Articulo Fermentados

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Research Article

Received: 16 November 2012 Revised: 9 April 2013 Accepted article published: 19 April 2013 Published online in Wiley Online Library: 17 June 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6184

Applicability of Lactobacillus plantarum IMDO

788 as a starter culture to control vegetablefermentations

Dorrit Wouters,a Silvia Grosu-Tudor,b Medana Zamfirb and Luc De Vuysta∗

Abstract

BACKGROUND: Fermentation of vegetables and fruits is a traditional preservation technique, e.g. in Eastern Europe. Althoughusually spontaneous fermentation processes are applied, the addition of lactic acid bacteria (LAB) starter cultures couldaccelerate processing and improve the consistency and quality of the end-products.

RESULTS: The application of Lactobacillus plantarum IMDO 788 as a starter culture strain for cauliflower and mixed vegetable

fermentations resulted in accelerated acidification as compared with the spontaneous fermentations. The strain dominatedthe background microbiota throughout the process, whereas the spontaneous fermentations were characterised by widelyvariable species diversity. During the spontaneous fermentations, almost all carbohydrates were converted into lactic acid,ethanol, mannitol and acetic acid, indicating the participation of both heterofermentative and homofermentative LAB species.During the starter culture-added fermentations, residual carbohydrates were found and lactic acid and ethanol were the mainend-metabolites.Vegetable-associatedaromas, ethyl acetate and isoamyl acetate were produced during all fermentations. Thehigh concentration of ethanol and the production of ethyl acetate and isoamyl acetate suggested the involvement of yeastsduring all fermentations.

CONCLUSION: Lactobacillus plantarum IMDO 788was an adequate starter culture strainfor vegetablefermentations, prevailingover endogenous LAB communities. Further optimisation of the starter culture formulation is necessary to avoid yeast growth.c 2013 Society of Chemical Industry

Keywords: vegetable fermentation; lactic acid bacteria; Lactobacillus plantarum; starter cultures

INTRODUCTIONFermented vegetables and fruits have been produced for

thousands of years mainly for preservation purposes, as fresh veg-

etables and fruits are very perishable.1,2 In addition, fermentation

deliversend-products thatare appreciated for their typical sensory

properties.3 Fermented vegetables and fruits of economic impor-

tance include sauerkraut and its Asian variant kimchi, cucumbers

and olives, although many fermentations performed on a small

scale or at household level are attracting interest as well.4 These

small-scale fermentations comprise a wide diversity of vegetables

and fruits such as artichokes,5 caper berries,6 carrots,7– 11

cauliflowers,11–13 cherries,14 eggplants,15 garlic,16,17 ginger,18,19

green beans,7 green tomatoes,11,20 leeks,21 mangoes,19 marrows,7

onions,22–25 peppers,26 pineapples27 and turnips28 and are often

performed in Eastern and Southern European countries.

At present, many vegetable and fruit fermentation processes

still rely on the naturally occurring microbiota of the

fresh raw materials, among which lactic acid bacteria (LAB)

overgrow the undesirable communities of Enterobacteriaceae,

Pseudomonadaceae and yeasts, at least when appropriate

fermentation conditions are set up.3 This involves the addition

of salt to the fresh vegetables and fruits (dry salting or brining),

after which the mixture is protected from light and oxygen

and left to ferment at temperatures between 16 and 35 ◦C.2

However, spontaneous fermentation processes suffer from a

lack of control, often resulting in end-products of inconsistent

quality.29 In thecase of cheese and fermented sausage production,

these problems are avoided by the use of starter cultures.29,30

Starter cultures are microbial preparations of a large number

of cells of one or more microbial strains, which are added to

the raw material to accelerate and control the fermentation

process.31 With regard to fermented vegetables and fruits,

however, several difficulties, e.g. the inability to inactivate the

endogenous microbiota by heattreatmentwithoutcausingdrastic

texture changes, and the desired succession of different micro-

organismsupon fermentation, somewhatimpede the introduction

of starter cultures in these cases.29,32 Nevertheless, an increasing

number of studies concerning starter culture use for fermented

∗ Correspondingto:Luc De Vuyst, ResearchGroup of IndustrialMicrobiologyand

Food Biotechnology (IMDO), Department of Bioengineering Sciences, Faculty

of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2,

B-1050 Brussels, Belgium E-mail: [email protected]

a Research Group of Industrial Microbiology and Food Biotechnology (IMDO),

DepartmentofBioengineeringSciences,FacultyofSciencesandBioengineering

Sciences, VrijeUniversiteitBrussel, Pleinlaan2, B-1050 Brussels, Belgium

b Instituteof Biology Bucharest(IBB),Romanian Academy,SplaiulIndependentei

296, 060031 Bucharest, Romania

J Sci Food Agric 2013; 93: 3352–3361 www.soci.org c 2013 Society of Chemical Industry



