Characterization of Lactobacillus strains and monitoring by RAPD-PCR in controlled fermentations of “Almagro” eggplants

International Journal of Food Microbiology 104 (2005) 325 – 335 www.elsevier.com/locate/ijfoodmicro

Characterization of Lactobacillus strains and monitoring by RAPD-PCR in controlled fermentations of bAlmagroQ eggplants Susana Sesen˜aa,*, Isabel Sa´nchezb, Llanos Palopa a

Departamento de Quı´mica Analı´tica y Tecnologı´a de Alimentos, Facultad de Ciencias del Medio Ambiente, Avda. Carlos III s/n, 45071 Toledo, Spain b Departamento de Quı´mica Analı´tica y Tecnologı´a de Alimentos, Facultad de Quı´micas, Avda. Camilo Jose´ Cela s/n, Ciudad Real, Spain Received 9 October 2003; received in revised form 12 March 2005; accepted 19 March 2005

Abstract The characterization of 23 Lactobacillus strains was performed. The strains were assayed for biogenic amine-forming capacity, hydrogen peroxide production, pectin esterase, cellulase and polygalacturonase production, growth rate, acidifying capacity and salt tolerance. Three strains were selected which belonged to the species, Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus fermentum. Different starter cultures prepared as combinations of these three strains were assayed in pilot scale fermentations and Randomly Amplified Polymorphic DNA (RAPD) analysis, using a previously selected random primer, was applied for monitoring the inoculated strains. The course of fermentations was similar in all batches but sensorial analysis of eggplants fermented using a mixed culture of the three strains displayed the best results, and no differences were obtained when compared with commercial eggplants. D 2005 Elsevier B.V. All rights reserved. Keywords: Eggplants; Lactobacilli; Starter culture; RAPD; Pickle

1. Introduction The manufacture of fermented vegetables has a long tradition in Spain and there is a wide variety of typical preparations, many of them still being produced using traditional technologies. The Almagro eggplant is a pickle elaborated with a native variety * Corresponding author. Tel.: +34 925 265 716; fax: +34 925 268 840. E-mail address: [email protected] (S. Sesen˜a). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.03.013

of eggplant, Solanum melongena L. var. esculentum depressum, in which a spontaneous fermentation by the indigenous microbial flora occurs. The seasonal nature of this crop is a source of difficulties for manufacturers, and the possibility of freezing these fruits after harvesting (Palop and Ballesteros, 2000), arose expectation in the sector. Sesen˜a et al. (2002) reported that there was no significantly adverse effect on the sensory acceptability of this pickle when blanched frozen fruits were used in its elaboration. However, some changes in the composi-

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tion of the microbial flora and a slight delay in the onset of the active fermentation stage occurred. The use of a selected starter was suggested as a solution to this problem. Sesen˜a et al. (2001) had assayed, using fresh fruits, three experimental starter cultures prepared as mixtures of three randomly chosen isolates obtained from spontaneous fermentations of Almagro eggplants (Sa´nchez et al., 2000). The results of that study showed some organoleptic differences in eggplants fermented with the different starters indicating that the contribution of each of the strains could not have been the same. The monitoring of inoculated strains was therefore considered necessary in order to know which strains were present during fermentation. For this purpose, the utilization of reliable genetic fingerprinting methods, able to distinguish a particular strain specifically and unambiguously (Ramos and Harlander, 1990), is essential, since conventional methods of identification, such as carbohydrate fermentation, have proven to have scarce power of discrimination and are time-consuming and laborintensive. Among the different typing methods reported to be useful for studying the dynamics of mixed cultures or for monitoring inoculated strains during fermentations (Garriga et al., 1996; Quiberoni et al., 1998; Mora et al., 2000; Blaiotta et al., 2001; Cocolin et al., 2001; Marcellino et al., 2001; Brennan et al., 2002), those based on the PCR products analysis, as Randomly Amplified Polymorphic DNA (RAPD), have been reported to be profitable. It has methodological simplicity, requires a short time, provides good levels of discrimination, and can be performed not only on purified DNA (Williams et al., 1990) but also on untreated (Mazurier and Wernars, 1992) or lysed cell without DNA extraction (Moschetti et al., 1998); therefore it is considered the most suitable method when a large number of strains (Vincent et al., 1998; Sa´nchez et al., 2004) must be analyzed. The development of starter cultures for food fermentations follows a multidisciplinary approach and requires not only an ecological study of the man made food ecosystems (Vogel et al., 2002), but also the characterization of useful technological and physiological features of the predominant strains, e.g. salt tolerance, acidifying capacity and biogenic amines production (Daeschel and Fleming, 1984; RuizBarba et al., 1994; Gardiner et al., 1998; Kimaryo et

