Quality and storage-stability of high-pressure preserved green beans

Journal of Food Engineering 54 (2002) 27–33 www.elsevier.com/locate/jfoodeng

Quality and storage-stability of high-pressure preserved green beans B. Krebbers *, A.M. Matser, M. Koets, R.W. Van den Berg Agrotechnological Research Institute (ATO), P.O. Box 17, 6700 AA, Wageningen, Netherlands Received 16 March 2001; accepted 23 September 2001

Abstract The effects of high-pressure technology on naturally present microbial flora, texture, color, ascorbic acid content and peroxidase activity of whole green beans were evaluated and compared to conventional preservation techniques. High-pressure processing (HPP) and two-pulse pressure treatment (pHPP) for achieving, respectively, pasteurization and sterilization on the product quality of intact green beans were under investigation. After 1-month storage there was no significant outgrowth of microorganisms for both HPP and pHPP. Both HPP and pHPP treatment showed a large retention of firmness and ascorbic acid for green beans compared to conventional preservation methods. pHPP resulted in marked changes in greenness. During storage after pressurization or conventional processing significant changes in ascorbic acid and color occurred. pHPP resulted in more than 99% inactivation of peroxidase, whereas after HPP 76% of the initial peroxidase activity remained and further decreased during storage. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: High pressure; Conventional processing; Green bean; Quality; Microbiology; Texture; Ascorbic acid; Color; Storage

1. Introduction Food processing under high pressure (HP) has been recently introduced as a mild alternative for some conventional pasteurization processes, e.g., the production of guacamole in the US and ham in Europe. Pressures lower than 600 MPa at ambient temperatures are industrially feasible conditions to inactivate food-spoiling micro-organisms and most detrimental enzymes, while minimally affecting sensory and nutritional quality (Smelt, Wouters, & Rijke, 1998; Yen & Lin, 1996). Since bacterial spores and some enzymes are extremely pressure resistant (Knorr et al., 1992), HP treatments may be more effective when combined with other processing variables, such as heating. Meyer (2000) has patented a HP process based on a two-pulsed pressure treatment at a minimum temperature of 70 °C aimed to sterilize foods, without adversely affecting flavors and minimally changing texture and color. Conventional processing, such as sterilization involves extensive thermal treatment caused by slow heat penetration to the core of the product and subsequent slow cooling. This thermal process induces quality *

Corresponding author. Tel.: +31-317-477566; fax: +31-317475347. E-mail address: [email protected] (B. Krebbers).

changes, such as off-flavor formation, texture softening and destruction of colors and vitamins. High-pressure techniques create new opportunities for replacing these conventional processes, while maintaining safety and quality. Effects of high pressure are generally marked as retention of color, flavor and fresh appearance as a result of the mild processing character (Cheftel, 1992). However, in real foods high pressure may cause several adverse biochemical changes, such as activation of enzymes and undesired browning reactions (Hendrickx, Ludikhuyze, Van den Broeck, & Weemaes, 1998; Matser, Knott, Teunissen, & Bartels, 2000). An array of food components, such as vitamins, fats or proteins may enter pressure-dependent chemical equilibria (Cheftel, 1992). Pressure conditions applied to obtain a microbial stable product may be insufficient to maintain preservation of color, texture or vitamins. In particular ascorbic acid (AA) is considered to be highly sensitive to losses during processing and storage, and is often used as a marker for product quality deterioration (Davey et al., 2000). Research on the influence of pressure on textural attributes of fruits and vegetables revealed that the products become softer (Eshtiaghi & Knorr, 1996; Knorr et al., 1992). However, most of those studies did not compare quality attributes to conventional processes. Matser et al. (2000) concluded

0260-8774/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 1 ) 0 0 1 8 2 - 0

