Effects of palm oil quality and packaging on the storage stability of dry vegetable bouillon paste

Food Chemistry 132 (2012) 1324–1332

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Effects of palm oil quality and packaging on the storage stability of dry vegetable bouillon paste Riikka Raitio, Vibeke Orlien, Leif H. Skibsted ⇑ University of Copenhagen, Department of Food Science, Faculty of Life Sciences, Rolighedsvej 30, DK-1930 Frederiksberg C, Denmark

a r t i c l e

i n f o

Article history: Received 7 February 2011 Received in revised form 8 November 2011 Accepted 28 November 2011 Available online 4 December 2011 Keywords: Storage stability Modified atmosphere packaging ESR spectroscopy Maillard reaction Lipid oxidation

a b s t r a c t Vegetable bouillon paste, prepared by dry-mixing of pre-produced dry ingredients with addition of semisolid palm oil, was stored at the slightly elevated temperature of 40 °C for up to 12 weeks for comparing modified atmosphere packaging (MAP; oxygen concentration initially below 0.5%) packaging with free availability of oxygen in order to identify the mechanisms leading to quality degradation. An increased browning and increased formation of volatile compounds related to Maillard reactions and oxidation of secondary lipid oxidation products were observed for the vegetable bouillon paste stored without limiting the oxygen availability. The use of palm oil (kept liquid by heating to 50 °C for one week) was further found to promote quality degradation as compared to fresh palm oil when the availability of oxygen was not limited by the packaging of the vegetable bouillon paste. For modified atmosphere packaging, the effect of the quality of the oil on storage stability of the product was less pronounced. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction A common way to produce dry foods, such as soup powders and bouillon pastes, is dry mixing of pre-produced dry ingredients, with subsequent addition of oil. In this type of food product, enzymatic (and microbial) spoilage is typically inhibited by a low water activity (aw) (Gibbs, 1986). Instead, lipid oxidation and Maillard reactions are generally considered as the major causes of the quality deterioration of food products characterised as having aw values below 0.6 (Labuza, 1971, 1980). Lipid autoxidation is a free radical chain reaction, leading to the formation of lipid hydroperoxides as primary oxidation products, and further to the formation of secondary lipid oxidation products, such as aldehydes and ketones. The secondary lipid oxidation products can cause off-flavours in foods and lipid oxidation can further affect the nutritional value and safety (Frankel, 2005). Maillard reactions are a complex set of reactions, typically occurring between carbonyl compounds and amino groups originating from proteins, peptides or amino acids. Maillard reactions may lead to formation of off-flavours, decreased nutritional value and discolouration.

Abbreviations: aw, water activity; ESR, electron spin resonance spectroscopy; FO, fresh palm oil; G, Gauss; GC–MS, gas chromatography–mass spectrometry; HO, heated palm oil; MAP, modified atmosphere packaging; NP, normal atmosphere packaging; POV, peroxide value; SPME, solid phase microextraction; VB, vegetable bouillon paste. ⇑ Corresponding author. Tel.: +45 3533 3221; fax: +45 3533 3344. E-mail addresses: [email protected], [email protected] (L.H. Skibsted). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.11.112

Discolouration typically occurs at the final stage of Maillard reactions as a result of the formation of melanoidins (Hodge, 1953; Nursten, 2005). Lipid oxidation and Maillard reactions may be further related. In studies with model systems, the lipid oxidation product malondialdehyde reacted with casein, resulting in browning (Adams, De Kimpe, & Van Boekel, 2008). Additionally, aldehydes from lipid oxidation can degrade amino acids in Strecker-type reactions (Hidalgo, Gallardo, & Zamora, 2005). In cream powder, a correlation between the formation of the secondary lipid oxidation product, hexanal, and Strecker aldehyde, 3-methyl butanal, has been observed (Andersson & Lingnert, 1998a). In turn, some Maillard reaction products can act as antioxidants against lipid peroxidation (Elizalde, Dalla Rosa, & Lerici, 1991; Nursten, 1980–1981). The quality of oil used in the preparation of food products can affect the oxidative stability of the final food product. The quality of fish oil has thus been found to be important for the oxidative stability of milk enriched with fish oil, and the initial peroxide concentration of fish oil was more important in determining the stability of fish oil-milk systems than was the level of unsaturation of fish oil (Let, Jacobsen, Frankel, & Meyer, 2003; Let, Jacobsen, & Meyer, 2005). In addition, lipid oxidation products readily react with proteins, in effect causing adverse changes to food quality (Schaich, 2008). Hence, the quality of oil used in food production also may become relevant in relation to protein degradation reactions, including Maillard reactions. Reduced oxygen concentrations in food packaging decrease the level of lipid oxidation (Andersson & Lingnert, 1997, 1998a; Lloyd,

