J Sci Food Agric 1996,70,224-230

Modified Atmosphere Packaging of Fresh Beansprouts Patrick Varoquaux,* Guy Albagnac, Christophe Nguyen The and Franqoise Varoquaux Institut National de la Recherche Agronomique, Stat ion de Technologie des Produits Vegetaux, Domaine St Paul Agroparc, BP 1200,84911 Avignon cedex 9, France (Received 10 October 1994; revised version received 15 June 1995; accepted 18 August 1995)

Abstract: Freshly harvested beansprouts displayed a respiration rate of about 1 mmol 0, kg- ' h-' at 10°C which was strongly dependent on temperature, a 10-fold increase being observed every 16'5°C (z = 16.5"C, ie Q,,, = 4-4). This commodity is also characterised by a high initial microbial load (about lo7 cells g-I). During storage at various temperatures from 1 to 20T, oxygen uptake rates dramatically increased with time and this phenomenon was well correlated with the development of aerobic microorganisms which reached lo9 cells g-' after 2 days at 20°C or 9 days at 1°C. Beansprouts were packaged in films, with permeabilities ranging from 950 to 200000 ml 0, m-2 day-' atm-', and stored at 8°C. Due to plant and microbial metabolism, oxygen concentrations decreased steadily within all packs until the onset of plant tissue decay. The latter occurred after 5-6 days with the least permeable films but did not occur within when the film permeability was over 100000 ml 0, m-' day-' atm-I. However, such films favoured brown discolouration, exudation texture and breakdown. The orientated polypropylene film (OPP) induced anoxic condition within 2 days and favoured anaerobic metabolism and necrosis of the sprouts. In all packages there was a rapid development of aerobic microorganisms and lactic acid bacteria that resulted in the.accumulation of acetate and lactate and a decrease in pH. Thus, it clearly appeared that tissue decay was enhanced by microbial activity. At 8"C, 0.24 m2 of film per kg of sprouts provided the optimal atmosphere composition (ie 5% oxygen and 15% carbon dioxide) when a film permeability of 50000 ml 0, m-' day-' atm-' was used. These conditions allowed a shelf-life of 4-5 days.

Key words : storage, respiration rate, microbial spoilage, quality, Vigna radiata.

INTRODUCTION

Modified atmosphere packaging (MAP) combined with chilled storage offers potential benefits for extending the shelf-life of fruit and vegetables by reducing microbial spoilage and some undesirable enzymatic activities, eg those responsible for enzymatic browning. In France, this technique has been extensively developed for mini- mally processed fresh salads (Varoquaux 199 1). When MAP is applied to living foods, a depletion of 0, and an accumulation of CO, occur within the pack. This is due to plant tissue respiration and limited film per- meability. A suitable equilibrated atmosphere, whose

* To whom correspondence should be addressed.

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

composition mainly depends on product respiration rate, temperature, fill weight, film surface and film per- meability, has to be defined for each commodity. For example, the shelf-life of shredded lettuce in polyethyl- ene packs filled with a 5% C0,-5% 0, mixture is double that of shredded lettuce in air (Ballantyne et a1 1988a). Using films of a too low permeability results in anaerobiosis and a high CO, content which, in turn, initiates anaerobic metabolism and the development of off-flavours (Lipton et al 1967; Ballantyne et al 1988b). In some cases, such 0,-deprived and C0,-enriched atmospheres may also favour the development of lactic acid bacteria, eg Leuconostoc, which are the prevailing microorganisms associated with the spoilage of grated carrots packaged in low permeability polypropylene

224

Modified atmosphere packaging of fresh beansprouts 225

films (Carlin et a1 1990a). However, literature concern- ing the storage and packaging of beansprouts is scarce. The main storage problem arises from high initial microbial loads, though guidelines for ensuring and checking microbial quality have been proposed by Brown and Oscroft (1989). Deterioration, as well as pro- duction of C 0 2 and ethylene during storage have been studied by Lipton et a1 (1981) who reported a limit of saleability of 8-5-2.5 days for storage temperatures ranging from 0 to 10°C, respectively. Beansprouts display a high respiration rate and have to be packaged in microperforated or microporous films (Day 1990). The purpose of this work was to study the influence of temperature and of modified atmosphere on physiologi- cal, microbial and organoleptic changes in beansprouts.

