influence of sodium chloride concentration on the controlled lactic acid fermentation of...

International Journal of Food Microbiology 53 (1999) 13–20www.elsevier.nl / locate / ijfoodmicro

Influence of sodium chloride concentration on the controlled lacticacid fermentation of ‘‘Almagro’’ eggplants

* ´Cristina Ballesteros, Llanos Palop , Isabel Sanchez

´ ´ ´ ´Departamento de Quımica Analıtica y Tecnologıa de Alimentos, Facultad de Quımicas, Universidad de Castilla-La Mancha,´Avda. Camilo Jose Cela s /n, 13071 Ciudad Real, Spain

Received 8 December 1998; received in revised form 2 August 1999; accepted 5 August 1999

Abstract

The effect of a commercial Lactobacillus starter and sodium chloride concentration on the fermentation of ‘‘Almagro’’eggplants (Solanum melongena L. var. esculentum depressum) was studied. The results of fermentation using added starterand varying salt concentrations (4, 6, and 10% w/v) in brine were compared with the results of spontaneous fermentationtaking place in brine with a salt concentration of 4%. Fresh fruits, medium in size (34–44 g), were used in all cases; all fruitswere blanched under identical conditions. Temperature in the fermenters was 32628C. The results obtained indicate thataddition of a suitable starter shortened the fermentation process, provided the salt concentration in the brine did not exceed6%. In the conditions tested, the eggplants obtained after fermentation were found to be of good quality though somewhatbitter which may explained by the starter employed. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Eggplant; Lactic acid fermentation; NaCl; pickled vegetables

1. Introduction many different factors, some of which are hard tocontrol. The microbial load of fresh fruits can be

The conventional method of fermenting vegetables highly variable and unquestionably exerts a decisiveused industrially is natural fermentation brought influence on the proper course of fermentation.about by the indigenous microbial flora of the Ambient temperature at harvest time during theproduct. This has numerous drawbacks that limit growing season, and particularly at season’s end, isprocess yields and affect final product quality, at the another factor exerting a major influence on normalsame time making it difficult to anticipate the course development of the natural flora during fermentation,of fermentation, since the process is dependent on and under unfavourable conditions fermentation may

stop or, in the worst case, even fail to start.To prevent defective, that is, nonlactic fermen-

tations, brines with high salt concentrations are*Corresponding author. Tel.: 1 34-26-295-300; fax: 1 34-26-sometimes used, because salt helps to regulate the295-318.

E-mail address: [email protected] (L. Palop) type and extent of microbial growth while at the

0168-1605/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 99 )00130-0

14 C. Ballesteros et al. / International Journal of Food Microbiology 53 (1999) 13 –20

same time preventing vegetables from turning soft. 2. Materials and methodsHowever, salt concentrations must not exceed certainlimits in order not to inhibit the growth of lactic acid 2.1. Materialsbacteria and slow the process down, which couldgive rise, to softening of the fruits due to the growth Freshly harvested ‘‘Almagro’’ eggplants (Solanumof certain microorganisms, such as moulds, and to melongena L. var. esculentum depressum) were usedavoid the need for subsequent removal of the salt, after conditioning by cutting off the spines and bractswhich could result in leaching and substantial losses so they did not project outwards from the fruits andin vitamins and other nutrients and involve the cutting the stalk. Fruits medium in size (between 34handling of highly polluting saline effluents (Jones, and 44 g) were used.1975). The eggplants that were to undergo spontaneous

Rodrigo et al. (1984a,b, 1985) proposed an alter- fermentation were not washed, whereas the fruitsnative for fermenting cucumbers that avoided certain selected for controlled fermentation were washedof these drawbacks, namely, addition of a suitable with pure water. All the eggplants were blanched. Astarter and anaerobic fermentation conditions. The total of 1.5 kg of eggplants was placed in 5-laddition of a starter previously adapted to the brine containers filled with water at room temperature. Theand fermentation conditions afforded certain advan- water was gradually heated to boiling and thentages, for instance, substantially shortening fermen- allowed to boil for 5 min. Approximately 24 mintation times, allowing the use of brines with lower elapsed from when the eggplants were placed in thesalt concentrations while avoiding the risk of un- containers until the water began to boil.controlled fermentations, and helping to improvequality while at the same time achieving uniform 2.2. Experimental designsensory and nutritional quality attributes in the finalproducts. All fermentations took place in rectangular, plastic