al., 2000) in order to select those with the highest potential for industrial applications. In this study, considering the characteristics of the fruits and the process of manufacture of fermented eggplants, the properties assayed have been the growth rate, the production of hydrogen peroxide and hydrolytic enzymes, salt tolerance, the acidifying capacity and the production of biogenic amines. The goals of this study are the characterization of the Lactobacillus strains isolated from a spontaneous fermentation for designing a starter culture able to colonize the fermentation process when blanched frozen eggplants are used. The suitability of RAPD analysis for monitoring inoculated strains in fermenting vegetables is also assessed.

2. Materials and methods 2.1. Strains and culture conditions A total of 23 strains of Lactobacillus, including 3 Lactobacillus brevis, 11 Lactobacillus plantarum, 5 Lactobacillus pentosus and 4 Lactobacillus fermentum obtained from brines of spontaneous fermentation of bAlmagroQ eggplants, were studied. Their phenotypic (Sa´nchez et al., 2000) and SDS-PAGE whole cell protein analysis (Sa´nchez et al., 2003) were carried out in our laboratory. All strains were grown in MRS broth (De Man et al., 1960) (Oxoid, England) at 37 8C under aerobic conditions and stored as frozen stocks in MRS broth containing 20% (v/v) glycerol (Panreac, Spain) at 80 8C. They were propagated twice before using. To prepare lyophilized cells, strains were grown in MRS broth for 18–20 h at 37 8C. Cells harvested by centrifugation (16,000g, 15 min at 4 8C) were washed twice with sterile saline solution before lyophilization (Kinetics Flexi-Dry, FTS Systems, New York, USA). 2.2. Characterization of the strains 2.2.1. Biogenic amine-forming capacity Amino acid-decarboxylase activity was assessed according to the method described by Bover-Cid and Holzapfel (1999). 1% (w/v) of each precursor

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amino acid, tyrosine di-sodium salt (Sigma, USA), lhistidine monohydrochloride (Sigma), l-ornithine monohydrochloride (Merck, Germany), and l-lysine (Merck), was added. Positive reactions were recorded when a purple color appeared on the plates or tyrosine precipitates disappeared around the colonies. 2.2.2. Hydrogen peroxide production It is known that several species of lactic acid bacteria produce hydrogen peroxide whose inhibitory effect on undesirable microflora has been reported (Reinheimer and Demkov, 1989; Ouwehand, 1998). Hydrogen peroxide was determined using spectrophotometric technique described by Reinheimer and Demkov (1989) measuring iodine released (I2) at 350 nm. Production was determined in sterile brine with the same composition employed in the fermentation of eggplants (Sesen˜a et al., 2001) to which 2% (w/v) of glucose (Cultimed, Spain) was added. Overnight cultures in MRS broth were inoculated (10%) in duplicate in tubes containing 10 ml of brine and incubated for 9 h at 37 8C. One of these cultures was used as control and after incubation 4000 U/ml of catalase (Sigma) was added. After 30 min incubation at room temperature, absorbance was read and the value was subtracted to that of the corresponding sample. The production was determined both in static and stirred (100 rpm) cultures in triplicate. Concentration was expressed as Ag/ml. 2.2.3. Determination of pectin esterase, cellulase and polygalacturonase activities The softening of fermented Almagro eggplants has been described as a defect by consumers. The presence of pectinolytic microorganisms has been associated with breakdown of tissues in other vegetables (Amoa-Awua and Jakobsen, 1995). Cellulase, pectin esterase and polygalacturonase activities were determined according to Amoa-Awua and Jakobsen (1995) except for a higher temperature of incubation (37 8C) and a longer incubation time (5 days). Pectin esterase activity appeared as precipitated finger-like outgrowths protruding from the colonies when a top agar plate containing pectin (10 g/l) was inoculated. Cellulase and polygalacturonase activities appeared as clearing zones in a red background when plates containing carboximethylcellulose (5 g/l) and polygalacturonic acid (10 g/l) respectively, were stained with