Lactobacillus plantarum IMDO 788 as a starter culture for vegetable fermentations www.soci.org

vegetable and fruit production are being published. Furthermore,

the search for appropriate starter cultures is directed towards

functional starter cultures, which are not only able to accelerate

and control the fermentation process but can also contribute to

food safety and/or offer sensory, technological, nutritional and/or

health-associated advantages.31 Goodcandidatefunctionalstarter

cultures are mainly wild-type strains that originate from the

naturalecosystem, as these usually exert more elaborate metabolic

activities in comparison with industrial bulk starters, often from

diversesources.31 This has indeedbeen shownforthefermentation

of carrots, green beans and marrows, forwhich an autochthonous

starter culture performed better with regard to acidification rate,

inhibition of undesired microbial communities, vitamin C content

andsensoryattributesin comparison withan allochthonousstarter

culture consisting of the same species.7 The use of starter cultures

also allows one to reduce the added salt concentration necessary

fore.g. cucumberand sauerkraut fermentations andto reduce the

biogenic amine content of sauerkraut and kimchi.33–39

The frequent appearance of Lactobacillus plantarum as the

prevailing LAB species during vegetable and fruit fermentations

makes this common species a promising candidate for starter

culture trials. In this direction, various Lb. plantarum strains havebeen successfully applied as starter cultures for olive, cucumber

and garlic fermentations.16,36,40–43 Also, Lb. plantarum IMDO 788

has been used as a starter culture for leek fermentations.44

Furthermore, several Lb. plantarum strains possess functional

properties such as thecapacity to degrade oleuropein, to produce

bacteriocins and/or certain flavouring compounds, to tolerate

salt and/or acid environments and to resist gastrointestinal

conditions.41,42,45 All these features make their application as

starter cultures even more interesting.

This study was aimed at the implementation and validation

of Lb. plantarum IMDO 788 as a starter culture for vegetable

fermentations in general. It is a competitive strain isolated from

spontaneously fermented leek that has been tested as a starterculture for fermented leek production.44 The work was performed

through an extensive study of the microbiology and metabolite

kinetics of fermentations with cauliflower and with a mixture of

carrot, cauliflower and green tomato carried out in Romania at

household level.

MATERIALS AND METHODSStarter culture preparation

Lactobacillus plantarum IMDO 788 was used as starter culture

throughout this study. The strain was originally isolated from

a spontaneous leek fermentation process and selected on the

basis of a comparison of 12 Lb. plantarum strains from fermentedleek owing to its fast growth and acidification as well as its

capability to produce diacetyl.21 The strain was stored at −80 ◦C

in de Man/Rogosa/Sharpe (MRS) medium (Oxoid, Basingstoke, UK)

supplemented with 250 g L−1 glycerol (Sigma-Aldrich, Steinheim,

Germany) as cryoprotectant.

For biomass production a monoculture fermentation with Lb.

plantarum IMDO 788 was carried out in MRS mediumat 30 ◦C o n a

200mL scale in glass bottles. After 12 h of fermentation, cells were

harvested through centrifugation (16 000× g, 20min, 4 ◦C) and

washed twice with saline (8.5g L−1 NaCl), each time followed by

centrifugation (16 000×g, 20 min, 4 ◦C). The fresh cell paste was

resuspended in 10 mL of saline, and 5 mL of this cell suspension

was subsequently added to the respective fermentation vessels.