Other researchers, including Etchells et al. (1973) containers (30 3 22 3 15 cm) with a capacity of 8 l,and Fleming (1982), also studied the effect of added open at the top, filled with 4 l of brine. With thestarter on fermentation of cucumbers, while Etchells exception of sodium chloride concentration, theet al. (1966), Christ et al. (1980), Leclaire (1981), composition of the brine was the same for all theGiesschner et al. (1982), Balatsouras et al. (1983), batches: 0.07% (w/v) citric acid (Panreac; Bar-Kandler (1983), and Ruiz-Barba et al. (1994) studied celona, Spain), 7.93% (v/v) vinegar (Consumer 68

˜the effect of starters in the fermentation of sauerkraut acidity; Logrono, Spain), and 0.05% (v/v) Tween 80and olives. (Sigma–Aldrich; Steinheim, Germany). The pH was

The literature has not disclosed any studies dealing 3.2.with the use of starters in the manufacture of A perforated sheet of plastic was fitted to the‘‘Almagro’’ eggplants, a widely consumed pickled containers in the form of a false lid at a level suitableproduct in our country. Indeed, published scientific for holding the fruits down and preventing themstudies dealing with the fermentation of ‘‘Almagro’’ from floating, thereby keeping them submerged ineggplants are extremely scarce in the literature the brine to approximate anaerobic conditions. The(Ballesteros et al., 1998). fruit to brine ration was 1:3 (w/v) in all the sample

The objects of this study were (1) to determine the batches.effect of using a commercial starter composed of The following sample batches were prepared:lactobacilli on the fermentation of ‘‘Almagro’’ egg-plants, comparing the results obtained with those of • Two fermenters containing brine with a low saltspontaneous fermentation, and (2) to determine the content (4% w/v of ground salt), without startermost appropriate sodium chloride concentration in (F1).the brine for the starter employed while at the same • Two fermenters containing brine with that sametime assessing the quality of the fermented eggplants salt content (4% w/v), with starter (F2).by means of instrumental and sensory analysis. • Two fermenters containing brine with 10% (w/v)

C. Ballesteros et al. / International Journal of Food Microbiology 53 (1999) 13 –20 15

of ground salt and starter (F4). That level is the the method described by Masure and Campbellsalt concentration that has customarily been used (1944), employing guaiacol as the substrate. Per-by Rodrigo et al. (1985) for the fermentation of oxidase is the most heat-stable enzyme present incucumbers. plants and vegetables (Scoth, 1975) and was there-

• Two fermenters containing brine with 6% (w/v) fore used to test the effects of the blanching pro-of ground salt and starter (F3). That salt con- cedure employed. The pH was determined using acentration was chosen as intermediate between pH meter Crison model 2002 (Crison; Barcelona,the two concentrations used in the other batches. Spain).

Represented values are the means of the valuesobtained for the two fermenters for each batch.The starter was added 2 days after the fruits were

placed in the brine. The inoculum of the starterculture was prepared by suspending 0.33 g of a

2.3.2. Microbiological analysesstarter culture for vegetables ‘‘Vege-Start 60’’ sup-Microbiological counts in the brine were per-plied by Hansen (Hørsholm, Denmark) in 10 ml of

formed using conventional methods (APHA, 1976).the brine used in each case and stirring for 10 min toLactobacillus counts were performed on Rogosa agarachieve good dispersion of the lyophilized starter.(Merck; Darmstadt, Germany) as described by ManThe cell suspension thus obtained was poured intoet al. (1960) and Enterobacteria counts on (BRBA)the fermenter. According to the manufacturer, theBrilliant Red Bile Agar (Difco; Detroit, MI, USA).starter was composed of a mixture of LactobacillusBRBA plates were incubated at 308C for 72 h, andspp., primarily Lactobacillus plantarum.Rogosa agar at 378C for 48–72 h under anaerobicAll the fermenters were kept in a chamber at aconditions (Gas Pack System, Oxoid; Basingstoke,controlled temperature of 32628C.Hampshire, UK). Decimal dilutions of the samplesFermentation was monitored by routine samplingwere prepared in a sterile sodium citrate (Panreac)of both the fruits and the brine.

˜solution (20 g/ l) as described by Nunez and Mar-´tınez-Moreno (1976).