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Hess, & Drake, 2009). However, very low oxygen concentrations are needed to avoid lipid oxidation (Karel, 1986), and in cream powder the detrimental effect of lipid oxidation on sensory stability has been observed already at oxygen concentrations of 0.3 ml/l after 25 weeks of storage at 30 °C (Andersson & Lingnert, 1998b). In relation to Maillard reactions, oxygen may both promote and hinder the progress of Maillard reactions (Nursten, 2005). Hence, it is not surprising that, for milk powder, anaerobic conditions have been shown to promote Maillard reactions (Thomsen et al., 2005), while soy sauce browning was found to be more enhanced due to the presence of oxygen (Hashiba, Okuhara, & Iguchi, 1981). This indicates the complexity of Maillard reactions and that the reactions occurring greatly depend on the composition of each particular food system, as well as other factors, such as temperature, pH and aw (Nursten, 1986). In the present investigations, the storage stability of vegetable bouillon paste was studied. This dry food system consisted of dry ingredients, such as salt, vegetable powders, spices and yeast extract powders mixed together and semisolid palm oil added as a lipid phase. The objectives of the study were to evaluate the effect of palm oil quality and packaging atmosphere on the storage stability of vegetable bouillon paste and to characterise the chemical reactions leading to quality deterioration in such dry food products prepared from dry ingredients and semisolid palm oil by mixing. 2. Materials and methods 2.1. Chemicals BaCl22H2O, hexane, 2-methyl butanal, pentanal, dimethyl disulphide, heptane, N-tert-butyl-a-phenyl-nitrone (PBN), and a-pinene were purchased from Sigma–Aldrich (Steinheim, Germany). HPLC grade chloroform was from Lab-Scan (Dublin, Ireland). HPLC grade isooctane, analytical grade methanol, dried methanol, NaCl and FeSO47H2O were from Merck (Darmstadt, Germany). NH4SCN was from Riedel-De-Haën (Seelze, Germany). Hexanal was obtained from Fluka (Steinheim, Germany). Fatty acid methyl ester standards were from Sigma–Aldrich (Steinheim, Germany). 2.2. Preparation of vegetable bouillon paste samples for storage experiment Vegetable bouillon pastes (VB) were prepared by dry-mixing of ingredients with addition of food grade semisolid palm oil (PO) (27%). The dry ingredients were mainly salt, maltodextrin, yeast extract, flavours, and spices. Two different kinds of bouillon pastes were prepared; one with fresh palm oil (FO) and one with heated oil (HO). The heated oil was prepared by storing palm oil in an open glass beaker at 50 °C for one week, with occasional mixing. Two different types of packaging were applied: 110 ml polypropylene containers (normal atmosphere packaging = NP) and modified atmosphere packaging (MAP). This led to four different bouillonpackaging combinations for storage tests: fresh oil-normal packaging (FO-NP), heated oil-normal packaging (HO-NP), fresh oil-modified atmosphere packaging (FO-MAP), and heated oil-modified atmosphere packaging (HO-MAP). For MAP, the bouillon paste was placed on a polystyrene plastic tray and packed in a 13  19.8 cmsized vacuum bag (polyamide/polyethylene 30/100 lm; permeability for O2 was 30 cm3/m3  d  bar) with Multivac A equipment (Multivac Sepp Haggenmüller GmbH & Co., Wolfertschwenden, Germany). In the MAP procedure, the sample was first evacuated before introducing the modified atmosphere of N2 (97%) and CO2 (3%). The O2 and CO2 concentrations in the MAP were measured at each sampling time with Checkmate 9900 equipment (PBI-Dansensor,