Experimental design

The vegetable fermentations carried out in this study were

performed at household level in Romania. They involved

a spontaneous fermentation with cauliflower alone (further

referred to as the spontaneous cauliflower fermentation) and a

spontaneous fermentation with a mixture of carrot, cauliflower

and green tomato (further referred to as the spontaneous

mixed vegetable fermentation), which served as the control

fermentations. In addition, two fermentations with the same

ingredients as the spontaneous ones (further referred to as the

starter culture-added cauliflower fermentation and the starter

culture-added mixed vegetable fermentation respectively) were

inoculated with Lb. plantarum IMDO 788 as starter culture strain

to obtain a final cell density of ca 6.0 log(colony-forming units

(CFU) g−1 vegetables). The fermentations were performed in 15 L

glass bottles, which were filled with whole (green tomato) or

cut (other vegetables) pieces of freshly harvested vegetables

that were submerged in tap water at room temperature, to

which 35 g L−1 NaCl, 20 g L−1 sucrose and/or starter culture were

added. All fermentations were initiated in the kitchen (ambient

temperature 25 ◦C)and left therefor 3 days, afterwhichthe bottles

were placed in the cellar (ambient temperature 16◦

C) until theend of the fermentations (5 weeks). Brine and vegetable particle

samples were withdrawn at specific time points throughout the

fermentations, namely at days 0, 1, 2, 3, 5, 8, 11, 14, 21, 28 and

35. All samples were immediately analysed for acidification by pH

measurements with an InoLab 720 pH meter (Wissenschaftlich-

Technische Werkstatten GmbH, Weilheim, Germany) and used for

plating andLAB isolation, whilepartsof thesampleswerestoredat

−20 ◦C and transported to Belgium for culture-independent and

metabolite target analyses.

Microbiological analysis

Isolation

For the isolation of LAB, 0.1 mL aliquots of appropriate decimaldilutions of the brine samples prepared in saline were plated on

MRS agar medium (Oxoid). Colonies (10%) from MRS agar were

picked up randomly, purified through plating after growth in MRS

medium, and 1.5 mL of each overnight culture was transferred

to cryovials containing glycerol (250 g L−1) for storage at −80 ◦C.

The isolates were transported on dry ice to Belgium for further

identification.

Bacterial DNA extraction

After having checked their catalase activity, 628 catalase-

negative MRS agar isolates (101, 213, 115 and 199 isolates

from the spontaneous cauliflower, spontaneous mixed vegetable,

starter culture-added cauliflower and starter culture-added mixedvegetablefermentationsrespectively) were grown in MRSmedium

overnight. Then 2 mL of each culture was centrifuged (8000×g,

15min,4 ◦C) and thecell pellets were subjected to DNAextraction

with a Nucleospin 96 tissue kit (Macherey Nagel GmbH, Duren,

Germany) following the manufacturer’s instructions.

For culture-independent analysis, total DNA was extracted

directly from the brine and vegetable particle samples as

described previously.11 Briefly, the actual DNA extraction from

cell pellets was carried out by a combination of enzymatic,

chemical and mechanical lysis of the cells, followed by

phenol/chloroform/isoamyl alcohol (49:49:1 v/v/v) extraction and

purification of the aqueous phase with a Nucleospin food kit

(Macherey Nagel) as described previously.11

J Sci Food Agric 2013; 93: 3352–3361 c 2013 Society of Chemical Industry wileyonlinelibrary.com/jsfa



www.soci.org D Wouters et al.

Culture-dependent identification of bacteria

Classification andidentification of theLAB isolateswere performed

by (GTG)5-PCR fingerprinting of their genomic DNA, numerical

analysis of the fingerprints using BioNumerics 5.1 software

(Applied Maths, Sint-Martens-Latem, Belgium) and verification

of the identity of the (GTG)5-PCR clusters through 16S rRNA gene

sequencing of representative isolates as described previously.11

Culture-independent community dynamics analysis and identification

Culture-independent analysis of the fermentation samples (brine

as well as vegetable particle samples), including community

dynamics and identification, was carried out through denaturing

gradient gel electrophoresis (DGGE) of PCR amplicons targeting

the V3 region of the 16S rRNA gene, using the forward 357f and

reverse 518r universal primer pair.46,47 A GC clamp was attached

to the 5 end of the forward primer. The DGGE conditions (such as

a 35–60% denaturing gradient) and analysis methods, including

DGGE bandsequencing and BLAST analysis of the sequences, were

as described previously.11

Metabolite target analysis

Carbohydrate consumption and end-metabolite production

Metabolite target analysis was performed on the brine samples

of the fermentations. Therefore cells and vegetable particles were

removed from the brines by a 15 min centrifugation at 8000×g

(4 ◦C). The resulting cell-free brine supernatants were analysed by

various chromatography techniques.

High-performance liquid chromatography was applied for the

determination of the concentrations of lactic acid, acetic acid and

ethanol as described previously.11 Calibration was performed with

external standards. Samples were analysed in triplicate and the

results arerepresentedas theaverages of these three independent

measurements. The errors on the measurements are represented

as standard deviations.High-performance anion exchange chromatography with

pulsed amperometric detection was used for the determination of

glucose, fructose, sucrose, glycerol and mannitol concentrations

as described previously.11 Quantifications were performed with

standard addition; the original concentrations in the cell-free

brine supernatants with corresponding errors were calculated as

described previously.48

Volatile aroma production

Static headspace gas chromatography coupled with mass

spectrometry was used for the determination of the evolution of

different volatile aroma compounds throughout the time course

of thefermentations. Forthis, 5 g of cell-freebrine supernatant wastransferred into a headspace vial, to which 1 g of NaCl was added

to enhance volatility.49 As an internal standard, a known amount

of 2-butanol (1mL L−1, Sigma-Aldrich) was added to each sample.