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that pressurization resulted in firmer mushrooms, compared to the blanched product. In addition to the initial effect of high pressure, more knowledge of the storage stability of pressure-treated whole products is essential with respect to microbiological shelf life, texture, color and nutrients, as research in this area is scarce (Palou et al., 2000; Sohn & Lee, 1998). Color and flavor deterioration of pressure-processed foods during storage has been attributed to residual enzyme activity (Cano, Hern andez, & De Ancos, 1997). During storage, enzymes such as lipoxygenase and peroxidase can induce negative changes in color and flavor of vegetables. Therefore, blanching is a commonly applied technique before freezing or sterilization. Peroxidase is a heat stable enzyme (Guenes & Bayindirli, 1993) as well as a pressure stable enzyme in green beans (Knorr, 1995). The aim of the present study was to investigate the effects of HP on the product quality and storage stability of whole green beans compared to conventional preservation techniques. We studied the effect of HPP at pasteurization conditions (HPP, 500 MPa, ambient temperature) and sterilization conditions as described by Meyer (pulsed high-pressure processing, pHPP); twopulse treatment at a minimum temperature of 70 °C (Meyer, 2000). The effects of these preservation technologies on natural present flora, texture, color, AA content and peroxidase activity of green beans were evaluated, also after storage.

2. Materials and methods 2.1. Plant material Green beans cvs. Amstel, were cultivated in Netherlands and purchased at a local auction. Green beans were processed a maximum of two days after purchase. Beans were stored at 6 °C and before further processing the ends of the pods were removed. 2.2. Processing conditions High-pressure treatments: Green beans were washed in tap water and vacuum-packed in polyethylene bags (10 cm
8 cm). High pressure treatments were performed in a Resato high pressure apparatus (volume 180 ml, maximum pressure 1000 MPa, maximum initial temperature 100 °C, Resato, Roden, Netherlands) at several pressure–temperature combinations. Pressure buildup rate was 10–15 MPa/s. After treatment, the samples were transferred to ice water. High-pressure treatment (HPP) was done at ambient temperature for 60 s at 500 MPa. Pulsed high-pressure treatment (pHPP) was done according to the Meyer patent (Meyer, 2000). The samples were preheated at 75 °C for 2 min, trans-

ferred to the high-pressure apparatus and processed at 75 °C, holding time 80 s at 1000 MPa, with a second pressure pulse of 1000 MPa after 30 s at 0.1 MPa. Due to adiabatic compression, the maximum temperature at the first pulse in the vessel for HPP and pHPP were, respectively, 45 °C and 105 °C. Conventional treatments: Green beans were blanched for 4 min at 90 °C. The succeeding canning process (CS) was conducted following a commercialized process. Portions (360 g) of beans were packed into glass jars (720 ml) and a 1% NaCl and glucose solution was added. Closed jars were sterilized at 118 °C for 30 min. For the freezing process (BF), beans were frozen at )20 °C after blanching under forced air in 10 min. For firmness and color measurements, beans were defrosted by microwave (4 min 180 W, 2450 MHz). High-pressure treatment (HPP), pulsed high-pressure treatment (pHPP), conventional sterilization (CS) and freezing process (BF) were performed in duplicate. After different processing treatments, the samples were either used for firmness and color measurements or frozen in liquid nitrogen and kept at )50 °C until biochemical analyses. Processed samples were also stored for 1 month at 20 °C for CS and pHPP or at 6 °C for raw (R) or HPP green beans. 2.3. Analysis 2.3.1. Firmness measurement The firmness of raw and processed beans was measured on 15 beans using a Texture Analyzer TA-XT2i (Stable Micro Systems, Godalming, UK) equipped with a Warner–Bratzler Blade. A green bean was placed on two parallel bars with a gap of 10 mm between them. The bean was fractured by a downward motion (10 mm/ min) of a steel blade with a thickness of 3 mm. The maximum force (top value in N) applied to break the beans was used to quantify the firmness. 2.3.2. Color measurement The color of the beans was calculated from 15 measurements with a Minolta Chroma meter within 10 min after opening of the jars or the pressurized bags. The L ; a ; b color space was used for determination of the color, with L representing the lightness, þa the red direction, a the green direction, þb the yellow direction, and b the blue direction. 2.3.3. Microbial determinations Reductions in the natural flora were investigated in whole green beans, processed under different conditions. Samples were aseptically transferred to a plastic bag, diluted three times in peptone physiological salt solution (PPS; 8.5 g/l NaCl and 1 g/l bacteriological peptone (Oxoid)) and homogenized by manually macerating for 1 min. Serial dilutions were made in PPS and plate count