MATERIALS AND METHODS

Mung beans (Vigna radiata, unknown cultivar) were sprouted at 20°C and 95-100 RH in a commercial farm at Marsanne (France). Immediately after harvesting, most radicles were mechanically removed and sprouts were cooled to 4°C under sprayed water. They were transported within 2 h to the laboratory.

Measurement of apparent respiration rates

Changes with storage duration under normal and temperature Beansprouts (380 L- 1 g) were poured into 1.5 litre glass jars equilibrated at the required temperature and stored in controlled temperature rooms at 1, 5, 8, 12 or 20°C for up to 9 days. Five sets of seven jars containing bean- sprouts were left open in the cold rooms. At time inter- vals, three jars per temperature were closed and gas samples (250 pl) were taken after 1, 3 and 5 h with an air-tight syringe through a silicone septum set with sili- cone glue in the jar lid and analysed by gas chromatog- raphy. Rubber gaskets and silicone septa on the jars were changed after each experiment to prevent any air leak. Apparent respiration rates (appRR) were calcu- lated by linear regression from 0, depletion curves and expressed as mmol O2 h-' kg-'. Mean values and standard deviations were measured on the three repli- cations. After the determinations, two jars for each tem- perature were opened and recycled, the last one was used for microbial counts.

Changes in appRR measured under normal air as a function ofJlm permeability and storage duration After atmosphere determinations, two packs per film permeabilities were used to measure the respiration rate of the MA packed beansprouts under normal air at 8°C. The duplicated determinations in air-tight glass jars were performed as described above.

Modified atmosphere packaging

Six films made of 35-pm-thick orientated polypropylene (OPP) were supplied by Sidlaw Packaging (Avon, UK). Film characteristics are summarised in Table 1. Pouches with an effective surface of 0-12 m2 were filled with 500 f 1 g beansprouts and stored at 8°C for up to 12 days. At time intervals gas composition was mea- sured in three packs, sampling 0-2 ml of the head space through a septum of alveolated rubber foam tightly stuck on the surface of the film. One pack per film per- meability was used for microbial and physicochemical determinations, the others were used to measure the apparent respiration rates of beansprouts under normal air at 8°C.

Gas analyses

Gas analyses were carried out by gas chromatography as previously described by Lopez-Briones et a1 (1993). The 0, and C 0 2 contents of the packs were expressed as a percentage of the total air volume. Argon was not separated from O2 resulting in a 0.8% absolute error on 0, measurements.

Microbial analyses

Three samples of 50 & 0.1 g sprouts were taken from the same jar or pouch, homogenised in 100 ml sterile water for 1 min in a Stomacher laboratory blender (Seward Lab, London, UK) and then filtered through a cloth to remove cell debris. The supernatant was serially diluted and aliquots were spread using the Spiral tech- nique (Model DS, Interscience, Saint Nom la Breteche, France). Total aerobic counts were carried out on tryp- tose agar medium incubated for 24 h at 30°C. Total counts of lactic acid bacteria (LAB) were made on MRS medium (De Man et a1 1960) with bromocresol-green (Carlin et a1 1990b). Incubations were carried out at

TABLE 1 Gas permeability of the films used throughout this study (values were calculated from the microperforation density and the permeability of the polymeric matrix; data supplied by the

manufacturer and verified by Lopez-Briones et al 1993)

Film reference Gas permeability supplier's code (ml m-' day-' atm-' at 25°C)

Oxygen Carbon dioxide

OPP 900 4 000 P + loo00 11000 14000 P i- 25000 26 000 29 000 P + 50000 51 000 54 000 P + 100000 101 000 104 000 P + 200000 201 000 204 000

226 P Varoquaux et a1

30°C in anaerobic jars under hypoxia (about 10% residual 0,) created with a candle. The mean values of the three microbial counts were expressed as colony forming units (CFU) per g of fresh weight.

