Available online at www.sciencedirect.

com

Food Control 19 (2008) 698705 www.elsevier.com/locate/foodcont

Application of high-pressure treatment on alfalfa (Medicago sativa) and mung bean (Vigna radiata) seeds to enhance the microbiological safety of their sprouts
mez Elena Pen as a, Rosario Go
a b

a,*

as b, Concepcio n Vidal-Valverde , Juana Fr

a de Productos La cteos, Instituto del Fr o (CSIC), C/Jose Antonio Nova is, 10, 28040 Madrid, Spain Departamento de Ciencia y Tecnolog as Sensoriales, Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva, 3, 28006 Madrid, Spain Departamento de Tecnolog Received 30 May 2007; received in revised form 12 July 2007; accepted 17 July 2007

Abstract The eect of several combinations of time, pressure and temperature applied on mung bean and alfalfa seeds, on the germination capacity as well as on the reduction of the native microbial load of sprouts developed from treated seeds was studied by using response surface methodology (RSM). The germination capability of mung bean seeds was unaected with increasing temperature and pressures up to 250 MPa. Increase of temperature from 10 to 40 C has a positive eect on the viability of alfafa seeds, which decreased however as pressure increased from 100 to 400 MPa. Enhanced reductions of total aerobic mesophilic bacteria, total and faecal coliforms and yeast and moulds populations were observed with increased pressure and temperature. The optimal treatment conditions for improving the safety of sprouts without impairing the germination capability of seeds were 40 C and 100 and 250 MPa for alfalfa and mung bean seeds, respectively. 2007 Elsevier Ltd. All rights reserved.
Keywords: High-pressure; Sprouts; Mung bean; Alfalfa; Microbiological safety

1. Introduction Sprouts are one valuable dietary supplement and are considered a natural healthy food by consumers in many parts of the world (Kurtzweil, 1999; Mwikya, Camp, Rodriguez, & Huyghebaert, 2001). The germination improves nutritional quality of seeds (Ghorpade & Kadam, 1989; Vidal-Valverde et al., 2002) because the lipids, carbohydrates and storage proteins are broken down to smaller and more digestible nutrients during this complex metabolic process (Vidal-Valverde & as, 1992; Ziegler, 1995). Furthermore, the levels of some Fr antinutritional factors decrease or even disappear during as, Diaz-Pollan, Hedley, & germination process (Fr

Corresponding author. Tel.: +34 91 549 23 00; fax: +34 91 549 36 27. mez). E-mail address: rgomez@if.csic.es (R. Go

Vidal-Valverde, 1995; Vidal-Valverde et al., 1998), while some compounds with antioxidant activity increase (Dobas, & Vidal-Valverde, 2007; Fr as, Miranda, Doblado, Fr lado, & Vidal-Valverde, 2005), phenomena that also contribute to improved nutritional quality of sprouts compared to seeds. However, over the last decade, sprouts have emerged as a recognized source of infectious foodborne outbreaks throughout the world (Bremer, Fielding, & Osborne, 2003). Microbial contamination of sprouts often begins with contaminated seeds, which are not properly disinfected before sprouting and they are the primary source in most of sprout-associated illness outbreaks (Mahon et al., 1997). Microorganisms on seeds can proliferate quickly under the favorable conditions during seed germination and subsequent sprout growth (water activity, temperature, pH and nutrients availability), and agar plate counts (APCs) of 108109 have been reported in alfalfa, bean or