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Ringsted, Denmark) and the air was measured as control. The sample sizes within both package types were 78 g (±4 g). The bouillon pastes were stored at 40 °C for up to 12 weeks and storage tests were done in duplicate. 2.3. Fatty acid composition The fatty acid compositions of the acylglycerols of the vegetable bouillon pastes were determined for both fresh bouillon paste and after 12 weeks of storage, and for palm oils used for the preparation of bouillon pastes. Fatty acids were determined by GC as the fatty acid methyl esters. Methylations were done directly on 40 mg of dry vegetable bouillon paste and on 5 mg of palm oil with 0.087 M sodium methylate solution at 60 °C for 40 min. The formed fatty acid methyl esters (FAME) were extracted with 1.5 ml of hexane, prior to analysis using gas chromatography (HP 6890 Series, Hewlett–Packard, Palo Alto, CA, USA; FFAP column (25 m  0.20 mm  0.33 lm) from Agilent Technologies, Waldbronn, Germany) with FID detection. The oven temperature programme was: 50 °C for 1 min; from 50 °C to 180 °C at 15 °C/min; from 180 °C to 195 °C at 2 °C/min, held for 15 min; from 195 °C to 240 °C at 4 °C/min, held for 10 min. Injected volume was 1 ll and split ratio was 1:25. Helium was used as carrier gas and the flow was 1 ml/min. The results were analysed with Chemstation software (Agilent Technologies) and fatty acid methyl esters were identified by comparison of retention times with known standards. 2.4. ESR spectroscopy on palm oil The susceptibility of fresh and heated palm oil to oxidation was measured with ESR spectroscopy, as described by Raitio, Orlien, and Skibsted (2011a, 2011b). PBN (1 mg/g oil) was used as spin trap and it was dissolved in the oil by mixing at 50 °C in a water bath for 20 min. Subsequently the oil samples containing PBN were placed in a 70 °C water bath for 5 h to enhance the formation of spin-adducts. After incubation at 70 °C the samples in ESR tubes were cooled to 20 °C in a water bath. After cooling, isooctane (1:1, w:w) was added to the samples in the test tubes and the oil-isooctane mixture was transferred to the ESR tube with 4 mm inner diameter (710-SQ-250M, Wilmad Glass, Buena, NJ, USA). The exact sample masses and heights in the ESR tubes were noted, from which the sample densities were calculated and used to obtain relative concentrations of spin-adducts; 6 min after removing from the 70 °C water bath, the samples were screened with the ESR spectometer (MiniScope MS 200, Magnettech, Berlin, Germany) equipped with X-band microwave supply. The ESR parameters applied were as follows: Centre field, 3336 G; sweep width, 80 G; sweep time, 60 s; modulation amplitude, 3.3 G and microwave power, 79 mW. Software analyses (Magnettech, Berlin, Germany) were employed to obtain the areas under the ESR spectra by double integration. 2.5. ESR spectroscopy of vegetable bouillon paste The relative free radical concentrations of vegetable bouillon pastes during storage were determined using ESR spectroscopy. For ESR analyses, bouillon paste was weighed into a long Pasteur pipette (glass) which was thereafter placed in a fused quartz ESR tube with 4 mm inner diameter and 1 mm wall thickness (710SQ-250M, Wilmad Glass). The exact sample mass in the pipette was measured and it was used to calculate relative free radical concentrations, as the whole sample was within the cavity of the ESR spectrometer. An ESR spectrometer (MiniScope MS 200, Magnettech, Berlin, Germany) equipped with X-band microwave supply was used. The microwave frequency of each spectrum was recorded with an external frequency metre (Agilent 53150A, Santa

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Clara, CA, USA) for the determination of g-values. The optimised parameters for ESR determinations were: centre field 3342.6 G, sweep width 75.6 G, sweep time 120 s, modulation amplitude 6000 mG and microwave power 1 mW. Software analyses (Magnettech, Berlin, Germany) were employed to obtain the area under the ESR spectra by double integration. The measurements were performed with microwave power with highest sensitivity without interference from saturation, under which conditions the double integrated area of the ESR spectrum is equal to radical concentration in the sample (Weil, Bolton, & Wertz, 1994). Software analysis (Magnettech, Berlin, Germany) was employed to determine the gvalues of the radicals from the ESR spectra. The relative free radical concentrations of different bouillon pastes were determined after 0, 2, 4, 8 and 12 weeks of storage at 40 °C, and the ESR determinations were done in duplicate. 2.6. SPME-GC–MS for analysis of volatiles Solid phase microextraction (SPME)-GC–MS was employed to determine the relative changes in volatiles formed in vegetable bouillon paste during the storage. The analyses were done in duplicate for the samples after 0, 4 and 12 weeks of storage. The 20 ml vials were used with 1 g (±0.1 g) of sample size. Before the extraction, the sample was equilibrated for 30 min at 37 °C with 500 rpm agitation. The headspace volatiles were extracted using a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 30/ 50 lm SPME fibre assembly (Supelco, Bellafonte, PA, USA) at 37 °C for 30 min. All the SPME-extractions were performed with the automated Combi PAL-system (CTC Analytics AG, Zwingen, Switzerland). After the extraction, the SPME-fibre was desorbed in the injection port of the gas chromatograph for 10 min. A GC–MS (Scan mode) (G1530A, Agilent 5973 Network Mass Selective Detector, Agilent Technologies), with DB-Wax capillary column (30 m  0.25 mm  0.25 lm) was used. The oven temperature programme for GC was: 45 °C for 10 min; from 45 °C to 240 °C at 6 °C/ min, held for 10 min. The split ratio was 1:10 and helium was used as carrier gas with a flow of 1 ml/min. The volatile compounds were identified by matching their mass spectra with those of a commercial database (Wiley275.L, HP product G1035A), using software of the MSD Chemstation (Agilent Technologies), and high quality of