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agar (PCA, Oxoid) plates were poured. Part of each sample was heated for 10 min at 70 °C in a water bath, to inactivate vegetative cells. Serial dilutions were made in PPS and PCA plates were poured. The plates were incubated in duplicate aerobically for 2 days at 30 °C. The confidence threshold was established as less than 10 CFU detected on the plate of the lowest applied dilution. 2.3.4. Ascorbic acid analysis Frozen green bean samples were ground in a DitoSama K55 mixer under continuous nitrogen supply. Further extraction was performed in line with Veltman, Sanders, Persijn, Peppelenbos, and Oosterhaven (1999). Total AA and dehydroascorbic acid content were measured using a Waters (Milford, USA) chromatograph model 510 with a Waters 486 UV–VIS detector (251 nm) according to the procedure described by Keijbets and Ebbenhorst-Seller (1990). A symmetry C-18 column (3:9
150 mm2 , particle size 5 lm, Waters), with a Sentry Guard column C-18 (Waters) was employed at 25 °C. 2.3.5. Peroxidase (POD) analysis Frozen green bean samples were ground in a DITOSAMA K55 mixer under continuous nitrogen supply. The ground samples were homogenized with a blender at 4 °C, five times for 10 s, at 10 s intervals, in 0.1 M Naphosphate buffer pH 7 (1:2 w/w), containing 2% (w/v) NaCl and 2% (w/v) glucose. The homogenate was filtered through two layers of cheesecloth. The filtrate was centrifuged for 10 min at 11,000 g, 4 °C. The supernatant was used for the enzyme analysis. POD activity was determined spectrophotometrically based on a method described by Childs and Bardsley (1975), using 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a coloring reagent. The increase in absorbance at 414 nm as a function of time was monitored using a Perkin Elmer Lambda 16 UV–VIS spectrophotometer. Measurements were performed in duplicate. 2.3.6. Statistical analysis Data sets were subjected to analysis of variance (ANOVA) to determine least significant differences (lsd) among processing treatments (p < 0:05).

Fig. 1. Typical temperature–pressure profile of pHPP, measured in the HP vessel.

In Table 1 results are shown for the counts of naturally present flora, vegetative cells and spores, of the raw, sterilized and pressure-treated green beans. Whereas complete inactivation of viable cells was found for conventional sterilization, in some cases a few colony forming units remained after HP treatment. It is not yet clear if these are survivors or due to minor contamination during handling. HPP green beans (500 MPa, 20 °C) resulted in complete inactivation of vegetative cells, i.e., a reduction of 4 log cycles, resembling a pasteurization treatment. In general total plate counts can be reduced 4–5 log cycles under these conditions for purees and juices. Guava puree appeared microbial safe for at least 60 days during cold storage (Yen & Lin, 1996). pHPP showed a reduction of spores and vegetative cells below the detectable levels, i.e., a reduction in viable cell number of at least 6 log cycles for the natural flora. These results confirmed the Meyer patent (Meyer, 2000), claiming a commercial sterilization (>6 log reduction of C. botulinum) of vegetable products under the applied conditions, while retaining substantially uncooked flavor and texture. Throughout storage at, respectively, 6 and 20 °C for HPP and pHPP green beans no significant outgrowth of Table 1 Number of natural flora (log CFU/g product) compared to raw green beans

3. Results and discussion 3.1. Effect on microbial reduction Pulsed high-pressure treatment (pHPP; 2 pulses of 1000 MPa, 75 °C) according to Meyer was applied and resulted in a temperature–pressure profile presented in Fig. 1.