Analyses were performed on an Agilent 6890 gas chromatograph

(Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent

5973 N mass spectrometer (Agilent Technologies) equipped with

an MPS2 Gerstel autosampler (Gerstel GmbH & Co. KG, Mulheim-

an-der-Ruhr, Germany). The capillary column was a DB-WAXetr

column (Agilent Technologies) with a length of 30 m, an internal

diameter of 0.25 mm and a film thickness of 0.5µm. Helium was

used as carrier gas at a flow rate of 1 mLmin−1. Samples were

equilibrated by agitation at 60 ◦C for 30 min prior to injection.

The injection port was in split mode (40:1 split ratio) and the

needle temperature was 150 ◦C. The injection volume was 1 mL

at a rate of 500 µL s−1. After optimisation, the oven temperature

programme consisted of an initial step at 40 ◦C for 5 min, followed

by a linear increase from 40 to 120 ◦C at 20 ◦Cmin−1 and a linear

increasefrom120to225 ◦Cat10 ◦Cmin−1.Finally,thetemperature

was kept constant at 225 ◦C for 5 min. The temperature of the

transfer tube was held at 280◦C. Detection was done with

an MS detector (ionisation energy 70 eV, 4.1 scans s−1, source

temperature 230 ◦C, scan range m / z 29–200). Identification of the

detectedcomponents was achieved by comparison with standard

compounds that were injected separately and with library data

(NIST 08 database, http://www.nist.gov).

Samples of each time point were analysed in triplicate and the

concentrations of volatile components of interestoriginating from

plant, bacterial or yeast metabolism are represented as arbitrary

units (AU) in relation to the internal standard (2-butanol). The

displayed values represent the means of the three independent

measurements and the respective standard deviations.

RESULTS

Acidification patterns The application of Lb. plantarum IMDO 788 as a starter

culture resulted in accelerated acidification of the vegetables

in comparison with the spontaneous fermentations (Table 1). The

pH values of the starter culture-added cauliflower and mixed

vegetable fermentations dropped within 1 day. Afterwards, only

slight decreases in the pH values occurred. In the spontaneous

fermentations, themajorpH drop occurred during thefirst 2 days,

after which the pH values decreased slightly.

LAB species diversity

The LAB species diversity was less elaborate during the starter

culture-added fermentations than during the spontaneous

fermentations. The starter culture-added fermentations weredominated by Lb. plantarum throughout the entire fermentation

processes. The (GTG)5-PCR fingerprints of the Lb. plantarum

isolates, except for those of some isolates appearing at the

end of the starter culture-added mixed vegetable fermentations,

concurred with the fingerprint of the applied starter culture,

Table 1. Acidification patterns in brine of vegetable fermentationsstudied

pH value

Time

(days)

Spontaneous

cauliflower

Starter

culture-added

cauliflower

Spontaneous

mixed

vegetable

Starter

culture-added

mixed vegetable

0 6.43 6.36 6.58 6.54

1 4.48 3.65 5.76 3.87

2 3.75 3.55 3.87 3.55

3 3.74 3.45 3.49 3.46

5 3.78 3.45 3.50 3.50

8 3.72 3.38 3.42 3.42

11 3.64 3.37 3.36 3.39

14 3.54 3.32 3.29 3.33

21 3.54 3.33 3.23 3.29

28 3.54 3.35 3.16 3.23

35 3.53 3.35 3.16 3.23

wileyonlinelibrary.com/jsfa c 2013 Society of Chemical Industry J Sci FoodAgric 2013; 93: 3352–3361




indicating that the starter culture strain Lb. plantarum IMDO 788

prevailed during these fermentations. In contrast, a succession of

micro-organisms occurredduring the spontaneousfermentations.