0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.07.010

E. Pen as et al. / Food Control 19 (2008) 698705

699

onion sprouts (Ghandi & Matthews, 2003; Lang, Ingham, & Ingham, 2000; Prokopowich & Blank, 1991). High microbial levels per se reduce the shelf-life and safety of sprouts, as described by the National Advisory Committee on Microbial Criteria for Foods (NACMCF, 1999). Food and Drug Administration (FDA) recommends the decontamination of seeds before sprouting. Several methods have been described for reducing microbial load on seeds, including heat treatment (Jaquette, Beuchat, & Mahon, 1996; Weiss & Hammes, 2003), exposure to ionizing radiation (Thayer, Rajkowski, Boyd, Cooke, & Soroka, 2003) and numerous chemical treatments such as chlorine or hypochlorite (Beuchat, Ward, & Pettigrew, 2001; Fett, 2002; Gill et al., 2003; Proctor, Hamacher, Tortorello, Archer, & Davis, 2001; Winthrop et al., 2003), hydrogen peroxide, ethanol (Piernas & Guiraud, 1997; Suzuki & Takizawa, 1997), ozone (Sharma, Demirci, Beuchat, & Fett, 2002), and commercial disinfectants. Despite of some of these methods can achieve P5 log reductions of pathogens on seeds, as recommended by NACMCF (1999), none of them eliminate completely pathogens inside seeds (Beuchat, 1997; NACMCF, 1999), and decreased however the germination capability of seeds. The development of a physical decontamination method that leaves no residues is needed, since chemical disinfectants are dicult to reconcile with the health food image of seed sprouts (Wuytack, Diels, Meersseman, & Michels, 2003). High-pressure (HP) could be a valuable alternative to chemical decontamination methods for inactivating pathogens, since it constitutes an excellent non thermal technique for food preservation because it reduces the microbial populations, maintaining sensorial and nutritional proper stamo, Lesmes, Otero, & Arroyo, 2000). ties of food (Pre HP treatment incorporating a combination of time, pressure and moderate heat could create hurdle eects that could signicantly reduce the microbial load in seeds and sprouts developed from pressurized seeds, work not yet reported. The purpose of this work was to evaluate the eects of HP, temperature and exposure time on germination capability of mung bean and alfalfa seeds and safety improvement of sprouts developed from treated seeds, by using response surface models. 2. Materials and methods 2.1. Plant material Alfalfa (Medicago sativa) and Mung bean (Vigna radiata var. Emmerald) seeds were provided by Man Fong Pacic Trading, S.A. (Spain) and stored at 4 C until their decontamination treatment. 2.2. High-pressure treatment Seeds were immersed for 3 h in distilled water at room temperature. The water was eliminated and then seeds were packed in polyethylene bags under vacuum and were sub-

mitted to 100, 250 and 400 MPa for 5, 10 and 15 min at 10, 25 or 40 C in a discontinuous high-pressure machine (ACB GEC, Alsthom, Nantes, France) with a hydrostatic pump and a steel-vessel of 2.35 L capacity (100 mm in diameter and 300 mm in height). These conditions of pressure, time and temperature were chosen according an experimental design using a response surface methodology (RSM). The vessel of the high-pressure machine was lled with water as uid of low compressibility. The temperature inside the vessel and the quick thermal equilibration was controlled by a circulating-thermostatic bath. In each experiment, the indicated pressure was achieved within 1 2 min, held for the period described above, and it was released to atmospheric pressure within 12 min. Control seeds were not pressurised but they were treated in similar way to experimental ones. 2.3. Germination Control and HP-treated seeds were germinated in a climatic cabinet (ASL Snijders Sci. International S.L., Tiburg, Holland) at 25 C for 5 days in darkness. Seeds were sprinkled with sterile distilled water every 12 h. Sprouts were obtained after 5 days and then the percentage of germination was determined by counting the number of germinating seeds. 50 seeds were germinated and germination was performed in duplicate for each sample. 2.4. Microbial analyses Microbiological analyses of sprouts obtained from control and pressurised seeds were performed. Microbial counts of sprouts from untreated seeds were used as initial values for calculating logarithmic reductions in microbial counts. Alfalfa and mung bean sprouts were mixed and stomached separately with buered peptone water (BPW) (Oxoid, Unipath, Ltd., Basingstoke, UK) at a ratio of product to medium 1:9. Then, 1 ml of each suspension was pour plated in triplicate on dierent media for the counting of the following microorganisms: Total aerobic mesophilic bacteria on tryptone soya agar (TSA), incubation at 30 C for 72 h; total and faecal coliforms on violet red bile agar (VRBA), containing lactose as carbohydrate source, incubation at 37 and 44 C, respectively, for 24 h; moulds and yeast on Sabouraud chloramphenicol agar, incubation at 23 C for 72 h. 2.5. Experimental design Response surface methodology (RSM) was used for investigating the eect of three independent variables (pressure, time and temperature) on ve response variables, (a) germination percentage, and reduction of: (b) total aerobic mesophilic bacteria, (c) total coliforms, (d) faecal coliforms and (e) moulds and yeast counts on mung bean and alfalfa sprouts from treated seeds compared to those from untreated seeds. The experiments were performed according

700

E. Pen as et al. / Food Control 19 (2008) 698705

Table 1 Levels of independent variables used in a response surface methodology design Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a b c

X1a 1 (5) 1 (15) 1 (5) 1 (5) 1 (15) 0 (10) 0 (10) 0 (10) 1 (15) 0 (10) 1 (15) 0 (10) 0 (10) 1 (5) 1 (15) 1 (5)