Biochemical analyses

Prior to analyses, 50 f 0-1 g sprouts were mixed with 50 ml distilled water in a Vorwerk mixer for 1 min at maximum speed, filtered over glass wool and stored frozen until analysed. Sugars (mono and disaccharides), organic acids and ethanol in the liquid phase were analysed on HPLC (Varian model, type 5000 fitted with a differential refractometer and a UV monitoring unit set at 210 nm). The ION 300 column (Biorad) was ther- mostated at 65°C and eluted with 0.05 M H,S04 at a flow rate of 0.4 ml min-'. Samples were filtered through 0.2 pm syringe filters (Dynaguard; Microgon Inc, Laguna Hills, CA, USA) and 10 pl of filtrate were injected.

Physical properties of the beansprouts

Assessment of the hypocotyl colour was performed using a Hunterlab 'Colorquestsphere' colorimeter (Hunter Associates Laboratories Inc, Reston, VA, USA) fitted with a specimen port of 2.5 cm in diameter and standardised with a white tile. Results were calculated with D65 illuminant and an observation angle of 0" in the L*a*b* mode of CIE. External luminance was mea- sured at five points on the surface of the sprout-covered support.

Firmness was measured with an Instron Universal Testing Machine, model TMSML (Instron Limited, High Wycombe, Bucks, UK). Thirty grammes of sprouts were compressed in an extrusion cell previously described by Varoquaux and Nguyen The (1994) at 50 mm min-' with a 500 dN load cell. Firmness in Newtons (N) was assessed at the point of biofailure.

RESULTS AND DISCUSSION

Physiological and microbial changes upon storage at different temperatures

Standard deviations calculated from the three determi- nations of appRR represented 5-15% of the corre- sponding mean values. Similarly, standard deviations for total aerobic counts varied from 6 to 13% of mean values. These ratios must be kept in mind since numer- ous superpositions prevented the report of error bars in Fig l(a) and (b).

After a lag period of 6 days, appRR at 1°C increased from 0.14 0.04 to 1.9 & 0.2 mmol kg-' day-' in 4 days. In all other cases appRR increased, reached a maximum and then decreased (Fig la). Total aerobic

10 , l a

0 1 2 3 4 5 6 7 8 9 10

lo . I

0 1 2 3 4 5 6 7 8 9 10

Storage duration (days)

Fig 1. Changes in (a) oxygen uptake rates, and (b) total aerobic counts during storage of beansprouts at various temperatures. Results are expressed as mmol h- ' kg-' and

CFU g- ' of fresh weight, respectively.

counts increased from 3.0 & 0.1 x lo7 to final values above lo9 CFU g-' with doubling times of 8 h (20°C) to 18 h (5°C). A lag period of 6 days was observed at 1°C and for all temperatures growth of microorganisms was synchroneous with the increase in appRR (Fig lb). Little or no development of lactic acid bacteria occurred at 1"C, but a rapid growth was observed without any lag at higher temperatures (results not presented). Apparent doubling times ranged from 5 h (ZOOC) to 12 h (8°C). This particular behaviour strongly suggested that the increase in appRR was mainly due to a substantial growth of microorganisms and that the subsequent decrease corresponded to a progressive death of plant tissues and a stabilization of microbial development.

Limits of lo7 aerobic microorganisms and lo6 coli- forms g-' of product immediately after packing were suggested as a criterion for acceptance of fresh bean- sprouts (Brown and Oscroft 1989). Samples used in this study appeared highly contaminated but Splittstoesser et a! (1983) reported aerobic plate counts on fresh bean- sprouts as high as 10' CFU g-'. These authors also found that washing beansprouts in water containing up

ModiJed atmosphere packaging of fresh beansprouts 227

to 120 pg litre-' of active chlorine was not efficient in reducing the initial number of microorganisms.