identification was required for each selected compound. The quantification of selected compounds was done using the area of the specific ion for each compound (Table 1). The results were expressed as relative concentrations (area/sample mass). The identification of pentanal, hexanal, 2-methyl butanal and a-pinene were confirmed by analysing a standard mixture of these compounds with the same GC–MS equipment and parameters. The liquid injection was applied for a standard mixture with 1 ll sample size and with split ratio 50:1. 2.7. Colour determinations The colour development of the vegetable bouillon pastes during storage was measured using the Hunter L⁄, a⁄ and b⁄ colour system with the BYK-Gardner colour guide (BYK-Gardner GmbH, Gerestried, Germany), in which L⁄-value indicates lightness (100 = white; 0 = black), a⁄-value indicates redness (+) or greenness (), and b⁄value refers to yellowness (+) and blueness (). Each sample was measured in triplicate after 0, 2, 4, 8 and 12 weeks of storage. The complete colour difference (DE) between the 0 and 12 week samples was further calculated with the following equation (Francis & Clydesdale, 1975): 2

DE ¼ ðDL2 þ Da2 þ Db Þ1=2

ð1Þ

2.8. Water activity (aw) determinations The water activities of vegetable bouillon pastes were determined at room temperature after 0, 2, 4, 8 and 12 weeks of storage with Aqualab CX2 equipment (Aqualab, Washington, USA). 2.9. Statistical analyses The significant differences between samples were analysed with two-sided heteroscedastic t-test with significance level P 6 0.05. 3. Results and discussion 3.1. Oxidative status of palm oil

Table 1 Volatiles analysed by SPME-GC–MS. Retention times (min) and target ions for integrations are presented. Grouping refers to the Fig. 4. Identification

2,4-Dimethylheptane 4-Methyloctane 2,4-Dimethyl-1-heptene 2-Methyl butanal 3-Methyl butanal Pentanal a-Pinene Aromatic compound e.g. toluene Hexanal b-Pinene Sabinene Ethylbenzene Deltacarene Limonene Gammaterpinene Ethylenebenzene Dihydro-2-methylfuranone 2,5-Dimethylpyrazine Acetic acid Propionic acid Butanoic acid Hexanoic acid

RT(min)

Target ion (m/z)

2.00 2.26 2.42 2.66 2.70 3.44 4.19 4.52 5.81 6.54 7.15 7.31 8.43 11.27 13.52 13.84 14.23 16.49 20.08 22.15 24.07 28.2

43 43 70 57 44 44 93 91 56 93 93 91 93 68 93 104 43 108 TIC 74 60 60

Group

A A A H H C I B C I I B I I I B G G E F D D

Fresh and heated semisolid palm oils (FO and HO, respectively) were used for the preparation of vegetable bouillon pastes for accelerated storage tests. The fatty acid compositions of FO and HO are presented in Table 2. The obtained fatty acid compositions for FO and HO were similar to the fatty acid compositions reported earlier for palm oil (Gee, 2007; Maclellan, 1983). The relative free radical concentrations in FO and HO were measured as spinadducts. Larger formation of spin-adducts was observed in HO

Table 2 Fatty acid compositions, peroxide values (POV), and relative concentrations of spin adducts (ESR analysis) of fresh semisolid palm oil (FO) and heated semisolid palm oil (HO).

Fatty acid (%) Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid Relative concentration of spin adducts POV (milliequivalent peroxides/kg oil) a

Fresh oil (FO)

Heated oil (HO)

0.9 ± 0.0 44.2 ± 0.0 5.0 ± 0.0 40.5 ± 0.0 9.4 ± 0.0 82 ± 4 0.013 ± 0.000

0.9 ± 0.00 44.5 ± 0.0a 5.0 ± 0.0 40.3 ± 0.0 9.4 ± 0.0 120 ± 13 0.033 ± 0.002a

Statistically significant difference between fresh and heated oil.