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0.1% Congo red (Scharlau, Barcelona, Spain). These assays were carried out in duplicate. In order to promote the enzyme induction, activation of microbial cultures was carried out by subculturing 5 times in MRS broth containing 0.1% (w/v) of pectin, carboxymethyl cellulose or polygalacturonic acid. All were purchased from Sigma. 2.2.4. Growth rate study Strains were inoculated (10%) in triplicate in MRS broth and incubated in aerobic conditions at 37 8C. Optical density (OD) at 660 nm was measured in a Beckman DU-530 spectrophotometer until the stationary phase. The average value of each measurement was used for the calculation of the specific growth rate (l) (h 1) using the equation l = d(ln OD660) / dt. 2.2.5. Acidifying capacity Strains were grown in the same conditions described above. pH was measured (Crison pH-meter; Crison, Barcelona, Spain) immediately after inoculation and after 24 h incubation and the acidifying capacity was expressed as DpH (Gatti et al., 1999). It was determined in triplicate. 2.2.6. Salt tolerance This was determined following the procedure described by Sa´nchez et al. (2000). Concentrations of NaCl assayed were 40 and 65 g/l, frequently used in industrial fermentations, and 80 g/l described by Montan˜o et al. (1992) as the upper limit to allow the growth of lactic acid bacteria without an excessive delay. 2.3. Eggplants processing Recently harvested eggplants (S. melongena L. var. esculentum depressum) with weights between 34 and 44 g were conditioned and processed according to Ballesteros et al. (1999). After blanching, fruits were frozen (Sesen˜a et al., 2002) and stored for 3 months. Batches were placed in rectangular, plastic containers (30  22  15 cm) with a capacity of 8 l, covered with a false lid with small holes. They were filled with 4 l of brine prepared as reported by Sesen˜a et al. (2001) and allowed to ferment at 30 8C F 2 8C for 8 days.

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2.3.1. Starter cultures and experimental design Two days after the eggplants were placed in the brine, the fermenters were inoculated. An appropriate quantity of each of the lyophilized strains was weighed out to ensure that the number of cells in the inoculum was the same in all the fermenters (about 105–106 cfu/ml of brine). This was suspended in 10 ml of brine and left to rehydrate for 30 min before added to the fermenters. After inoculation, the fermenters were stirred manually to distribute the cells. Inoculated sample batches were fermented in duplicate and two un-inoculated batches were used as controls. Fermentation was monitored by chemical and microbiological analysis of the brine. At the end of the 8-day period, 0.03% (w/v) potassium disulphite (Panreac, Spain) was added to the brine to stop fermentation and 24 h later, the eggplants were removed and packed as described by Sesen˜a et al. (2002). 2.4. Microbiological and chemical analysis Samples of brine were taken daily under aseptic conditions using sterile 10-ml plastic test tubes. Lactic acid bacteria (LAB) counts were performed on MRS agar plates after incubation in aerobic conditions at 37 8C for 48 h, and counts were expressed as colony forming units (cfu) per milliliter of brine. Concentrations of d-glucose/d-fructose and lactic acid were determined using enzymatic tests (Boehringer Mannheim, Germany). The pH was measured using a Crison model 2002 pH-meter (Crison, Spain). NaCl concentrations (%) were determined using a Chloride Analyser Sherwood mod. 926 (Sherwood Scientific Ltd. Cambrigde, UK). Average values for all parameters were calculated from samples obtained from duplicated fermenters of every batch. 2.5. RAPD analysis The starter cultures were monitored throughout the fermentations using RAPD analysis. Twenty well-developed colonies were picked up from MRS plates of each day of fermentation and analyzed as described later. The occurrence of each inoculated strain was calculated as the number of colonies with the specific RAPD pattern divided by the total number of colonies