Within the range tested, the influence of temperature on appRR fitted Arrhenius' law. In addition, a good linear relation (Rz = 0.99) was observed between the decimal logarithm of appRR and the temperature in "C (results not shown). This allowed the calculation of the thermal coefficient ( z ) and Q , , from the slope of the line. A 10-fold increase in appRR was observed every 16.5"C ( z = 16.5"C), this corresponds to a Qlo of 4-4. Very similar values were reported for carbon dioxide pro- duction rates of beansprout under air (Lipton et a1 1981). This indicated a strong dependence of beansprout metabolism on temperature. Generally, metabolic pro- cesses increase two- to three-fold for a 10°C rise in tem- perature (Zagory and Kader 1988). For instance a Qlo of 2.8 was reported for respiration rate of shredded endive under normal air (Chambroy 1989). The experi- mental values obtained for beansprouts were sur- prisingly much higher (el, = 4.4). Due to the very high initial bacterial contamination (about 5 x lo7 CFU g - l), microorganisms may contribute significantly to the initial appRR. Thus, this discrepancy may be explained by an apparent Q l o value of microorganisms much higher than that of plant tissues. Since it is almost impossible to produce non-contaminated sprouts or to measure accurately the appRR of contaminating micro- organisms under real conditions, none of the experi- mental results support this assumption directly.

Storage under modified atmospheres

A set of 15 packs were prepared with each film. For each sampling date, ie after 2, 5 , 7, 9 and 12 days of storage at 8"C, three randomly chosen packs per film were analysed for atmosphere composition. Subse- quently, respiration rates, microbial counts, biochemical analysis and quality assessment were determined for the contents of each pack. Almost identical results were obtained with P + 100000 and 200000 and, therefore, data corresponding to the latter were omitted from the figures.

After sealing the packs, internal oxygen is consumed by the respiration of both plant tissues and micro- organisms with carbon dioxide production. The latter may also arise from fermentative metabolism which occurs under hypoxic or anoxic conditions. Changes in internal oxygen concentration as a function of the per- meability of films and storage time are shown in Fig 2. As expected, the lower the gas permeability of films, the higher the apparent rates of oxygen decrease within the packs. Usually, oxygen concentrations decrease to asymptotic limits corresponding to an equilibrium between the rates of oxygen consumption and diffusion through the film. As a first approximation, the recipro- cal of final concentrations should be proportional to the

b 0

- * - IK-B-~OK--A--~SK.-K- SOK-100K

6o 5

0 2 4 6 8 10 12 14

30 8 L

0 2 4 6 8 10 12 14

Storage duration (days)

Fig 2. Changes in surrounding atmosphere composition during storage at 8°C of beansprouts packaged in micro-

perforated polypropylene films.

reciprocal of the permeability of films (Lopez-Briones et al 1993). Results obtained with beansprouts did not fit this model since a steady state was not reached in any of the treatments.

When using unperforated OPP film, the maximal dif- fusion rate of oxygen was 23 ml per pack and per day which was far from fulfilling the requirements for the residual respiration of sprouts and associated micro- organisms (about 115 ml per pack per day). The small oxygen input was immediately consumed and apparent anoxia was obtained within 5 days. With very high per- meability films (P + 100000 and 200000) changes in oxygen concentration were very slow and linear (Fig 2). With films of intermediate permeability (P + 10000 to 50 000) oxygen concentration decreased to about 5% and then increased after 6-9 days. The decrease in oxygen consumption was probably associated with a progressive death of the plant tissues. This phenomenon did not occur when gas permeability exceeded lo5 ml 0, m-' day-' atm-' (P + 100000 and 200000). Mea- suring changes in appRR of the sprouts at 8"C, after restoring normal atmosphere, confirmed the progressive

228 P Varoquaux et a1

decrease in oxygen uptake rate after 5-9 days for bean- sprouts packed with films of low or medium per- meability (Fig 3). With P + 100000 and 200000 oxygen consumption rate increased to 3 L 0.25 mmol kg-' h-' during the first 9 days and then remained const ant.