Lactobacillus plantarum was the main species found during the

spontaneous cauliflower fermentation (Table 2). Also, Leuconostoc

mesenteroides, Lactobacillus pentosus and, to a lesser extent,

Lactobacillus brevis were important members of the fermentation

microbiota of the spontaneous cauliflower fermentation. At the

start of this fermentation, Lb. plantarum and L. mesenteroides

prevailed (Fig. 1). After 5 days of fermentation, the share of

L. mesenteroides decreased and Lb. pentosus together with Lb.

plantarum took over. After 5 weeks of fermentation, Lb. plantarum

was the prevailing LAB species in the spontaneous cauliflower

fermentation. Except for Lb. pentosus, the same LAB species as

those found during the spontaneous cauliflower fermentation

were present in the spontaneous mixed vegetable fermentation

(Table 2). However, the presence of Lb. plantarum was not

as abundant. The most frequently isolated species during the

spontaneous mixed vegetable fermentation was L. mesenteroides,

followed by Lb. brevis. In addition, Leuconostoc citreum was often

isolated from this fermentation. Together with L. mesenteroides,

it prevailed at the onset of the spontaneous mixed vegetablefermentation (Fig. 1). After 5 days, a shift of the fermentation

microbiota took place, with Lb. brevis and L. mesenteroides as the

prevalentLAB species.Upon furtherfermentation, L.mesenteroides

disappeared and Lb. brevis together with Lb. plantarum took over

the fermentation process.

PCR-DGGE gels of brine and vegetable particle fermentation

samples were similar for all fermentations carried out; an overview

of the PCR-DGGE analyses of the brine samples of the different

fermentations is presented in Fig. 2. Culture-independent analysis

of thestarterculture-added fermentations exposed theubiquitous

presence of Lb. plantarum. Culture-independent analysis of

the spontaneous fermentations revealed the presence of L.

mesenteroides, Lb. brevis and Lb. plantarum, with L. mesenteroides

and Lb. plantarum as the prevailing LAB species throughout

the spontaneous cauliflower fermentation and Lb. plantarum

together with Lb. brevis as the prevailing LAB species from day 8

onwards during the spontaneous mixed vegetable fermentation.

The presence of L. mesenteroides during the spontaneous mixed

vegetable fermentation was restricted to the first 8 days of the

fermentation. During the initial phase of the spontaneous mixed

vegetable fermentation, L. citreum, Weissella soli and Lactococcus

lactis were present as well. The latter two species, however, were

not found during the culture-dependent diversity analysis. Besides

LAB, Pseudomonas species and a Psychrobacter species were

found at the start of the spontaneous mixed vegetable and the

spontaneous and starter culture-added cauliflower fermentations

respectively.

Metabolite kineticsCarbohydrate consumption and end-metabolite production

At thestart of allfermentations, sucrose was thesolecarbohydrate

in the brine samples (Fig. 3). Upon further fermentation, a

clear distinction could be made between the carbohydrate

consumption patterns for the spontaneous and starter culture-

added fermentations. Sucrose was depleted at days 1 and 2 of

the spontaneous mixed vegetable and spontaneous cauliflower

fermentations respectively, whileitwas onlydepletedafter2 weeks

during the starter culture-added mixed vegetable fermentation.

During the starter culture-added cauliflower fermentation, only

part of the sucrose was used. Glucose and fructose were present

in nearly equimolar concentrations (±13mmolL−1)atday1ofthe

spontaneous fermentations, after which fructose was consumed

gradually, and it was depleted after 11 days and 3 weeks of the spontaneous cauliflower and spontaneous mixed vegetable

fermentations respectively. During the spontaneous cauliflower

fermentation, glucose concentrations increased slightly until day

11 of the fermentation, after which it was further consumed to

become depletedin week 3. During the entire spontaneous mixed

vegetable fermentation, the glucose concentrations remained

nearly constant. Glucose and fructose concentrations of the

starter culture-added fermentations were initially low. During

fermentation, they first increased slightly, reaching their maximal

values at day 11, and decreased again upon further fermentation.

Glucose and fructose were depleted after 3 weeks of the starter

culture-added cauliflower fermentation, whereas only glucose

was depleted at the end of the starter culture-added mixedvegetable fermentation. Lactic acid was a major end-metabolite

of carbohydrate metabolism during all fermentations. However,

final lactic acid concentrations of the spontaneous and starter

culture-added cauliflower fermentations were approximately

twice those of the spontaneous and starter culture-added

mixed vegetable fermentations. Ethanol was produced during

all fermentations, reaching divergent final concentrations. Acetic

acid and mannitol were produced during both spontaneous

Table 2. LAB species diversity of vegetable fermentations studied as revealed through (GTG)5-PCR fingerprinting of genomic DNA of 628 isolates. The numbers represent the percentages of the to tal amount of isolates of the particular fermentations

Species

Spontaneous cauliflower

(101 isolates)

Starter culture-added

cauliflower (115 isolates)

Spontaneous mixed

vegetable (213 isolates)

Starter culture-added

mixed vegetable (199 isolates)