X2b 1 (10) 1 (40) 1 (40) 1 (10) 1 (40) 0 (25) 0 (25) 0 (25) 0 (10) 1(10) 0 (10) 0 (25) 1 (40) 0 (25) 0 (25) 1(40)

X3c 1 (100) 1 (400) 1 (100) 1 (400) 1 (100) 1 (100) 0 (250) 1 (400) 1 (100) 0 (250) 1 (400) 0 (250) 0 (250) 0 (250) 0 (250) 1(400)

ture and time. Treating each factor separately would be very time consuming. Besides, if several factors play a role, their interactions would not be discernible even if they were dominant. The use of experimental factorial design and of the response surface methodology is suitable for studying the main and interaction eects of the factors on the response variables. Multiple regression analysis of the experimental data obtained following the experimental design shown in Table 1, gave second-order polynomial equations for the ve response variables studied. The coecients of the equations and their signicance are given in Tables 2 and 3. 3.1. Germination of mung bean seeds treated with combinations of HP and temperature for several exposure times according to the experimental design The regression model for germination of mung bean seeds showed that the linear factor of pressure was signicant (p 6 0.05) on the capacity of germination of seeds and the value of R2 (0.9811) indicated that the model could explain about 98% of the variations in the percentage of germination (Table 2), meaning only 1.8% of the variation was due to other factors not included in the model. The predicted response surface plot presented in Fig. 1a shows that no inuences were observed for mung bean seed viability (that is the ability of seeds to germinate) with increasing temperature and pressure up to 250 MPa. In contrast, seed viability was found to decrease at higher pressures, regardless of the temperature during the treatment. 3.2. Microbial load on mung bean sprouts from HP-treated seeds The predictive models for all microbial groups studied were considered adequate because they showed values of R2 > 0.75 (Table 2), that indicates the aptness of the models. The values of R2 for total and faecal coliforms, moulds and yeast, indicated that only about 3.1%, 6.9% and 1.2% of the total variation, respectively, were not explained by the respective models. However for total aerobic bacteria about 19.4% of the variability in the response was not explained by the model. The linear and quadratic terms of pressure and temperature, and the interaction term between these both factors (Table 2) had eect on the levels of the studied microbial groups on mung bean sprouts. The linear and quadratic terms of time had also signicant inuence on moulds and yeast populations (p 6 0.01) and on total and faecal coliforms counts (p 6 0.05). The interaction terms between time and temperature/pressure were also signicant (p 6 0.05) for faecal coliforms, moulds and yeast. Fig. 2 shows the graphical representations of the regression equations for the reduction of these microbial populations on mung bean sprouts obtained from treated seeds, as a function of pressure and temperature at constant time

time (min). temperature (C). pressure (MPa).

to a central composite face-centered composite design. Three levels of each independent variable (pressure, time and temperature) and sixteen combinations of these three variables were chosen following the design (Table 1); two replications of each experimental condition were performed. The low, middle and high levels of each variable were designated as 1, 0, and +1, respectively, and are given in Table 1. Statgraphics Plus 5.1 (Statistical Graphics Corporation, Inc., Rockwille, MD, USA) software was used for statistical analysis. Results were averages of three independent determinations. The following quadratic polynomial equation was used to express responses as a function of independent variables: X X X Y bo bi X i bii X 2 bij X i X j i where Y represents the dependent variable to be modelled; b0, bi, bj and bij represent the coecients of the model; and Xi and Xj represent the independent variables. The goodness of t of the models obtained was evaluated by R2 (multiple determination coecient), the Fischer F-test (and the derived p-values) and the standard errors of the estimate. Three-dimensional surface plots were drawn to illustrate the eects of the independent variables on the dependent ones. Analysis of variance was performed for each response variable using the full models where p-values indicated if the terms were signicant. Lack of t determined whether the selected model appeared adequate to describe the observed data or not. None of the predicted models had a signicant lack of t. 3. Results High-pressure treatment of seeds is a three-dimensional process consisting of a combination of pressure, tempera-