Changes in carbon dioxide within packs (Fig 2) were almost mirrored by those of oxygen, a linear increase in carbon dioxide occurring with the most permeable films (P + 100000 and 200000). For films of intermediate permeability (P + 10000 to 50000) carbon dioxide increased to about 20% after 9 days of storage and then slightly decreased. In some cases (OPP, P + 10000) values far above 20% were reached indicating that the metabolism was mainly fermentative.

Development of microorganisms during storage under modified atmospheres

The increase in oxygen uptake was associated with the development of mesophilic aerobic microorganisms. After 9 days (Fig 4) total aerobic counts ranged from 1.2 f 0.1 to 7.8 f 0.6 x lo9 CFU g-' and were signifi- cantly higher when using very permeable films (P + 100000 and 200000). In addition, MAP had pro- nounced adverse effects on growth rates of the aerobic flora.

Doubling times of microorganisms (about 30 h) were two times higher than those found for beansprouts

stored under air at 8°C. A lag-time of about 4 days was observed with films of low permeability suggesting the development, after an adaptation period, of micro- organisms on fermentation products, ie lactate, acetate and ethanol.

Lactic acid bacteria (results not shown) increased from 3.4 f 0.2 x lo3 CFU g-' to about lo9 CFU g-' in 9 days. Final growth (1.2 f 0.1 x lo9 to 1-8 f 0-2 CFU g-') was not really affected by the permeability of films. Corresponding doubling times (1 1 h) were almost identical to the value calculated for lactic acid bacteria growing on the product stored in air at the same tem- perature. A Gram-positive and heterofermentative lactate producing bacterium was isolated and found to be prevalent. Based on its morphology and fermenta- tion pattern, it was tentatively identified as a Leuco- nostoc species. Leuconostoc mesenteroides is responsible for the spoilage of grated carrots packed in poly- propylene films (Carlin et al 1990a,b). However, it does not proliferate in grated carrots unless low 0, or high COz contents trigger an increase in exudation from plant tissues. Beansprouts appear to be a very unstable product since it favours the growth of Leuconostoc under any atmospheric conditions.

Analysis of beansprouts stored under modified atmospheres

During storage at 8°C the pH of beansprout homoge- nates remained very close to 5.5 for 5 days. A slight

- - 1K + 10K - A- - 25K *- 50K -1OOK

4

0 2 4 6 8 10 12 14

Storage duration (days)

Fig 3. Changes in respiration rate (assessed under normal air) of beansprouts packaged in microperforated polypropylene films and stored at 8°C. Results are expressed as mmol h-' kg-' of fresh weight.

Modijied atmosphere packaging of fresh beansprouts

- - 1K -1OK - A - 25K 4- 50K -1OOK

229

0 2 4 6 8 10 12 14

Storage duration (days)

Fig 4. Growth of aerobic mesophilic microorganisms on beansprouts packaged in microperforated polypropylene films and stored at 8°C. Results are expressed as CFU g-' of fresh weight.

decrease to 5-2 then occurred within 7 days with films of high permeability (P + 100000 and 200000). With lower permeability films, pH decreased to final values between 4.2 and 4.5 (results not shown).

The acidic pH was associated with the accumulation of lactate and to a lesser extent of acetate (Table 2). Significant amounts of ethanol (5-6 mmol kg-I fresh weight) were also present in homogenates and fermenta- tion balances obtained with P + films were consistent with the prevailing action of heterofermentative lactic acid bacteria. The very high amount of ethanol produc- ed when using OPP (40 mmol kg-' of fresh weight) clearly indicated a shift of beansprout metabolism to an ethanolic fermentation.

TABLE 2 Fermentation products in beansprout homogenates after 9 days of storage at 8°C and packed in films of various per-

meabilities

Film reference Concentration supplier's code (mmol kg - fresh weight)

Lactate Acetate Ethanol ~~ ~ ~ ~

OPP 38 5 41 P + 10000 18 11 6 P + 25 000 17 11 6 P + 50000 5 P + 100000 12 6 6 P + 200000 6

__ -

__ -

Initially beansprouts contained about 9 g kg-' of mono- and disaccharides, mainly fructose, glucose and sucrose. Sugar content increased immediately after packaging and reached 12 g kg-' within 5 days and was independent of film permeability (data not shown). Hydrolysis of starch, located in the plant cotyledons, is likely to be responsible for this phenomenon. The oligo- saccharide content then decreased due to both plant tissue metabolism and microorganisms. Values of 10-5, 8.5, 5.5 g litre-' were found after 7, 9 and 12 days of storage respectively at 8°C.