Lb. plantarum 52.5 93.9 18.3 96.0

Lb. plantarum fingerprint type (FT) 2 — — 0.9 3.5

L. mesenteroides 23.8 3.5 28.2 0.5

L. mesenteroides/pseudomesenteroides 1.0 — 0.5 —

Lb. pentosus 13.9 3.6 — —

Lb. brevis 7.9 — 23.0 —

Lb. brevis FT2 — — 2.3 —

L. citreum 1.0 — 12.7 —

L. citreum/lactis/garlicum — — 11.3 —

L. citreum/lactis — — 1.4 —

Unidentified — — 1.4 —





100

50

0

100

50

0

100

50

0

100

50

0

0 20 8 10 12 13 13 6 7 7 5# isolates

% %

% %

Time (d) 0 1 2 3 5 8 11 14 28 35

2 22 32 25 2020 24 24 21 12 11# isolates

Time (d)

# isolates

Time (d)

2 13 13 13 15 12 15 11 7 6 8

# isolates 4 26 22 24 21 18 27 17 18 11 11

A

C

B

D

0 1 2 3 5 8 11 14 21 28 3521

0 1 2 3 5 8 11 14 28 35 Time (d) 0 1 2 3 5 8 11 14 21 28 3521

Figure 1. Temporal evolution of different LAB species in (A) spontaneous cauliflower, (B) starter culture-added cauliflower, (C) spontaneous mixedvegetable and (D) starter culture-added mixed vegetable fermentations: , Lactobacillus plantarum (100%); , Leuconostoc mesenteroides (100%); ,Lactobacillus pentosus (100%); , Lactobacillus brevis (100%); , Leuconostoc citreum (100%); , Leuconostoc mesenteroides/pseudomesenteroides; ,Leuconostoc citreum/lactis/garlicum; , Leuconostoc citreum/lactis; , unidentified. The total number of isolates per time point is given below each bar.Different fingerprints of the same species are represented by the same pattern in grey shades.

A B

A

D

B

E

C

4 D

B

E

A

1

C

C

A

D

B

E

D

B

E

A

C 2

3

1

4

C

A

D

B

E

C

1

23

5

6

7

8

8

8

88

8

D

1

D

B

E

A

C

L 0 1 2 3 5 8 11 14 21 28 35 L L 0

0

1

1

2

2

3

3

5

5

8

8

11

11

14

14

21

21

28

28

35

35

L

L0 1 2 3 5 8 11 14 21 28 35L

Figure 2. PCR-DGGE (35–60% denaturing gradient from top to bottom of gels) of brine samples of (A) spontaneous cauliflower, (B) starter culture-added cauliflower, (C) spontaneous mixed vegetable and (D) starter culture-added mixed vegetable fermentations. The closest relatives of thefragments sequenced (% of identical nucleotides compared with sequences retrieved from GenBank database; accession number) were (1) Lactobacillus plantarum (100%; AB362386.1), (2) Leuconostoc mesenteroides (100%; AB723680.1), (3) Lactobacillus brevis (100%; JX003598.1), (4) Psychrobacter sp.(100%; JX005863,1), (5) Leuconostoc citreum (100%; JQ712017.1), (6) Lactococcus lactis (100%; JQ965756.1), (7) Weissella soli (100%; GU470977.1) and (8)Pseudomonas spp. The reference ladders consisted of (A) Lb. plantarum LMG 6907 T , (B) Lactobacillus fermentum LMG 6902 T , (C) L. mesenteroides LMG6987 T , (D) Pediococcus pentosaceus LMG 13561 and (E) L. lactis LMG 6890 T .





A B

C D

Figure 3. Metabolite kinetics of (A) spontaneous cauliflower, (B) starter culture-added cauliflower, (C) spontaneous mixed vegetable and (D) starterculture-added mixed vegetable fermentations. Left axis: concentrations (mmol L−1) of glucose ( ), fructose ( ), sucrose ( ) and glycerol ( ). Right axis:concentrations (mmol L−1) of lactic acid ( ), ethanol ( ), mannitol ( ) and acetic acid ( ).

fermentations. The final acetic acid concentrations were similar,

whereas the final mannitol concentration was higher for

the spontaneous mixed vegetable fermentation than for the

spontaneous cauliflower fermentation. During fermentation, part

of the mannitol was again consumed, resulting in maximal

mannitol concentrations at days 2 and 11 of the spontaneous

cauliflower and spontaneous mixed vegetable fermentationsrespectively. Mannitol and acetic acid were nearly absent

throughout the starter culture-added fermentations. At the start

of all fermentations, glycerol was present in low concentrations.

During fermentation, these glycerol concentrations increased

only slightly. Since vegetable fermentations are complex,

heterogeneous and dynamic ecosystems, it was not possible to

calculate mass balances.