Raw HPP (500 MPa, 20 °C) Conventional sterilization pHPP (2
1000 MPa, 75 °C)

Viable count

Spore count

6.1 1.7 <1.6a <1.6b

3.5 1.7 <1.6 <1.6c

a 1.6 log CFU/g product ¼ detection level (<10 CFU/plate in the lowest dilution). b 1 CFU on 1 plate out of 6 plates. c 6 CFU on 1 plate out of 6 plates.

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Table 2 The effect of high-pressure and conventional treatments on the average color L -, a -, b -values for green beans directly after processing

R BF HPP CS pHPP

L

a

b

41.5e 32.0bc 33.4cd 30.0ab 26.6a

)16.1bc )17.9a )16.9ab )1.2d )1.1e

22.1d 18.0bc 20.4d 17.2b 14.8a

Different superscripts (a, b, c, d, e) indicate significant differences between mean values of treatments (n ¼ 15; p < 0:05).

total mesophilic (an) aerobic count or spores was measured. The raw beans were spoiled after 1 week of storage at 6 °C. 3.2. Effect on color Color differences between conventionally and pressure-processed beans were determined directly after processing (L -, a - and b -values in Table 2) and during storage (a -value in Fig. 2). Table 2 shows that treatments did have a significant effect on the Hunter values. L -, a - and b -values of blanched and frozen beans were significantly lower than the green bean control, representing a more intense green color. This decrease was also measured for the HPP-beans, resembling the appearance of mildly heat-treated beans. It is well known that application of high pressures (100–800 MPa) causes permeabilization of plant and microbial cells (Dornenburg & Knorr, 1993; Prestamo & Arroyo, 1999). This will cause damage to the choloplasts, resulting in leakage of chlorophyll into the intercellular space. This phenomenon is probably the cause of the (initial) more intense bright green color on the surface of the HPP beans. During conventional sterilization the beans showed a clear increase in a -value to 1.2; a visible color change from green to olive-green. Similar changes were measured for pHPP treatment of green beans. These results indicate that HP in combination with higher tempera-

Fig. 2. Effects of storage after high-pressure and conventional treatments on a -value of green beans.

tures, even for a short time, can already be detrimental for greenness. Van Loey et al. (1998) studied the effects of pressure and temperature on chlorophyll degradation in a broccoli-extract. They have also found significant reductions of chlorophyll when pressure was combined with temperatures higher than 50 °C. The color loss is due to conversion of labile chlorophylls a and b to yellow-olive colored pheophytin. Storage significantly affected the a -value of raw, frozen (BF) and HPP-treated beans (Fig. 2). During storage the green contribution to the color (a ) of the raw and HPP-treated beans gradually decreased and turned into a pale yellow/green color. Raw green beans became spotted after 1-month storage, due to microbial spoilage. HPP-treated beans were more uniformly discolored compared to raw beans, but had an unacceptable appearance after 1-month storage at 6 °C. There was no additional change in color during storage for pHPP-treated beans and conventionally sterilized beans. Negative effects on the color after HPP treatment were probably caused by residual activity of enzymes such as lipoxygenase, peroxidase or chlorophyllase. Effects of storage on green color after pressurization have been studied, but only in purees and extracts of, e.g., guacamole (Palou et al., 2000; Sohn & Lee, 1998; Van Loey et al., 1998). They have also observed decreases in greenness during storage. 3.3. Effect on firmness In Fig. 3 the firmness of green beans as a result of different processing conditions and after 1-month storage is shown. The firmness of green beans decreased dramatically after conventional sterilization; only 3% of the original firmness of fresh beans remained. The most important softening process during sterilization is believed to be temperature-dependent b-elimination of pectin (Saijaanantakul, Van Buren, & Downing, 1989). Both pHPP and HPP resulted in a significantly higher firmness compared to the conventional steriliza-

Fig. 3. Effects of processing and storage (31 days) on the firmness of green beans.