Deterioration of beansprouts stored under modified atmospheres

Major symptoms of deterioration are the darkening of sprouts and the development of sliminess and musty fla- vours (Lipton e t a1 1981).

Since the first aspect of deterioration was a visual loss in brightness, only the L* parameter was considered. Initial luminance of the beansprouts was about 84% and remained constant for 12 days except with the two most permeable films. A significant decrease to 75% of the L* parameter was observed after 5 or 9 days for products packed with P + 200000 and P + 100000, respectively. Changes in product appearance during storage at 8°C were also assessed hedonistically by a panel of 10 untrained tasters who were not accustomed to beansprouts. Due to their brown colour and the pres- ence of abundant exudates, samples packed in the

230 P Varoquaux et a1

P + 200000 film were rejected after 2 days while those packed in the P + 100000 film were rejected after 4 days due to brown discolouration. On a visual basis, beansprouts packed in the other films were not found to be significantly different and were acceptable up to 6 days. Therefore, in order to prevent the brown dis- colouration of the sprouts, the film permeability to oxygen should be limited to 50000 ml m-’ day-’ atm-’. Beansprouts packed in OPP and P + 10000 were rejected after 2 and 4 days, respec- tively, because of off-odours.

Initially firmness of beansprouts was 150 f 10 dN and remained constant during storage except when sprouts were packed with P + 100000 and P + 200000. With these films, firmness decreased to about 100 dN after 7 days at 8°C. Firmness of beansprouts is usually described by the consumer as ‘crunchy’ which refers to an adequate level of tissue turgor and therefore to low flexibility. The procedure used here for determining firmness is based on shearing and extrusion and so is rather inadequate to assess small changes in tissue turgor. This may explain why few or no changes were observed except when product deterioration was advanced.

CONCLUDING REMARKS

Microbial growth is a major cause of beansprout spoil- age and could explain, in part, for the unusual behav- iour of oxygen uptake rate with time and temperature. Usually MAP associated with chilled storage permits the extension of shelf-life by preventing dehydration, limiting oxidation of phenolics and reducing the growth of aerobic mesophilic microorganisms. In the case of beansprouts, the development of lactic acid bacteria, whatever the atmosphere consumption, limits the effi- ciency of this technique. The packaging film should be permeable enough to avoid a carbon dioxide content exceeding 20% (threshold of the switch to anaerobic metabolism), since this will enhance plant tissue decay and the deterioration rate. In contrast, the permeability has to be sufficiently low to reduce the oxygen concen- tration, thereby preventing the development of a brown discolouration. The results indicate that the optimal film permeability is about 50000 ml O2 m-’ day-’ atm-’ and that, under these conditions, the shelf-life is approximately 5 days at 5°C and 9 days at 1°C. Lipton et al(l981) stated that a limit of 9 days can be obtained when sprouts are stored at 0°C. However, when considering commercial chilled storage condi- tions, an average storage temperature of about 8°C would appear to be more realistic. In this case, as stated by Lipton et al (1981), the normal shelf-life of bean- sprouts under air is only 2.5 days. Nevertheless it may be extended to 5 days with suitable modified atmo- spheres which never reached a steady state, not even a

transient one, whatever the film permeability. Since no model to date takes into account the incidence of microbial growth and cell death on gas exchanges in packed plant tissues, it is not possible to calculate an estimate of beansprout respiration rate under MAP. Conversely, modelling atmosphere changes within fruit and vegetables packages should take into account the respiration of the aerobic flora.

ACKNOWLEDGEMENT

This work was partly supported by the EU-AIR Project No. 920125.

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