picked up, and expressed as a percentage (Ruiz-Barba et al., 1994). Colonies were lysed following the fast lysis method described by Veyrat et al. (1999) except for a higher mutanolysin (Sigma) concentration (5 U) and a longer incubation time (2 h). The primer of arbitrary sequence OPL-05 (5VACGCAGGCA-3V) obtained from Genotek (Spain), was used. Amplification reactions were performed using a Thermal Cycler 2400 (Perkin Elmer Co., USA). 20 Al of amplification products were analyzed by electrophoresis (70 A for 2 h, without cooling) in 1.5% (w/v) D-1 Low-EEO agarose (Pronadisa, Madrid) in 1 TBE-buffer (45 mM Tris–base, 89 mM boric acid, 2.5 mM EDTA; pH 8.3) gels containing (0.5 Ag/ml) of ethidium bromide and made visible by transillumination at 254 nm. DNA molecular weight marker (100 bp Ladder; Biotools, Spain) was used as a molecular size standard and normalization reference. Photographs of RAPD-PCR gels were obtained with a KODAK DC290 Zoom Digital Camera (Eastman Kodak Company, USA). The similarity of the patterns was expressed by the Pearson product moment correlation coefficient (r), and clustering was performed by the unweighted pair group method using average linkage (UPGMA; Gel Compar, Comparative Analysis of Electrophoresis Patterns, Version 4.0, Applied Maths/Kortrijk, Belgium) (Vauterin and Vauterin, 1992). A reproducibility study to determine the minimum percentage of similarity needed to assign the isolates to the starter strains was carried out as described by Sa´nchez et al. (2004). 2.6. Sensory evaluation A descriptive sensory analysis was carried out 3 months after canning the samples. A panel consisting of 10 trained members evaluated one eggplant from each batch and 2 commercial samples (1 and 2). The sensory attributes analyzed were those put forward by Sesen˜a et al. (2001). Tasters used a 100-mm unstructured scale to rate the perceived intensity. Each sample was tasted twice in two different sessions and the mean score of the panel for each sample was recorded. All tests were performed in a tasting room that fulfilled the requirements of ISO Standard 8589-88 (1988).

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2.7. Statistical analysis Multiple comparison of means (Student–Newman– Keuls test) using a significance level of a V 0.05 was applied to the results of the sensory analysis to determine the significance of differences in the mean values for each of the attributes for the different batches. Statistical analysis was performed using the SPSS program for Windows (version 10.0) (SPSS, 2000).

3. Results 3.1. Characterization of the strains and design of the starter cultures The results of the determination of biogenic amineforming capacity (Table 1) indicated that 7 of the 23 strains studied were able to decarboxylate some of the assayed amino acids, in particular ornithine. For spe-

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cies, it should be noted that while the L. brevis strains were able to decarboxylate some of the amino acids none of the L. plantarum strains had this capacity, with the exception of L. plantarum 4G23, which decarboxylated ornithine. The strains of L. pentosus showed a similar behavior to those of L. plantarum and only a slight change in color in the plates of llysine was observed for the strain 7G36. The decarboxylating capacity of ornithine of L. fermentum and L. brevis isolates was strain-dependent. None of the 23 strains tested produced histamine. All the strains grew at the lower concentrations of NaCl (40 and 65 g/l), although those of L. fermentum showed a weak growth at 65 g/l (results not shown). Furthermore, as reported by Hugas et al. (1993), all the strains of L. plantarum and L. pentosus grew at the concentration of 80 g/l. With respect to the production of hydrogen peroxide, we observed that in all samples, I2 was produced after the colorimetric reaction (results not shown). This led us to believe that oxidizing compounds dif-

Table 1 Results of characterization of isolates from spontaneous fermentation of bAlmagroQ eggplantsa Strain