B. Krebbers et al. / Journal of Food Engineering 54 (2002) 27–33

tion process, up to 60% of the original firmness of the raw beans. This relative large retention of firmness after pressurization may partly be due to the lower temperature and shorter time to which the beans have been exposed, resulting in less b-elimination of pectin. Besides the milder conditions applied, the larger retention of the firmness may also be attributed to activation or retention of pectin methylesterase (PME) activity. Stute, Eshtiaghi, Boguslawski, and Knorr (1996) and Kasai, Okamoto, Hatae, and Shimada (1997) also hypothesized that the improved texture properties of vegetables after pressurization could be due to the enzymatic demethylation of pectins, followed by the formation of calciumpectate complexes. Results were comparable to Quaglia, Gravina, Paperi, and Paoletti (1996) in green peas, who also found that pressure level, treatment time and temperature did not have any major additional softening effect. Basak and Ramaswamy (1998) showed a loss in texture, as a result of HP (100–400 MPa) for some vegetables up to 67%, ascribed to expeditious action of pressure. They have found a recovery of firmness to the initial level during long pressure holding time (60 min), also ascribed to the firming action of PME. Blanching, freezing and subsequent defrosting by microwave also appeared detrimental for firmness as less than 10% of the initial firmness remained. In Fig. 3 storage results show that pressure-treated beans retained their firmness, independent of storage temperature. Conventionally treated beans remained relatively soft, whereas raw beans lost their firmness. This decrease may be a result of breakdown of pectins or due to microbial contamination. 3.4. Effect on AA content and peroxidase activity Table 3 summarizes the effect of pressurization, freezing, sterilization and storage on the AA content. Losses in AA were largest after CS (up to 90%). HPP did not significantly affect AA content compared to raw beans. In previous studies, HP treatments of purees and juices at ambient or low temperatures also appeared to have little effect on the AA content (Ogawa, Fukuhisa,

Table 3 Effects of high pressure and conventional treatments on the changes in the average ascorbic acid content for green beans during storage (in% of the initial content of the fresh material)

R BF CS HPP pHPP

Initial

7 days

31 days

100  7.2 79.4  7.6 9.5  8.5 92.1  1.2 75.8  8.4

75:4  7:2 79:4  9:1 n.d. n.d. < 0:01

n.d. 79:2  9:8 < 0:01 < 0:01 < 0:01

n.d. ¼ not determined.

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Kubo, & Fukumoto, 1990; Sancho et al., 1999; Yen & Lin, 1996). AA retention of green beans after pHPP treatment was 76%; this decrease was probably due to (short) exposure to heat and pressure. This was also derived by Van den Broeck, Ludikhuyze, Weemaes, Van Loey, and Hendrickx (1998), who showed that pressure and temperature acted synergistically in tomato and orange juice. Several publications implicate a matrix dependent effect of pressure on vitamin C breakdown, influencing susceptibility to vitamin C breakdown (Taoukis et al., 1998; Van den Broeck et al., 1998). BF resulted in an equal loss of AA as in pHPP-treated beans. Only relatively few data are available on vitamin C effects by pressurization on whole intact pressure-treated foods (Quaglia et al., 1996; Sancho et al., 1999). Quaglia et al. (1996) have found that an increase in pressure up to 900 MPa resulted in a higher level of retained AA. Storage of green beans resulted in major decreases of AA to negligible contents in HPP after 1 month. Equivalent treatments showed similar trends in AA breakdown. Sancho et al. (1999) also showed that decreases in AA content of untreated and pressurized (400 MPa) strawberry pulp during 1-month cold storage were similar. Yen and Lin (1996) reported that after highpressure treatment (600 MPa) the AA decline during storage was less explicit than in pasteurized (90 °C) avocado puree. However, in these references the concentration of AA was higher than observed in our experiments. This may be due to, besides differences in product and stability of endogenous enzymes, higher residual tissue oxygen in whole products compared to purees or juices. Only frozen storage resulted in retention of AA. Ambient storage of pHPP showed, similar to its equivalent CS, only traces of AA. In earlier literature (Cain, 1967; Sistrunk, 1980) decreases of AA after canning of vegetables were not that explicit; storage at 18 °C did not result in any major additional reduction (10–20%) of vitamin C over two years. The main reason for this difference is that in our work and over the last decade more advanced and accurate chromatographic methods have been used for determination of AA (Veltman et al., 1999). Other reducing compounds, formed during storage after intensive processing, may interfere with the spectro-photometric method used in the past, resulting in a over-estimation of vitamin C contents (Davey et al., 2000). In general differences in rates of breakdown may be explained by several factors, such as storage temperature, residual activity of oxidative enzymes (such as peroxidase and ascorbate oxidase), oxygen level in the tissue, presence of light, pH, water activity and also available catalysts, e.g., metal ions. HPP showed residual activity of POD (75%), even after 1-month storage (Table 4). The observed AA