E. Pen as et al. / Food Control 19 (2008) 698705

701

Table 2 Coecients of the second-order polynomial equations and signicance of each model and dependent response variables in mung bean sprouts obtained from HP-treated seeds Germination % R2 model Intercept b0 Linear b1 b2 b3 Quadratic b11 b22 b33 Interaction b12 b13 b23 0.9811 53.5551 5.8137 1.2545 0.2079* 0.3207 0.0310 0.0007 0.0250 0.0025 0.0014 Aerobic mesophilic bacteria counts reduction 0.8062 0.7825 0.2305 0.0990* 0.0127** 0.0121* 0.0012* 0.00002** 0.00005 0.000001 0.00002* Total coliforms counts reduction 0.9693 2.1997 0.2772* 0.5805** 0.0114** 0.0115* 0.0116** 0.00003** 0.0003 0.00006 0.0006** Faecal coliforms counts reduction 0.9310 1.4100 0.2386* 0.2298** 0.0020** 0.0135* 0.0079** 0.00001* 0.0070* 0.0006* 0.0002* Moulds and yeast counts reduction 0.9880 2.0615 0.3734** 0.1531** 0.0116** 0.0128** 0.0021* 0.000006** 0.0011* 0.0002* 0.0001**

Independent variables: Time (X1), Temperature (X2) and Pressure (X3). * p 6 0.05. ** p 6 0.01.

Table 3 Coecients of the second-order polynomial equations and signicance of each model and dependent response variables in alfalfa sprouts obtained from HP-treated seeds Germination % R2 model Intercept b0 Linear b1 b2 b3 Quadratic b11 b22 b33 Interaction b12 b13 b23 0.9877 122 2.1621 1.5926 0.9901* 0.1283 0.0031 0.0016* 0.0233 0.0007 0.0016* Aerobic mesophilic bacteria counts reduction 0.9825 2.4032 0.0388* 0.2340** 0.0023** 0.0020 0.0025** 0.000007** 0.0002 0.00007* 0.0001** Total coliforms counts reduction 0.9706 3.2235 0.0867* 0.3580** 0.0032** 0.0046 0.0082** 0.0000** 0.0008 0.0002 0.0002** Faecal coliforms counts reduction 0.9120 6.5664 0.5324 0.3040** 0.0143** 0.0280 0.0045* 0.00002* 0.0012 0.00002 0.0006** Moulds and yeast counts reduction 0.9660 2.7842 0.1925** 0.1460** 0.0005** 0.0099 0.0024* 0.0000001 0.0013 0.00005 0.0002**

Independent variables: Time (X1), Temperature (X2) and Pressure (X3). * p 6 0.05. ** p 6 0.01.

(10 min). The counts of aerobic mesophilic bacteria (Fig. 2a), total and faecal coliforms (Fig. 2b and c, respectively) were reduced with increasing pressure and temperature during the treatment of seeds. The increase of pressure had a more pronounced eect on inactivation of these bacteria when temperatures >25 C were used and the highest synergic eect between pressure and temperature was observed at 40 C. Thus, sprouts from seeds treated at this temperature and relatively low pressures (250 MPa) show higher inactivation of total aerobic mesophilic bacteria, total and faecal coliforms (2.0, 2.4 and 2.0 log units, respectively) than those developed from seeds treated at 10 C and 400 MPa (0.4 and 1 log unit, respectively). The pressure had more inuence than the temperature on moulds and yeast counts (Fig. 2d).

3.3. Germination of alfalfa seeds treated with combinations of HP and temperature for several exposure times according to the experimental design The regression model for germination of alfalfa seeds was considered adequate, because the value of the determination coecient (R2) (0.9877) ensured a satisfactory adjustment of the quadratic model to the experimental data. The linear and quadratic factors of pressure, as well as interaction term between pressure and temperature had signicant inuence (p 6 0.05) for germination of alfalfa seeds (Table 3). A decrease of germination percentage of seeds was observed when pressure increased from 100 to 400 MPa, and the lowest values were obtained at pressures about 250 MPa (Fig. 1b). The increase of temper-

702

E. Pen as et al. / Food Control 19 (2008) 698705

100 80 60 40 20 0 10

15

20 25 30 35 Temperature (C)

40

100

200

300

400

Pressure (MPa)

110 90 70 50 30 10 -10 10

15

20 25 30 35 Temperature (C)

40

100

200

300

400

Pressure (MPa)

Fig. 1. Response surface plot of the percentage of seed germination as a function of pressure and temperature, while the time of pressure treatment was kept constant (10 min). (a) Mung bean sprouts; (b) alfalfa sprouts.