Identification

010 3G22 08 3G33 7G43 7G45 5G17 7G31 5G22 5G28 7G24 6G310 3G34 4G23 7G38 7G36 5G15 6G48 7G27 2G26 6G14 06 3G45

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

a b c d

brevis brevis brevis plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum pentosus pentosus pentosus pentosus pentosus fermentum fermentum fermentum fermentum

l-lysine

l-histidine

l-tyrosine

l-ornithine

H2O2b

DpHc

l (h

+ +

ND ND ND ND ND ND ND ND ND 9.8 F 0.1 ND ND ND ND ND ND ND ND ND 7.2 F 0.3 83.8 F 0.8 ND 11.9

2.15 F 0.45 2.20 F 0.23 2.09 F 0.32 2.42 F 0.15 2.41 F 0.25 2.35 F 0.31 2.23 F 0.14 2.27 F 0.09 1.89 F 0.21 2.35 F 0.13 2.00 F 0.22 2.26 F 0.14 2.37 F 0.11 2.37 F 0.09 2.25 F 0.08 2.34 F 0.41 2.19 F 0.25 2.30 F 0.33 2.36 F 0.42 1.92 F 0.07 1.81 F 0.15 1.86 F 0.21 1.90 F 0.13

0.53 F 0.02 0.39 F 0.04 0.64 F 0.05 0.50 F 0.10 0.50 F 0.09 0.44 F 0.04 0.48 F 0.03 0.55 F 0.12 0.69 F 0.08 0.53 F 0.04 0.51 F 0.13 0.51 F 0.03 0.55 F 0.07 0.37 F 0.08 0.57 F 0.04 0.69 F 0.11 0.48 F 0.12 0.48 F 0.06 0.41 F 0.08 0.41 F 0.02 0.41 F 0.06 0.67 F 0.04 0.55 F 0.09

+

+ w

+ +

Symbols: ( ), negative; (w), weakly positive; (+) positive; ND = not detected (V 0.5 Ag/ml detection limit of the method). Values obtained using stirred cultures. Acidifying capacity measured as DpH after 24-h incubation in MRS. Specific growth rate l = d(ln OD660) / dt.

1 d

)

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log cfu/ml

8

6

4

2

0 0

1

2

3

4

5

6

7

8

9

Time (days)

Fig. 1. Lactic acid bacterial counts during fermentation. Represented values are the means F standard deviation of the values obtained for the two fermenters for each batch. U = batches I and II; oUo = batches III and IV; DUD = batches V and VI; 5U5 = uninoculated fermenters. The arrow indicates the time of inoculation.

ferent to hydrogen peroxide were produced during incubation, thus affecting the results. It was the reason to introduce controls, as described in Materials and methods. The results of this study (Table 1) showed that the capacity to produce hydrogen peroxide is a property inherent to the strain itself. The amounts produced varied when static (results not shown) or stirred cultures were assayed and in all cases the amounts were higher in the stirred cultures. Three of the four strains of L. fermentum and one of L. plantarum produced hydrogen peroxide (Table 1) which is an interesting property for the control of undesirable microbial flora growth. The strain L. fermentum 6G14 produced the highest amount. The acidifying capacity varied with the strains (Table 1), with decreases in pH ranging from 1.81 to 2.42 units. L. fermentum strains showed a more homogenous behavior, and were the least acidifying strains, which corresponds to their nature as an obligate heterofermentative species. By contrast, the L. plantarum strains, except strain 5G22, were the most acidifying.The values of specific growth rate shown in Table 1 varied from 0.37 h 1 to 0.69 h 1. Beal et al. (1994) reported similar values for L. plantarum species using the same culture conditions. None of the 23 strains showed any pectin esterase, cellulase or polygalacturonase activity. A strain of each species was selected on the basis of its technological properties. Strain L. fermentum

6G14 was selected because it produced a high amount of hydrogen peroxide; the strain L. plantarum 3G33 having the highest acidifying capacity and L. brevis 3G22 was randomly chosen because all L. brevis strains had a similar behavior. The decarboxylating capacity of the ornithine observed in the strain 3G22 was not considered to be a serious impediment since this strain had been isolated from a food and the low protein content of eggplants makes it unlikely that any toxic concentrations of putrescine would be reached (Stratton et al., 1991).

