-Coumaric acid glucoside

Ferulic acid glucoside

Brown linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates

56.23 ± 1.56e 9.77 ± 1.27a 44.48 ± 4.32e 35.16 ± 1.62d

0.591 ± 0.123a 0.149 ± 0.019a 0.589 ± 0.103a 0.133 ± 0.029a

3.648 ± 0.186a 0.521 ± 0.042a 1.886 ± 0.076a 0.435 ± 0.031a

Yellow linseed Alkaline soluble protein Acid soluble protein Soluble dietary fiber Acid soluble carbohydrates

110.1 ± 11.5f 15.7 ± 1.22b 33.70 ± 1.90cd 25.2 ± 2.71c

0.636 ± 0.178a 0.245 ± 0.010a 0.355 ± 0.170a 0.137 ± 0.019a

2.935 ± 1.138a 0.514 ± 0.025a 1.015 ± 0.559a 0.273 ± 0.050a

Different letters within the same column indicate significant differences (p < 0.05).

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methodology applied, showed an effective extraction of SDG, especially resulting into a high concentration of SDG in the alkaline soluble protein fraction. Stability of purified linseed SDG, added to different dairy and bakery products, has been shown (Hyvärinen et al. 2006a,b) during food processing and storage of the final products. Strandås et al. (2008b) investigated the lignan content of commercial breads prepared with linseed as food ingredient and found SDG concentrations in the range of 15 to 157 mg/100 g dry bread, indicating no significant influence of bread making procedure on lignan contents. Applying the SDG rich, alkaline soluble protein fraction as food ingredient, the final product may be characterized by bioactive properties and could have a function in the prevention of chronic diseases (Eeckhaus et al., 2008). Further research is required to visualize the physiological effects of the final products to evaluate their potential health benefit in human nutrition. The total extracted amounts of p-coumaric acid and ferulic acid of all fractions were smaller than 1.1 mg/g, indicating a lower efficiency of the applied fractionation methodology as for SDG. 4. Conclusions The fractionation process for de-oiled linseed meal presented in this study could be proven to result in an acid soluble protein fraction with relatively high viscosity values, usable as food ingredient adding viscosity properties to a processed food. Alkaline soluble protein fraction showed emulsification and foaming properties comparable to egg yolk and therefore, it is applicable as an excellent replacement for egg protein in bakery products. The production of this fraction in industrial scale is of special interest because of high protein yield and the enriched concentration of SDG as a bioactive food compound. Acknowledgements This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernaehrungsindustrie e.V., Bonn). Project AiF 14447 BG. This research was partly supported by Iijima Food Science Foundation, Japan. We also thank the support of international collaboration work by Tokyo University of Marine Science and Technology. References Alix, S., Marais, S., Morvan, C., Lebrun, L., 2008. Biocomposite materials from flax plants: preparation and properties. Composites: Part A 39, 1793–1801. American Association of Cereal Chemists, 2000a. Approved Methods of the AACC, 10th ed. AACC, St. Paul, MN (Method 46–23. Nitrogen Solubility Index). American Association of Cereal Chemists, 2000b. Approved Methods of the AACC, 10th ed. AACC, St. Paul, MN (Method 56–20. Hydration Capacity of Pregelatinized Cereal Products). American Oil Chemists’ Society (AOCS), 1998. In: Official Methods on Recommended Practices of the AOCS, fifth ed. AOCS Official Method Ba 11– 65. Champaign, IL. Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry 54, 484–489. Chung, M.W.Y., Lei, B., Li-Chan, E.C.Y., 2005. Isolation and structural characterization of the major protein fraction from NorMan flaxseed (Linum usitatissimum L.). Food Chemistry 90, 271–279. Dev, D.K., Quensel, E., 1986. Functional properties and microstructural characteristics of linseed flour and protein isolate. LWT-Food Science and Technology 19, 331–337. Dev, D.K., Quensel, E., 1988. Preparation and functional properties of linseed protein products containing differing levels of mucilage. Journal of Food Science 53, 1834–1837. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugar and related substance. Analytical Chemistry 28, 350–356. Eeckhaus, E., Strujis, K., Possemiers, S., Vincken, J.-P., De Keukeleire, D., Verstraete, E., 2008. Metabolism of the lignan macromolecule into enterolignans in the gastrointestinal lumen as determined in the simulator of the human intestinal

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