ature from 10 to 40 C caused the opposite eect and the application of a combination of 100 MPa and 40 C on seeds produced a percentage of germination close to that of untreated seeds. 3.4. Microbial load on alfalfa sprouts from HP-treated seeds The models for aerobic mesophilic, total and faecal coliforms, moulds and yeast were considered adequate because of they were signicant with a relationship between the variables at the 98.2%, 97.0%, 91.2% and 96.6% condence levels, respectively (Table 3). The linear and qua-

dratic terms of temperature and pressure and the interaction between both terms had a signicant eect for total aerobic bacteria and total and faecal coliforms counts on sprouts. The linear term of time was also signicant for total aerobic and total coliforms populations (p 6 0.05) (Table 3). The time, temperature and pressure linear terms and interaction terms between temperature and pressure were highly signicant (p 6 0.01) on the moulds and yeast reduction model, while the temperature quadratic term had signicant eect at p 6 0.05. The predicted response surface plots given in Fig. 3ad show the reduction of the described microbial groups in alfalfa sprouts developed from treated seeds. A reduction of total aerobic counts on sprouts was observed when the temperature increased up to 40 C and no signicant dierences were found at this temperature by the pressure applied (Fig. 3a). Fig. 3bd shows, respectively, that the total and faecal coliforms, and moulds/yeast loads of sprouts decreased as pressure applied to seeds increased, in most extent when the treatments were performed at 40 C. Reductions of total coliforms ranged between 2.5 and 5.0 log units were shown by alfalfa sprouts developed from seeds treated at 40 C, and pressures between 100 and 400 MPa, compared to reductions of 60.5 log units on sprouts from seeds treated at these pressures and below room temperature. No signicant dierences were found on the reduction of faecal (Fig. 3c) and moulds/yeast populations (Fig. 3d) between the treatments at 10 and 40 C when seeds were pressurised at 100 MPa. However, dierences between both temperatures were observed at higher pressures. Reductions on faecal populations of 3.4 and 4.7 log units of sprouts from seeds treated at 40 C and 250 and 400 MPa, respectively were observed (Fig. 3c) compared to reductions of 1.8 and 2.5 log units for the

% germination

% germination

2.4 2 1.6 1.2 0.8 0.4 0 10

15

20 25 30 Temperature (C)

35

40

100

200

300

400

Pressure (MPa)

5.4 4.4 3.4 2.4 1.4 0.4 -0.6 10

Log reduction

Log reduction

400 15 20 25 30 Temperature (C) 200 35 40 100 300 Pressure (MPa)

c
4 Log reduction 3 2 1 0 10 15 200 35 40 100 300 Pressure (MPa) 400 25 30 Temperature (C) 20

d
5 Log reduction 4 3 2 1 0 10 15 20 25 30 35 Temperature (C) 400 300 200 100 Pressure (MPa)

40

Fig. 2. Response surface plot for microbial counts reduction in mung bean sprouts as a function of pressure and temperature, while the time of pressure treatment was kept constant (10 min). (a) Total aerobic mesophillic microorganism; (b) total coliforms; (c) faecal coliforms; (d) moulds and yeasts reductions on sprouts from treated seeds compared to the respective from untreated seeds.

E. Pen as et al. / Food Control 19 (2008) 698705

703

Log reduction

4 3 2 1 0 10 15 20 25 30 35 Temperature (C) 100 200 300 400 Log reduction

40

Pressure (MPa)

5.5 4.5 3.5 2.5 1.5 0.5 -0.5 10

15

20 25 30 35 Temperature (C)

40

100

200

300

400

Pressure (MPa)

c
5.5 4.5 3.5 2.5 1.5 0.5 -0.5 10 Log reduction

d
4 Log reduction 3 2 1 0 10 15 20 25 30 Temperature (C) 200 35 40 100 300 Pressure (MPa) 400

15

20 25 30 35 Temperature (C)

40

400 300 200 100 Pressure (MPa)

Fig. 3. Response surface plot for microbial counts reduction in alfalfa sprouts as a function of pressure and temperature, while the time of pressure treatment was kept constant (10 min). (a) Total aerobic mesophillic microorganism; (b) total coliforms; (c) faecal coliforms; (d) moulds and yeasts reductions on sprouts from treated seeds compared to the respective from untreated seeds.