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Table 4 Residual activity (% of activity in raw beans) of POD in green beans after different treatments and storage Initial R BF CS HPP pHPP

a

100 <0.1d <0.1d 75b <0.1d

7 days b

69.2 <0.1d <0.1d n.d. 0.9d

31 days 27.2c <0.1d <0.1d 29.5c 1.0d

n.d. ¼ not determined. Different superscripts (a, b, c, d) indicate significant differences between mean values of treatments (n ¼ 2
2; p < 0:05).

breakdown was a result of enzymatic degradation, probably coinciding with chemical degradation. Kinetic studies should distinguish between the quantitative importance of chemical or enzymatic contribution. POD appeared to be baro-resistant in several fruits and vegetables compared to other endogenous enzymes, even at elevated temperatures (60 °C) (Lemos, Oliveira, Van Loey, & Hendrickx, 1999; Rastogi, Eshtiaghi, & Knorr, 1999), probably due to its small molecular size (35 kDa). After processing and 1-month storage of pHPP beans, no considerable activity of POD could be detected (<1%). Therefore, AA breakdown after pHPP was mainly a result of chemical breakdown, due to damaged cell membranes and subsequent increased diffusion and reaction rate of substrates. More research is required to explore the mechanisms involved in AA breakdown.

4. Conclusions After HPP treatment, pasteurization of green beans was achieved. HPP green beans showed retention of color, firmness and AA compared to raw beans. Whereas HPP showed considerable residual POD activity, conventional treatments and pHPP resulted in inactivation. No measurable regeneration of POD occurred during storage. pHPP showed similar reductions of vegetative cells and spores as in heat-sterilized green beans. Firmness and AA levels were much higher in pHPP than in the equivalent conventional sterilization, whereas green color losses were similar. Microbial shelf life of HPP and pHPP could be extended for at least 1 month at, respectively, 6 and 20 °C. Both HPP and pHPP have potential to substitute conventional preservation techniques, such as blanching, pasteurization, sterilization or freezing of green beans, with improved textural, nutritional properties and presentation without added processing water. Firmness was retained after HPP and pHPP treatment during storage. Results showed that shelf-life studies were indispensable in proving the merit of HP technology, especially with regard to green color and nutritional

attributes. These characteristics showed deterioration after storage of HPP and pHPP green beans, similar to raw and conventional-sterilized green beans, respectively.

Acknowledgements This study is part of a project between ATO B.V., Stork Food and Dairy Systems and Unilever Vlaardingen, which is financially supported by the program ‘EET’ of the Dutch Ministry of Economic Affairs. For experimental support and stimulating discussions we would like to thank S. Hoogerwerf, R. Meyer, R. Moezelaar, E. Schijvens and F. Van Den Wall.

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