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(120 ± 13) than in FO (82 ± 4). This result was in line with our previous work (Raitio et al., 2011a, 2011b). 3.2. Storage stability of vegetable bouillon paste The level of O2 in the MAP packages increased during storage time, but still remained below the normal atmospheric levels. The level of CO2 decreased in the MAP packages and it was not detectable after 8 weeks of storage (Fig. 1). The changes in the composition of headspace atmosphere in MAP can be explained by the permeability of packaging material, which allowed slow diffusion of O2 into the MAP packages. The fatty acid composition of the vegetable bouillon pastes (Table 3) was very close to that of palm oil (FO and HO, Table 2.) except for a little higher proportion of linoleic acid and a smaller proportion of palmitic acid. The fatty acid composition remained almost unchanged during the 12 week storage in MAP-stored bouillon pastes but, in NP-stored bouillon pastes, the proportion of linoleic acid decreased and proportion of stearic acid increased during storage. This may be an indication of lipid oxidation; however, the observed changes were relatively small. The POV of the vegetable bouillon pastes (Table 3) were larger than those obtained for FO and HO, which may indicate that the samples were oxidised during fat extraction prior to POV analysis. Alternatively, the peroxides in bouillon pastes did not originate solely from the palm oil but, e.g., from proteins from other ingredients. POVs were found to be very variable and, in general, the varying peroxide values may be explained by a further transformation to secondary lipid oxidation products, which apparently is different for different storage conditions. All the bouillon pastes got darker and redder during the storage (Fig. 2A and B.). The colours of fresh bouillon pastes were measured before applying the MAP; however, an observed instant darkening followed packaging under modified atmosphere. This caused approximately 11% decrease in the L⁄-value and 10% decrease in b⁄-value, while a⁄-value was not affected. When the colour changes

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caused by MAP were taken into account in calculations of the total colour change (DE) for bouillon pastes (Table 3), the results clearly indicated that the colours of MAP bouillon pastes were more stable over the storage period than were the colours of NP bouillon pastes. The DE-values furthermore showed that, in the HO bouillon pastes, colour changes were greater than those in FO bouillon pastes. The combination of HO-NP was the most detrimental for colour of bouillon paste as all the colour attributes changed the most in this bouillon paste (Fig. 2A–C). The free radical concentrations decreased during storage in all bouillon pastes (Fig. 3A). The steepest declines of free radical concentrations were observed between 0 and 2 weeks of storage. Significant decreases in free radical concentrations were further observed between 2 and 4 weeks of storage in all bouillon pastes except HO-MAP and this decrease was steeper in NP bouillon pastes than in FO-MAP bouillon paste. Beyond four weeks of storage, the relative free radical concentrations in all bouillon pastes were stable, and in MAP bouillon pastes the relative free radical concentrations were slightly higher than those in bouillon pastes stored in NP. A decrease in free radical concentrations has earlier been observed in cappuccino powder (Becker, Madsen, & Skibsted, 2009). In cappuccino powder, the decrease of free radical concentrations was linked to the increased water content and aw values, leading to increased mobility of molecules in powder, allowing the radicals to reach the steady state-concentrations. Similarly, Jensen and Risbo (2007) have observed a decrease in free radical concentration due to increased relative humidity during storage and subsequent moisture absorption by different dry products. However, water activities of vegetable bouillon pastes (Fig. 2D) were stable during the first two weeks of storage, when the highest decline in free radical concentrations was observed. Thus, it was not possible to relate the decreased free radical concentrations to increased molecular mobility in the bouillon pastes. The packaging had an effect on the free radical concentrations of vegetable bouillon pastes, as seen in Fig. 3A. Although the decrease in free radical concentrations between 0 and 2 weeks of

Fig. 1. Concentrations (%) of O2 and CO2 in the headspace of MAP during storage. d = O2 in the MAP packaging of fresh oil bouillon paste, s = O2 in the MAP packaging of heated oil bouillon paste, j = CO2 in the MAP packaging of fresh oil bouillon paste, h = CO2 in the MAP packaging of heated oil bouillon paste.

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Table 3 Fatty acid compositions, peroxide values (POV), and total colour change (DE) of vegetable bouillon pastes (VB) at the beginning of storage experiments and after 12 weeks of storage at 40 °C.

Fatty acid (%) Myristic acidb Palmitic acid Stearic acidb Oleic acidb Linoleic acidb POVb (milliequivalent peroxides/kg oil)