sprouts from seeds treated at these pressures and 10 C. Similar synergic eects of temperature and pressures >100 MPa were observed for moulds/yeast populations (Fig. 3d), where reductions of 2 and 3.7 log units on sprouts from seeds treated at 40 C and 250 and 400 MPa, respectively, were found, compared to 0.9 and 1.8 log reductions for the sprouts from seeds treated at these pressures and 10 C. 4. Discussion To minimise the risk associated with human pathogens on sprouted seeds, several intervention strategies have been explored. Particular focus has been placed on seed decontamination, critical control point in sprout production, since indigenous and contaminant populations of seeds grow to high levels during the microbiologically favourable warm, moist and nutrient-rich conditions of sprouting (NACMCF, 1999). However, none of the strategies have proved totally successful (Jaquette et al., 1996; Lang et al., 2000; Linton & Patterson, 2002) and, besides, the microbicide eect is at the expense of seed germination (Ariefdjohan et al., 2004). Response surface methodology and the experiments design applied in the present work by combining pressures (100400 MPa), times of exposure (515 min) and temperatures (1040 C) revealed themselves to be ecient in the determination of the optimal conditions to maintain the germination capability of the mung bean and alfalfa seeds and improve the safety of the developed sprouts. Treatment of seeds incorporating a combination of time, pressure and moderate heat seems to create hurdle eects that signicantly reduce the microbial load in seeds and sprouts developed from them. The results predicted by

the models after statistical analysis showed acceptable correlation with those obtained empirically (data not shown). Temperature and pressure interaction response surfaces plots for the germination percentage, clearly demonstrate no inuences for the viability of alfalfa and mung bean seeds with increasing pressures up to 100 and 250 MPa, respectively, decreasing at higher pressures. The opposite eect was observed for alfafa seeds by increasing the temperature. Ariefdjohan et al. (2004), reported that pressurised alfalfa seeds at 40 C (275575 MPa for 2 min or 475 MPa for 28 min) took longer to germinate, achieving germination rate of up to 34%, while 95% of the control seed germinated. The authors observed under a light microscope that the coats of treated seeds at the described conditions were damaged, showing cracks or completely broken seeds. Wuytack et al. (2003), reported a higher germination rate than Ariefdjohan et al. (2004) for seeds pressurised in sterile water, results attributed by the authors to the softening of seed coats by water, alleviating the coat damage and thus improving the germination capability. As would expected, enhanced microbial reductions in mung bean and alfalfa sprouts occur with increasing the level of pressure applied on seeds, but impairing their capability of germination at the highest pressures. On the other hand, treatments of seeds at 40 C enhances the eectiveness of pressure for killing the microbial groups studied in mung bean seeds, with the exception of moulds/yeast, microorganisms however also aected in alfalfa seeds by the synergic eect of both parameters. A set of optimal treatment conditions found in the present work for improving the sprouts safety without impairing the germination capability of seeds would be 40 C for 10 min and 100 and 250 MPa for alfalfa and mug bean seeds, respectively. Lower total aerobic, total and fecal

704

E. Pen as et al. / Food Control 19 (2008) 698705

coliforms, moulds and yeast counts (3.0, 2.5, 3.5 and 2.0 log units) were found on alfalfa sprouts treated at the described conditions, compared to controls sprouts The values were 2.0, 4.0, 3.0 and 2.8 log for mung bean sprouts from treated seeds. Ariefdjohan et al. (2004) observed reductions of 1.4 and 2 log units of Escherichia Coli 0157 inoculated on alfalfa seeds after their pressurisation at 575 MPa and 475 MPa at 40 C for 2 and 8 min, respectively. The nding of the present work related with the microbicide eect of pressure/temperature combinations is higher than those described by other authors in dierent foods when room or lower temperature was used. Arroyo, stamo (1997) found a reduction of 1 log units Sanz, and Pre of viable aerobic mesophilic bacteria on lettuce and tomato treated at 350 MPa at 20 C for 10 min and reductions of 24 log units in lettuce, tomato, asparagus, onions, cauliower and spinach processed between 200 and 400 MPa for 30 min at 5 C. A decrease of about 1 log unit of total and faecal coliforms counts in tofu treated at 400 MPa at stamo et al. (2000). 5 C for 30 min was observed by Pre Mun oz, de Ancos, Sanchez-Moreno, and Cano (2006) observed 4 log reduction in total aerobic mesophilic counts on mung bean sprouts after treating them at 400 MPa (25 C, for 2 min). According with McClements, Patterson, and Linton (2001) the pressure causes changes in cell morphology, inhibition of genetic mechanisms and disruption of ribosomes, although the primary site for pressure-induced microbial inactivation is the cell membrane by modications in its permeability and ion exchange. In addition, HP causes changes in biochemical reactions and denaturation of proteins included key enzymes (Linton & Patterson, 2002). The combination of these factors might be responsible for both microbial inactivation and impaired germination capability induced by high-pressure observed by us. The present work provides evidence that pressure can be used as an ecient tool to improve the safety of mung been and alfalfa sprouts. However, at high pressures, microbial inactivation occurred as the expense of seed germination, since this process was severely reduced by HP treatment. Thus, the treatment conditions selected for each seed to maintain a targeted germination percentage, which was >90% in this study, clearly reduced the microbial load in sprouts but failed to produce a microbial reduction high enough to secure microbial safety of sprouts (reductions P5 log). Further research by using antimicrobial or disinfectants compounds and relatively low pressures is needed in order to optimise the combined hygienisation processing of seeds to achieve a P5 log reduction on sprouts (Michaelsen, Med, & Friis, 1998) without impairing the germination capability of seeds, work now in progress at our laboratory. Acknowledgements This work was supported through an Institute Danonenancied grant and through the project AGL2004-00886 from the Spanish Ministry of Science and Technology.