Volatile aroma production

The aroma profile of the vegetable fermentations studied was

composed of a combination of different volatile compounds,

including vegetable-associated sulfur- and non-sulfur-containing

compounds (Fig. 4) and compounds linked to bacterial and yeastmetabolic activities (Fig. 5). Methanethiol, allyl isothiocyanate,

methyl thioacetate and dimethyl disulfide were the sulfur-

containing compounds present in all fermentations studied.

During the first days of the fermentations, methanethiol

concentrations increased, reaching maxima at day 3, after which

these concentrations againdeclined. Similar trends were foundfor

the concentrations of dimethyl disulfide and allyl isothiocyanate,

with the difference that the maximal allyl isothiocyanate

concentrationswereonlyreachedbetweenday5andweek2ofthe

fermentations. The concentrations of methylthioacetate increased

during the late fermentation phaseof the spontaneous and starter

culture-added cauliflower fermentations, whereas during the

starter culture-added mixed vegetable fermentation the methyl

thioacetate concentration increased gradually throughout the

entire fermentation. During the spontaneous mixed vegetable

fermentation, the maximal methyl thioacetate concentration was

reached after 5 days, after which it decreased again. Furthermore,

non-sulfur-containing vegetable-associated aroma compounds

such as eucalyptol and γ -terpinene were continuously present in

all fermentations, and 2-methyl-2-propenenitrile concentrationsincreased during all fermentations. Theimpact of thefermentation

microbiota on the aroma of the fermented vegetables was

reflected in the presence of ethyl acetate and isoamyl acetate

during all fermentations studied. Ethyl acetate concentrations

gradually increased throughout the spontaneous and starter

culture-added cauliflower fermentations and the starter culture-

added mixed vegetable fermentation, whereas isoamyl acetate

was produced in the late stages of these fermentations. During

the spontaneous mixed vegetable fermentation, the increase in

the concentrations of ethyl acetate and isoamyl acetate occurred

in the initial phase of this fermentation. In addition, 2-methyl-

1-propanol (spontaneous cauliflower fermentation and both

starter culture-added fermentations) and traces of 1-propanol

(spontaneous mixed vegetable fermentation and both starter

culture-added fermentations) werefound.Diacetylproduction did

not occur during any of thefermentations studied.Among thefour

fermentations studied, the concentrations of the volatile aroma

compounds of the spontaneous mixed vegetable fermentation

were much lower than those of the other fermentations.

DISCUSSION The present study focused on the implementation and validation

of Lb. plantarum IMDO 788 as a starter culture for different

types of vegetable fermentations. It has been shown before

that Lb. plantarum can be applied as a starter culture for





A B

DC

Figure 4. Evolution of vegetable-associated sulfur- and non-sulfur-containing compounds during (A) spontaneous cauliflower, (B) starter culture-addedcauliflower, (C) spontaneous mixed vegetable and (D) starter culture-added mixed vegetable fermentations. Left axis: concentrations (AU) of methylthioacetate ( ), dimethyl disulfide ( ), 2-methyl-2-propenenitrile ( ) and allyl isothiocyanate ( ). Right axis: concentrations (AU) of methanethiol ( ).

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Figure 5. Evolution of volatile aroma compounds linked to bacterial and yeast metabolic activities during (A) spontaneous cauliflower, (B) starterculture-added cauliflower, (C) spontaneous mixed vegetable and (D) starter culture-added mixed vegetable fermentations. Concentrations (AU) of ethylacetate ( ), isoamyl acetate ( ), 2-methyl-1-propanol ( ) and 1-propanol ( ).

many kinds of vegetable and fruit fermentations.16,36,40–43

Also, leek fermentations can be started successfully with

Lb. plantarum IMDO 788 as a starter culture strain.44 For

its general evaluation, a cauliflower and a mixed vegetable

fermentation were inoculated with this starter culture strain,

and its performance was compared with spontaneous vegetable

fermentations containing the same raw materials. The course

of the latter fermentations was similar to previously performed

spontaneous cauliflower and mixed vegetable fermentations

in Romania, during which LAB species prevailed and major

acidification occurred after 3 days of fermentation, resulting in

final pH values of approximately pH 3.5.11 However, with regard to





the composition of the fermentation microbiota, the spontaneous

cauliflower fermentation of the present study differed from the

previously performed cauliflower fermentation, as Lb. pentosus, L.