2.3. Analytical methods Counts were expressed as colony forming units(cfu) per ml of sample.

2.3.1. Chemical analysesSolid samples were homogenized in a blender for

2 min. An amount of ca. 4 g was weighed out, 25 ml 2.4. Instrumental texture analysisof absolute ethanol was added, and the mixture wasstirred for 1 h. The mixture was then filtered, and 10 Eggplant texture was measured using an Instronml of Carrez solution [300 g/ l of ZnSO and 150 g/ l Universal Testing Machine mod. 4301 (Instron; High4

of K Fe(CN) 1:1] were added to the filtrate, after Wycombe, Buckinghamshire, UK). Penetration tests4 6

which the mixture was made up to 50 ml with water. were carried out on ten samples from each fermen-After centrifuging (7000 3 g for 10 min) the super- tation batch. The instrumental parameter measured innatant was decanted and used in the analyses. Brine these tests was ‘‘firmness’’ in Newtons (N). Mea-samples were analysed directly. surement conditions were as follows: cross-head

Reducing sugars were determined by means of the speed, 50 mm/min; force, 1 KN; cylindrical cross-D-glucose / D-fructose enzymatic test (Boehringer head 5 mm in diameter and maximum displacementMannheim; Mannheim, Germany) as described by up to 80% of fruit width. Multiple comparison ofBeutler (1984) and Kunst et al. (1984). D- and means (Student–Newman–Keuls test) with a level ofL-lactic acid in the brine was determined using significance of 0.05 was performed using programenzymatic tests (Boehringer Mannheim) based on the 4D from the BMDP statistical package (Dixon,methods described by Gawenh and Bergmeyer 1988) to determine whether or not the differences in(1974) and Gutmann and Wahlefeld (1974). Per- the firmness measurements for each batch wereoxidase (E.C.1.11.1.7) activity was determined using significant.


2.5. Sensory analysis able between 9 and 11 days of fermentation. Nomajor differences were apparent but the rate of sugar

Samples from all batches, coded with three-digit consumption appeared to be faster in batches pre-random numbers, were presented to an untrained 15 pared with added starter culture.member panel in randomized order. The panelists In the brine (Fig. 2), diffusion of the reducingwere requested to assign an overall score on the basis sugars out of the fruits caused the sugar content toof their evaluations of sample taste and texture. The increase in the early phases of the process. Thelevels of quality were set as follows: 0 to 1, maximum sugar content attained higher levels inunacceptable; 1 to 2, very low grade; 2 to 3, low batches F1 (no starter added) and F4 (starter and agrade; 3 to 4, acceptable; 4 to 5, good; 5 to 6, very high salt concentration used) ((1.7 g/ l) than ingood; and 6 to 7, excellent. The statistical analysis of batches F2 and F3 (starter and low salt concen-sensory evaluation data was effected by analysis of trations used) (0.5–0.7 g/ l). This can be explainedvariance (SPSS; Package version 6.0.1; SPSS, 1994) by the fact that, after the starter had been added toand the Student Newman–Keuls test. the fermenters, diffusion of the sugars continued, but

consumption by microorganisms exceeded diffusion,preventing such high levels from being attained. It is

3. Results and discussion reasonable to think that if not for these conditions(use of starter and low salt concentrations), the sugar

The glucose and fructose measurements for the concentration attained in the brine would have beenfresh and blanched eggplants show that the blanching the same in all cases, since the characteristics of theconditions employed (heating in ambient temperature fresh eggplants used were similar in all cases. Thewater to boiling followed by boiling for 5 min) diffusion rate decreased with increasing sodiumbrought about a 20% decrease from 1.306 to 1.052 g chloride concentration in the brine, as shown by theglucose 1 fructose per 100g in the sugars but were lower slope of the first section of the curve.effective in inactivating the peroxidase and thus in After the peak, sugar concentration fell off rapidly,preventing subsequent browning in the eggplants beginning immediately after addition of the starter in(results not shown). the batches with low salt concentrations in which

Fig. 1 depicts the changes in reducing sugar starter was used (batches F2 and F3) and concludingconcentration in the fruits during fermentation. In all on the third day of fermentation, whereas in batchescases the concentration decreased being nondetect- F1 and F4 the process did not conclude until 2 days

Fig. 1. Changes in reducing sugars in fruits during fermentation. (j) 5 F1; (♦) 5 F2; (m) 5 F3; ( ) 5 F4. F1: noninoculated fermenter with4% (w/v) NaCl. F2, F3 and F4: inoculated fermenters with 4%, 6% and 10% (w/v) NaCl respectively.