References
Ariefdjohan, M. W., Nelson, P. E., Singh, R. K., Bhunia, A. K., Balasubramaniam, V. M., & Singh, N. (2004). Ecacy of high hydrostatic pressure treatment in reducing Escherichia coli 0157 and Listeria monocytogenes in alfalfa seeds. Journal of Food Science, 69, 117120. stamo, G. (1997). Eect of high pressure on Arroyo, G., Sanz, P. D., & Pre the reduction of microbial populations in vegetables. Journal of Applied Microbiology, 82, 735742. Beuchat, L. R. (1997). Comparison of chemical treatments to kill Salmonella on alfalfa seeds destined for sprout production. International Journal of Food Microbiology, 34, 329333. Beuchat, L. R., Ward, T. E., & Pettigrew, C. A. (2001). Comparison of chlorine and a prototype produce wash product for eectiveness in killing Salmonella and Escherichia coli 0157:H7 on alfalfa seeds. Journal of Food Protection, 64, 152158. Bremer, P. J., Fielding, T., & Osborne, C. M. (2003). The safety of seeds and bean sprouts: risk and solution. New Zealand Food Journal, 3, 3340. as, J., & Vidal-Valverde, C. (2007). Changes in vitamin C Doblado, R., Fr content and antioxidant capacity of raw and germinated cowpea (Vigna sinensis var. carilla) seeds induced by high pressure treatment. Food Chemistry, 101, 918923. Fett, W. F. (2002). Reduction of the native microora on alfalfa sprouts during propagation by addition of antimicrobial compounds to the irrigation water. International Journal of Food Microbiology, 72, 1318. as, J., Diaz-Pollan, C., Hedley, C., & Vidal-Valverde, C. (1995). Fr Evolution of trypsin inhibitor activity during germination of lentils. Journal of Agricultural and Food Chemistry, 43, 22312234. as, J., Miranda, M. L., Doblado, R., & Vidal-Valverde, C. (2005). Fr Eect of germination and fermentation on the antioxidant vitamin content and antioxidant capacity of Lupinus albus L. var Multolupa. Food Chemistry, 92, 211220. Ghandi, M., & Matthews, K. R. (2003). Ecacy of chlorine and calcinated calcium treatment of alfalfa seeds and sprouts to eliminate Salmonnella. International Journal of Food Microbiology, 87, 301306. Ghorpade, V. M., & Kadam, S. S. (1989). Germination. In D. K. Salunkhe & S. S. Kadam (Eds.), Handbook of world food legumes: nutritional chemistry, processing technology, and utilization. Boca Raton, FL: CRC Press. Gill, C. J., Keene, W. E., Mohle-Boetani, J. C., Farrar, J. A., Waller, P. L., Hahn, C. G., et al. (2003). Alfalfa seed decontamination in a Salmonella Outbreak. Emerging Infectious Diseases, 9, 474479. Jaquette, C. B., Beuchat, L. R., & Mahon, B. E. (1996). Ecacy of chlorine and heat treatment in killing S. stanley inolulated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage. Applied and Environmental Microbiology, 62, 22122215. Kurtzweil, P. (1999) Food and Drug Administration. Washington, DC. <http://www.fda.gov/fdac/features/1999/199_sprt.html>. Lang, M. M., Ingham, B. H., & Ingham, S. C. (2000). Ecacy of novel organic acid and hypochlorite treatments for eliminating Escherichi coli 0157:H7 from alfalfa seeds prior to sprouting. International Journal of Food Microbiology, 58, 7382. Linton, M., & Patterson, M. F. (2002). High pressure processing of foods for microbiological safety and quality. Acta Microbiologica et Immunologica Hungarica, 47, 175182. Mahon, B. E., Ponka, A., Halla, W., Komatsu, K., Beuchat, L., Shiett, S., et al. (1997). An international outbreak of Salmonella infections caused by alfalfa sprouts grown from contaminated seeds. Journal of Infectious Diseases, 175, 876882. McClements, J. M. J., Patterson, M. F., & Linton, M. (2001). The eect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. Journal of Food Protection, 64, 514522. Michaelsen, K. F., Med, S., & Friis, H. (1998). Complementary feeding: A global perspective. Nutrition, 14, 763766.