1 2 3 4 5 6 7 bp

bp

1000---900---800---700---600---500---400----

---- 1000 ---- 900 ---- 800 ----700 -----600 -----500 ---- 400

300----

----300

Fig. 2. RAPD-PCR fingerprints for L. plantarum 3G33 (lane 2), L. fermentum 6G14 (lane 3), L. brevis 3G22 (lane 4) and unidentified isolates (lanes 5 and 6). (Lanes 1 and 7) DNA molecular size markers (100 bp Ladder; Biotools).

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We did not include any strain of L. pentosus due to that their behavior was very similar to that of L. plantarum strains. Three different parallel fermentations and an uninoculated control were performed in duplicate. Batches I and II were inoculated with L. plantarum 3G33 because this homofermentative species is the most frequently used in commercial starters for pickles; batches III and IV were inoculated with a mixture of homofermentative and heterofermentative strains, L. plantarum 3G33 and L. fermentum 6G14. Batches V and VI were inoculated with a mixed culture of L.

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plantarum 3G33, L. fermentum 6G14 and L. brevis 3G22. 3.2. Microbial growth in fermentation brines The course of fermentation in all batches as monitored by the chemical parameters pH, concentrations of d-glucose/d-fructose, lactic acid and NaCl (data not shown), was similar to that reported by Ballesteros et al. (1999). The population of LAB (Fig. 1) was built up to high levels (108 cfu/ml) as soon as the fermenters

A 100 80 60

% = (no. of isolates of each strain / total no. of isolates) × 100

40 20

L. plantarum 3G33 Unidentified

0 1

2

2,5

3

4

5

6

7

8

B 100 80 60 40 L. fermentum 6G14 L. plantarum 3G33 Unidentified

20 0 1

2

2,5

3

4

5

6

7

8

C 100 80 60 40

L. brevis 3G22 L. fermentum 6G14 L. plantarum 3G33 Unidentified

20 0 1

2

2,5

3

4

5

6

7

8

Time (days) Fig. 3. Occurrence of the inoculated strains calculated from the RAPD analysis of 10 colonies picked up from MRS plates of each day of fermentation and of each batch. A: batches I and II; B: batches III and IV; C: batches V and VI (The strains used as starter cultures are shown).

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Table 2 Sensory evaluation results Eggplants from batches

I and II III and IV V and VI Commercial 1 Commercial 2

Scores* for the following attributes Wine Spiced Odour Odour Hardness Rubbery Spiced Salty Acid Pungent Aftertaste Aftertaste Overall vinegar odour intensity quality texture flavour taste taste taste intensity quality score odour 2** 2 5 3 4

3 3 6 4 7

4a 3b 6c 4a 6a

3 5 7 5 7

5 4 5 5 5

7 5 6 5 9

5 6 5 3 5

5 5 5 4 6

6 6 5 4 5

3 2 3 2 2

6 6 7 3 7

4 5 5 5 4

53 52 65 47 65

a,b,c

Different indices indicate statistical significant difference at a V 0.05. * Average value of the batches. The positive attributes are wine vinegar odour, spiced odour, odour intensity, odour quality, hardness, spiced flavour, salty taste, acid taste, pungent taste, aftertaste intensity and aftertaste quality, which are measured on a scale from 10 (very desirable) to 0 (undesirable). Rubbery texture is a negative attribute, which is measured on a scale from 10 (not detectable) to 0 (very pronounced). Higher overall scores indicate higher overall quality. ** No indices means no significant differences at a V 0.05.

were inoculated and about 4 days later when a starter had not been added. These counts remained constant until the end of the fermentation. RAPD patterns of the strains used as starters (Fig. 2) were clearly different from each other, which allowed an easier differentiation. A similarity level (r) of 0.81 was obtained in the reproducibility study and used in the assignment of the isolates to the corresponding strains. Monitoring of starter cultures through fermentations is shown in Fig. 3. In the samples taken before inoculation (days 1 and 2 in Fig. 3) all the isolates showed profiles different from those of the strains of the starters and were designated as unidentified isolates. Some of the patterns corresponding to these isolates were also obtained in the samples taken on other fermentation days. In the days following inoculation, the dominance of these unidentified isolates was especially noteworthy in batches V and VI, although it was never higher than 20%. As expected, when a unique strain was inoculated, an absolute dominance was observed throughout practically all the days of the process (Fig. 3A). When a mixture of cultures of L. plantarum and L. fermentum (Fig. 3B) or of L. plantarum, L. fermentum and L. brevis (Fig. 3C) were used, the presence of all the inoculated strains was confirmed in the samples taken just a few hours after inoculation (day 2,5 in Fig. 3). However, 24 h after inoculation a clear predominance of L. fermentum and the almost total disappearance of L. plantarum were observed in both cases. L.