Fig. 2. Evolution of reducing sugars in brine during fermentation. (j) 5 F1; (♦) 5 F2; (m) 5 F3; ( ) 5 F4. For explanation of F1–F4 seeFig. 1.

later. In addition, in batch F4 a residual sugar level maintain the sugar concentration. Next, as a conse-of about 0.4 g / l was observed a the end of fermen- quence of selection of the microorganisms in thetation. starter because of the high salt concentration in the

The response in batch F4 between days 2 and 4 of brine, consumption ceased and sugar concentrationthe process was interesting because, although starter increased, because diffusion from inside the fruitswas used in that batch, the behaviour was different continued. On day 4, fermentation of sugarsfrom that of the other batches in which starter was reinitiated, yielding a final amount of lactic acidadded. After addition of the starter, a small amount equivalent to that in the remaining batches.of reducing sugars was consumed, as indicated by As depicted in Fig. 3 production of lactic acidthe presence of a small amount of lactic acid (Fig. began immediately after addition of the starter to the3). The amount consumed was equivalent to that batches with low salt concentrations, corroboratingdiffused during the same period, which acted to the results already described above for the reducing

Fig. 3. Evolution of lactic acid in brine during fermentation. (j) 5 F1; (♦) 5 F2; (m) 5 F3; ( ) 5 F4. For explanation of F1–F4 see Fig. 1.


sugars. In the batch in which no starter was used, lactic acid in the brine, which suggested that fermen-onset of lactic acid production took place 2 days tation of the sugars took place concomitantly in the

˜later. In all batches, it was observed that when sugars fruits and in the brine, as reported by Montano et al.were nondetectable in the brine, the lactic acid (1992) in the fermentation of olives.content still continued to rise slightly. The changes in pH (Fig. 4) were wholly consistent

Rodrigo et al. (1986) reported similar findings in with those already described for the reducing sugarscucumber fermentation and attributed the lactic acid and the lactic acid. Initially the pH underwent anlevels to fermentation of the malic acid present in increase due to exchanges between the fruit and thevegetables in substantial amounts, that acid being the brine, and after addition of the starter it fell to amain organic acid in cucumbers (McFeeters et al., value of around 3.4 where it stayed until the end of1982), or to fermentation of the tartaric acid that is fermentation, even though lactic acid productionalso present, though in smaller amounts. Rodrigo et continued. This finding is again in agreement withal. (1986) also reported that malic acid levels in the results reported by Rodrigo et al. (1986) forcucumbers normally fell to zero after the fourth to controlled fermentation of cucumbers. That pH leveleleventh day of fermentation. The presence of an is considered to be a factor contributing to preserva-inducible enzyme, malolactic enzyme, that is able to tion of the finished product. In the batch in which nobreak malic acid down into lactic acid and CO starter was used, the decrease took place 2 days later.2

(Kunkee, 1967; Shultz and Radler, 1973; Radler, Conversely, in the batch in which added starter was1975) in some species of lactic acid bacteria supports combined with a high salt concentration, though thethat idea. The explanation in our case might be pH began to fall on the day after the starter wassimilar, but this needs to be confirmed by analysing added, the decrease was much less pronounced,the malic acid and tartaric acid contents in the indicating that fermentation was not as active or waseggplants. hindered by the salt concentration used.

The final amount of lactic acid recorded in the Counts of lactic acid bacteria in the brine, in thebrine was around 2g/ l in all the batches. Between 51 batches in which starter was combined with a low

8 9and 69% of the lactic acid present was the D(2) salt concentration rose to high levels (10 –10 cfu /form. ml) immediately after addition of the starter. In the

The trend for lactic acid in the fruits (data not batch in which no starter was used, similar levelsshown) was parallel to that described above for the were attained though it took two more days, and in

Fig. 4. Changes in pH during fermentation in brine. (j) 5 F1; (♦) 5 F2; (m) 5 F3; ( ) 5 F4. For explanation of F1–F4 see Fig. 1.


Christ, C., Lebeault, J.M., Noel, C., 1980. Preparation of Sauer-the batch that used starter with the high salt con-kraut US Patent 4.242.361, Washington, DC.centration, counts did not attain those same high

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H.U. (Ed.), Methoden der Enzymatischen Analyse, Verlagplants in the batch to which no starter was added asChemie, Weinheim, Germany, pp. 1538–1541.‘‘very good’’. Though ratings for the eggplants in the

Giesschner, K., Buckenhuskes, H., Gail, R., Schneider, M., 1982.batches with added starter combined with a low salt ¨Neues verfahren zur herstellung von garfrischen sauerkraut incontent (4 and 6%) also received good ratings, they ¨kleinverpackungen. Ind. Obst.-Gemuseverwet 63 (3), 51–55.were considered somewhat ‘‘bitter’’. The eggplants Gutmann, Y., Wahlefeld, A.W., 1974. L( 1 ) lactat Bestimmung mit

Lactat-Dehydrogenase and NAD. In: Bergmeyer, H.U. (Ed.),in the batch in which added starter was combinedMethoden der Enzymatischen Analyse, Verlag Chemie,with a high salt content were rated as ‘‘very salty’’;Weinheim, Germany, pp. 1510–1514.and bitter flavours were not perceptible, perhaps

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