E. Pen as et al. / Food Control 19 (2008) 698705 nchez-Moreno, C., & Cano, M. P. (2006). Mun oz, M., de Ancos, B., Sa Evaluation of chemical and physical (high-pressure and temperature) treatments to improve the safety of minimally processed mung bean sprouts during refrigerated. Journal of Food Protection, 69, 23952410. Mwikya, S. M., Camp, J. V., Rodriguez, R., & Huyghebaert, A. (2001). Eects of sprouting on nutrient and antinutrient composition of kidney beans (Phaseolus vulgaris var. Rose coco). European Food Research and Technology, 212, 188191. National Advisory Committee on Microbial Criteria for Foods. Food and Drug Administration. (1999). International Journal of Food Microbiology, 52, 123-153. Piernas, V., & Guiraud, J. P. (1997). Microbial hazards related to rice sprouting. International Journal of Food Science and Technology, 32, 3339. stamo, G., Lesmes, M., Otero, L., & Arroyo, G. (2000). Soybean Pre vegetable protein (tofu) preserved with high pressure. Journal of Agricultural and Food Chemistry, 48(7), 29432947. Proctor, M. E., Hamacher, M., Tortorello, M. L., Archer, J. R., & Davis, J. P. (2001). Multistate outbreak of Salmonella serovar Muenchen infections associated with alfalfa sprouts grown from seeds pretreated with calcium hypochlorite. Journal of Clinical Microbiology, 39, 34613465. Prokopowich, D., & Blank, G. (1991). Microbiological evaluation of vegetable sprouts and seed. Journal of Food Protection, 54, 560562. Sharma, R. R., Demirci, A., Beuchat, L. R., & Fett, W. F. (2002). Inactivation of Escherichia coli 0157:H7 on inoculated alfalfa seeds with ozonated water and heat treatment. Journal of Food Protection, 65, 447451. Suzuki, T., & Takizawa, T. (1997). Sprouted vegetables seeds sterilizing method, and sprouted vegetables cultivating method. U.S. Patent No. 5615518.

705

Thayer, D., Rajkowski, K., Boyd, G., Cooke, P., & Soroka, D. (2003). Inactivation of Escherichia coli 0157: H7 and Salmonella by gamma irradiation of alfalfa seed intended for production of sprouts. Journal of Food Protection, 66, 175181. as, J. (1992). Changes in carbohydrates during Vidal-Valverde, C., & Fr germination of lentils. Zeitschrift fu r Lebensmittel Untersuchung und Forschung, 194, 461464. as, J., Sierra, I., Bla zquez, I., Lambein, F., & Kuo, Vidal-Valverde, C., Fr Y. H. (2002). New Functional legume foods by germination: eect on the nutritive value of beans, lentils and peas. European Food Research and Technology, 215, 472477. as, J., Sotomayor, C., Diaz-Pollan, C., Fernandez, Vidal-Valverde, C., Fr M., & Urbano, G. (1998). Nutrients and antinutritional fractors in faba beans as aected by processing. Zeitshcrift fu r Lebensmittel Untersuchung und Forschung A, 207, 140145. Weiss, A., & Hammes, W. P. (2003). Thermal seed treatment to improve the food safety status of sprouts. Journal of Applied Botany, 77, 152155. Winthrop, K. L., Palumbo, M. S., Farrar, J. A., Mohle-Boetani, J. C., Abbott, S., Beatty, M. E., et al. (2003). Alfalfa sprouts and Salmonella Kottbus Infection: A Multistate Outbreak following inadequate seed disinfection with heat and chlorine. Journal of Food Protection, 66, 1317. Wuytack, E. Y., Diels, A. M. J., Meersseman, K., & Michels, C. W. (2003). Decontamination of seeds for seed sprout production by high hydrostatic pressure. Journal of Food Protection, 66, 918923. Ziegler, P. (1995). Carbohydrate degradation during germination. In J. Kigel & G. Galili (Eds.), Seed development and germination (pp. 447474). New York: Marcel Dekker.