FO-VB 0 weeks

HO-VB 0 weeks

FO-NP-VB 12 weeks

HO-NP-VB 12 weeks

FO-MAP-VB 12 weeks

HO-MAP-VB 12 weeks

1.1 ± 0.1 43.4 ± 0.3 4.4 ± 0.1 40.7 ± 0.2 10.5 ± 0.3 0.100 ± 0.048

1.1 ± 0.0 43.8 ± 0.4 4.5 ± 0.0 40.4 ± 0.1 10.2 ± 00 0.140 ± 0.035

1.0 ± 0.0 43.9 ± 0.1 4.7 ± 0.1a 40.5 ± 0.1 9.9 ± 0.1a 0.118 ± 0.012

1.1 ± 0.0 43.9 ± 0.1 4.6 ± 0.1a 40.5 ± 0.1 10.1 ± 0.0a 0.124 ± 0.023

1.1 ± 0.0 43.8 ± 0.1 4.5 ± 0.1 40.3 ± 0.1a 10.3 ± 0.0 0.115 ± 0.008

1.1 ± 0.0 44.1 ± 0.1 4.4 ± 0.1 40.2 ± 0.2 10.2 ± 0.0 0.099 ± 0.012a

12.0

15.5

6.3

8.5

DE a b

Change between 0 and 12 weeks statistically significant. Statistically significant difference between samples from NP and MAP after 12 weeks storage.

Fig. 2. Colour and water activity (aw) of vegetable bouillon pastes during storage at 40 °C. (A) Hunter L⁄-value, (B) Hunter a⁄-value, (C) Hunter b⁄-value, (D) aw. N = FO-NP, j = HO-NP, M = FO-MAP, h = HO-MAP.

storage was identical to those of both MAP and NP bouillon pastes, the free radical levels in MAP bouillon pastes were higher at the storage times of 4, 8, and 12 weeks and the differences were statistically significant when pooled results for MAP bouillon pastes were compared to pooled results of NP bouillon pastes. The observed ESR spectra of bouillon pastes were typical for spectra of dry systems consisting of only one line; thus no hyperfine structures were observed. Our previous studies with cauliflower soup powder, prepared similarly vegetable bouillon pastes, indicated that the ESR spectrum of dry foods could be accounted for by addition of ESR spectra of dry ingredients. This indicated that

the observed spectrum was a superposition of two or more overlaying spectra (Raitio et al., 2011a, 2011b). For these reasons the identification of types of radicals in bouillon pastes was very tentative. Still, the difference in g-values at the later stages of storage is an indication of the different radical species of bouillon pastes stored in MAP and NP (Fig. 3B). The g-value is specific for each type of radical and indicates the relationship between magnetic field and the frequency of electromagnetic radiation under the resonance condition. At the storage times of 4, 8, and 12 weeks a difference in g-values between MAP and NP bouillon pastes was observed (Fig. 3B), which result resembles to the relative free radical

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Fig. 3. (A) Relative free radical concentrations, and (B) g-values of vegetable bouillon pastes during storage at 40 °C. N = FO-NP, j = HO-NP, M = FO-MAP, h = HO-MAP.

concentrations (Fig. 3A). However, standard deviations were large for the observed g-values, but the differences were statistically significant at 4, 8, and 12 weeks of storage. At the beginning of the storage, all the bouillon pastes had the g-value of 2.0053, indicating N-centered radicals (Schaich, 2002), and the g-values decreased similarly between 0 and 2 weeks in all bouillon pastes. After this, the g-values of MAP bouillon pastes remained at the same level while the g-values of NP bouillon pastes gradually decreased. Similarly, Thomsen, Lauridsen, Skibsted, and Risbo (2005) observed a decrease in g-values of milk powder during storage and this was related to the late stage Maillard reactions as an increased browning was simultaneously observed. In the present study the changes in colour and g-values were primarily observed in the NP bouillon pastes, and thus in the presence of normal atmospheric concentrations of oxygen, and no increases in free radical concentrations were observed in relation to formation of browning. The studies of Mitsuda, Yasumoto, and Yokoyama (1965) suggested the presence of free radicals in the end products of Maillard reactions, melanoidins. However, it has also been suggested that the formation of melanoidins is free radical-assisted and the free radicals do not locate in the melanoidins. For this reason, the ESR intensities do not necessarily increase due to formation of browning (Namiki & Hayashi, 1975). The relative concentrations of selected volatiles (Table 1) were followed during the storage of bouillon pastes. The volatile concentrations increased during storage in all bouillon pastes; however, the volatile profiles of bouillon pastes in normal package and in MAP after 12 weeks of storage were quite different. The formation of branched alkanes and alkenes (Fig. 4A) dominated in the normal package bouillon pastes while they were formed much less in MAP bouillon pastes. In MAP bouillon pastes, aromatic compounds were formed during storage, while these compounds were absent in NP-bouillon pastes (Fig. 4B). The formation of aromatic compounds in MAP may be explained by the use of polystyrene trays on which the bouillon pastes were placed within MAP. The degradation products of polystyrene may have migrated to the bouillon paste, as was seen for yoghurt stored in polystyrene cups (Frederiksen, Haugaard, Poll, & Becker, 2003). Similarly, we suggest that the increases of relative concentrations of branched alkanes and alkenes in NP bouillon pastes may result from the packaging, as the degradation products of polypropylene. The relative concentrations of pentanal and hexanal, which are formed in lipid oxidation, increased during the first 4 weeks of storage in all bouillon pastes (Fig. 4C). After 12 weeks, the relative concentrations of these secondary lipid oxidation products were higher in bouillon pastes prepared with heated oil in both types of packaging and the highest concentration was found in the