brevis was present along the whole process (Fig. 3C) but its dominance was much lower than that of L. fermentum. 3.3. Sensorial evaluation Sensorial analysis of eggplants is a difficult task because of the numerous attributes to be evaluated and the differences among them. While some of the attributes should be strong (odour intensity, odour quality, spiced odour or spiced flavour), others should be weakly present (pungent taste or salty taste), and other should be absent (e.g. rubbery texture). Table 2 shows the results of the main evaluated attributes. Eggplants from batches V and VI and the Commercial 2 obtained an identical overall score. The eggplants from these batches reached the highest scores for over half of the attributes evaluated. Those of batches I and II and III and IV very similar to each other and their overall score was higher than the Commercial 1. However, the results of the Student–Newman– Keuls test (Table 2) revealed that there were no significant differences in the attributes assessed, except for the odour intensity.

4. Discussion The results of this study show differences in properties among strains and confirm the necessity reported by others authors (Ramos and Harlander,

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1990; Mora et al., 2000; Bover-Cid et al., 2001) of assessing its potential before selecting a strain as a starter culture for food fermentations. The findings concerning the production of biogenic amines support the limited capability to decarboxylate amino acids of LAB from vegetables reported by Daeschel et al. (1987). A special predisposition to produce biogenic amines has been found in L. brevis strains, in contrast to L. plantarum. The disability of L. plantarum strains to produce biogenic amines makes them safer as starter cultures. Some authors (Beutling, 1996; Bover-Cid et al., 2001) state that the capacity to produce biogenic amines might be strain dependent rather than being related to specific species. However, it seems to be more common among strains of certain species such as L. brevis or L. curvatus than in L. plantarum or L. sakei strains, which are generally described as not producers of biogenic amines (Silla, 1998; Bover-Cid et al., 2001). It should be pointed out that the isolates identified as L. pentosus and L. plantarum showed a very similar behavior in many of the properties tested, as corresponds to closely related species with highly similar phenotypes (Torriani et al., 2001). We have found that RAPD is an adequate method for monitoring inoculated strains in bAlmagroQ eggplants fermentations. However, it must be appointed that well-defined experimental conditions are needed to obtain a highly reproducible amplification of random DNA fragments. A fact observed with certain frequency in the patterns of the isolates obtained from brines of final days of fermentation was the disappearance of some of the bands. Then, the strong and reproducible bands were only considered (Hayford et al., 1999). This simplification could not be applied when monitoring of industrial fermentations is being carried out, as the presence of wild strains of the same species is practically guaranteed. The results of the sensorial analysis indicated that the best starter was that composed of a mixture of the strains L. plantarum 3G33, L. brevis 3G22 and L. fermentum 6G14. The use of this starter would assure the maintenance of the typical organoleptic characteristics of this product even using frozen fruits. The eggplants elaborated with it were identical or of even better quality than some of those currently marketed. Its application in industry should allow the manufacturing of this pickle out of crop, although some

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aspects related to the craftsmanship nature of the manufacturing industries should be solved. The use of starter cultures requires suitable hygienic conditions and technology in the facilities in order to facilitate the colonization of the inoculated cultures (Etchells et al., 1973; Daeschel and Fleming, 1984), which would require additional efforts and cost.

Acknowledgments This work was co-financed by the Direccio´n General de Ensen˜anza Superior e Investigacio´n Cientı´fica (Ministerio de Educacio´n y Cultura) and European Funds for Regional Development (FEDER) (1FD971991). S. Sesen˜a was supported by a grant of the Junta de Comunidades de Castilla-La Mancha (Spain).

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