HO-NP bouillon paste. At 12 weeks of storage, the difference between HO and FO bouillon pastes was statistically significant, for both hexanal and pentanal results. This indicated that the oxidative status of palm oil used for preparation of bouillon pastes had more significance than had the packaging atmosphere for the oxidative stability of bouillon pastes. This result was surprising as the availability of oxygen in the packaging has earlier been found to correlate with hexanal formation and thus lipid oxidation, during storage in various dry food products (Andersson & Lingnert, 1997, 1998a; Jensen, Danielsen, Bertelsen, Skibsted, & Andersen, 2005; Lloyd et al., 2009). The explanation for our observations for bouillon paste may be the increase in oxygen concentration in MAP during storage (Fig. 1) as, in general, very low oxygen concentrations are required to limit the oxidation (Karel, 1986). The increases in the butanoic and hexanoic acid concentrations during storage were observed in all bouillon pastes (pooled results in Fig. 4D). These short aliphatic acids can form in the oxidation of secondary lipid oxidation products (Frankel, 1982). Butanoic and hexanoic acids were not observed in the FAME analyses. This indicated that their formation during storage was not related to the lipolytic activity, as the base-catalysed methylation only forms fatty acid methyl esters from acylglycerols (Ackman, 1998). The storage of bouillon pastes in NP promoted the formation of butanoic and hexanoic acids, and the highest concentration of these compounds after 12 weeks of storage was observed in HO-NP bouillon paste. This can be considered as evidence of more enhanced lipid oxidation in NP bouillon pastes, although the formation of secondary oxidation products seems to be more dependent on the oil quality than on the packaging. The analyses of volatiles were done only at the storage times of 0, 4, and 12 weeks. Hence, it is possible that the concentrations of secondary oxidation products in NP bouillon pastes were higher between 4 and 12 weeks of storage but decreased to the observed levels due to further oxidation reactions. Butanoic and hexanoic acids had characteristic sweaty flavour (Belitz & Grosch, 1999); thus they may contribute to the flavour degradation of bouillons. A clear effect of packaging atmosphere was observed on the formation of acetic acid, propionic acid, butanoic and hexanoic acids, and Maillard reaction products (Fig. 4D–G, respectively). In contrast, the relative concentrations of methyl butanals (Fig. 4H) were unaffected both by palm oil quality and packaging atmosphere and increased in all bouillon pastes during the storage period. The large difference in relative concentrations of acetic acid between MAP and NP bouillon pastes at 4 weeks of storage may be a result of the MAP procedure, as the samples were evacuated before the new atmosphere was introduced. This evacuation likely removed some of the volatile compounds initially present in the samples

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Fig. 4. Relative concentrations of volatiles in vegetable bouillon pastes during storage at 40 °C determined by SPME-GC–MS. (A) Branched alkanes and alkenes, (B) aromatic compounds, (C) Secondary lipid oxidation products, (D) butanoic and hexanoic acid (E) acetic acid, (F) propionic acid, (G) Maillard reaction products, (H) methyl butanals, (I) terpene compounds. The volatiles in each group are presented in Table 1. N = FO-NP, j = HO-NP, M = FO-MAP, h = HO-MAP.

(Andersson & Lingnert, 1998a). A similar tendency was observed in the case of other short aliphatic acids (Fig. 4D and F), and terpene compounds (Fig. 4I). In contrast, the concentrations of secondary lipid oxidation volatiles (Fig. 4C) and methyl butanals (Fig. 4H) were unaffected. As the effect of MAP on the volatiles of fresh bouillon pastes was not separately evaluated, the increases and/ or decreases in the volatile concentrations between 4 and 12 weeks of storage were further calculated (Table 4). These results

Table 4 Changes (%) in the relative concentrations of volatiles in vegetable bouillon pastes between 4 and 12 weeks of storage. Superscripts after volatile group refer to Fig. 4 and Table 1.

Lipid oxidation (C) Butanoic and hexanoic acid (D) Acetic acid (E) Propionic acid (F) Maillard reaction products (G) Methyl butanals (H) Terpene compounds (I)

FO-NP

HO-NP

FO-MAP

HO-MAP

3 24 28 30 210 37 4

11 37 32 36 299 40 23

0 7 43 29 50 39 14

6 8 44 29 44 38 14

indicated that methyl butanals were affected neither by the oil quality or the packaging atmosphere; lipid oxidation was affected by the oil quality, and the other volatiles were affected by packaging atmosphere. It should be noted that the concentration of acetic acid in all studied bouillon pastes was high throughout the storage period. Due to the large size of the acetic acid peak in GC–MS chromatograms, it was not possible to integrate the acetic acid peak very precisely. In addition, a small peak of acetic acid was observed in the control analysis (empty vial). For these reasons the results for acetic acid should be considered as tentative. The formation of acetic acid and propionic acid in NP bouillon pastes may have resulted from the Maillard reaction, particularly after sugar fragmentation (Davídek, Robert, Devaud, Vera, & Blank, 2006; Nursten, 2005). The sugar fragmentation may involve oxidative reactions (Nursten, 2005). This can explain the higher formation of propionic acid in NP-stored bouillon pastes than in MAP-stored bouillon pastes. In all bouillon pastes, formation of dihydro-2-methylfuranone and 2,5-dimethylpyrazine was observed during storage. The NP enhanced the formation of these compounds, and again the highest concentrations after 12 weeks of storage was observed in

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HO-NP bouillon paste, as was also found for propionic acid. Furanones and pyrazines are typical Maillard reaction products. The pyrazine formation may involve Strecker degradation of amino acids by dicarbonyls, and may further proceed through oxidation of dihydropyrazines to corresponding methylpyrazines (Mottram, 1994). This may be one explanation of why this compound was formed to a greater extent in NP bouillon pastes than in MAP bouillon pastes. The results of analysis of volatiles related to Maillard reactions supported the more intense browning of NP bouillon pastes during storage and confirmed the importance of Maillard reactions for the storage stability of vegetable bouillon paste. The results of analysis of 2- and 3-methyl butanals in bouillon pastes were surprising, as no differences in methyl butanal concentrations between bouillon pastes were observed during the storage period (Fig. 4H). These compounds are regarded as Strecker aldehydes and are formed from amino acids by degradation by dicarbonyl compounds. In Maillard reactions, sugar fragmentation may lead to the formation of dicarbonyls, along with the formation of short chain carboxylic acids (Nursten, 2005). However, in the present study, the formation of propionic and acetic acids was more enhanced in NP bouillon pastes, while methyl butanals were formed to a similar degree in all bouillon pastes. This indicated that, in NP bouillon pastes, competitive reactions for methyl butanal formation were present for the compounds formed, along with short chain carboxylic acids. Andersson and Lingnert (1997) observed a correlation between a high formation of hexanal and a high formation of 3-methyl butanal in cream powder. This was suggested to be due to the participation of hexanal in the Maillard reaction, leading to the formation of dicarbonyls, which further reacted with amino acids, forming methyl butanals in Strecker degradation reactions. In general, the interactions between oxidising lipids and amino acids/ proteins are well documented (Adams et al., 2008; Hidalgo & Zamora, 1993; Karel, Schaich, & Roy, 1975; Schaich, 2008; Schaich & Karel, 1976; Yong & Karel, 1978). The use of slightly oxidised palm oil (HO) in the preparation of vegetable bouillon enhanced the formation of short chain carboxylic acids, Maillard reaction products, and discolouration in the NP bouillon pastes. This is strong evidence of interrelations of lipid oxidation and protein degradation in vegetable bouillon paste, although the methyl butanal formation was not affected by the oil quality. The adverse effect of HO on bouillon stability (regarding lipid oxidation, Maillard reactions and interrelations between lipid oxidation and protein degradation reactions) was not as pronounced in MAP as it was in NP. Thus, greater stability of bouillon was obtained by using the MAP. The use of HO was clearly found to be detrimental on the stability of vegetable bouillon when stored in NP. Similarly, in the studies by Let et al. (2003, 2005) the quality of fish oil was found to be important for the oxidative stability of fish oil-milk emulsions. In comparison to fish oil, palm oil is considered to be of high stability (Matthäus, 2007). Still, even the small changes in the oxidative status of palm oil were enough to cause adverse effects on the stability of vegetable bouillon paste. Furthermore, the oil quality clearly affected non-enzymatic browning in vegetable bouillon paste when oxygen was freely available in the packaging.

4. Conclusions The storage stability of vegetable bouillon paste, prepared by mixing of preproduced dry ingredients with addition of semisolid palm oil, was studied. Not much is known about the mechanisms leading to quality degradation in such dry food products. In the present study, the effects of palm oil quality and packaging method on the stability of vegetable bouillon paste were evaluated. The

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