mesenteroides and, in particular, Lb. plantarum prevailed during

the different fermentation stages instead of Weissella kimchii and

Lb. brevis. Yet, such a sequential prevalence of L. mesenteroides

and Lb. plantarum has been shown for cauliflower fermentations

performed according to a traditional recipe in Northern Greece

before.13 The spontaneous mixed vegetable fermentation of thepresent study harboured fewer Weissella species compared with

the one performed before.11

The application of the Lb. plantarum IMDO 788 starter culture

strain affected the fermentation courses in different ways. For

example, its addition in high numbers ensured a nearly immediate

prevalence of this strain, which was maintained throughout

the entire starter culture-added fermentation processes. Such

a persistence of the added starter culture strain is one of the

main criteria for its applicability, which has been demonstrated

for the use of Lb. plantarum starter culture strains in the

production of, among others, kimchi and olives before.50,51 As

a result of the fast prevalence of Lb. plantarum IMDO 788 in

the fermentations studied, the pH dropped very rapidly duringday 1 of the fermentations, which is beneficial for the quality of

the end-products, as rapid acidification minimises the influence

of the background microbiota that may contain spoilage and

pathogenic bacteria.7,26,42 Regarding carbohydrate consumption

patterns, one of the main influences of the application of the

Lb. plantarum IMDO 788 starter culture strain was the low

presence of mannitol and acetic acid due to a low abundance

of heterofermentative LAB species. In contrast, residual sucrose

was retrieved for a long time during the starter culture-added

fermentations, even until the end of the starter culture-added

cauliflower fermentation. Residual glucose and fructose were

found in the case of the spontaneous and starter culture-added

mixed vegetable fermentations respectively. The presence of

residual carbohydrates may cause problems with regard to thestability of the end-products, since it may lead to secondary

fermentation by yeasts, as has been shown during carrot

fermentations. 8 Secondary fermentation by yeasts can result in

tissue softening due to pectinolytic activity and in the production

of off-flavours through lipolytic and saccharolytic activities.3,52

Nevertheless, yeasts are not always considered undesirable, as

they can contribute to the generation of a versatile aroma profile,

as, for instance, in the case of fermented olives, artichokes

and caper berries.5,6,53,54 Yet, it remains unclear if yeasts are

desirable in vegetable and fruit fermentations or whether they are

primarily a threat to the stability and quality of the end-products.

Indications of a strong involvement of yeasts in the fermentations

studied were reflected in the ethanol concentrations, which weregenerally much higher than those measured in the case of

Romanianvegetablefermentationsthatwerestudiedpreviously. 11

Aroma analysis revealed this large participation of yeasts in the

fermentation processes as well, as ethyl acetate and isoamyl

acetate were among the main aroma compounds found. Since

such esters may provide fruity notes, their presence may be

desirable, although excessive concentrations lead to off-flavours.

Also, glycerol formation during these fermentations pointed

towards the activity of yeasts. In many other cases, however,

the addition of a starter culture is sufficient for microbial control

of the fermentation process, as has been shown, for instance, for

kimchi fermentations.55 Presumably, the addition of sucrose at

the start of the fermentations is one of the main reasons for the

presence of yeasts, since sucrose is not preferred as an energy

sourceby the added Lb. plantarum starter culture strain. However,

in view of a fast prevalence of LAB, the addition of carbohydrates

occurs occasionally at the start of traditional vegetable and fruit

fermentations, such as in the case of olive, onion and Thai mixed

vegetable and fruit fermentations.19,25,56 For example, for the

fermentation of Bella di Cerignola table olives, the addition of a

starter culture consisting of multiple strains of Lb. plantarum and

of small amounts of glucose assures a correct fermentationcourse,

which includesa decreasein the pHto a safevalue and the control

of yeast growth, whereas the addition of glucose alone stimulates

yeast growth.56 Besides the contribution of yeasts to the aroma of

fermented vegetables, theraw ingredientsand metabolic activities

of LAB influence the final aroma. Sulfur-containing compounds

such as methanethiol and allyl isothiocyanate and the non-sulfur-

containing compound 2-methyl-2-propenenitrile belong to the

breakdown products of glucosinolates, present in all cabbage

types; they are partly responsible for the characteristic aroma of

cabbages.57 Besides through the formation of lactic acid, acetic

acid, mannitol and/or ethanol, the influence of the metabolic

activities of LAB on the aroma of fermented vegetables could be

expressed through the presence of volatile compounds such as1-propanol and methanethiol, of which the former is presumably

produced via lactic acid conversion and the latter via amino acid

conversions.58,59 Overall, no clear influence of the application of

the Lb. plantarum starter culture strain was noticed with regard

to the aroma of the end-products, as no diacetyl production,

characteristic for this strain, was noticed.

To conclude, the applied starter culture strain Lb. plantarum

IMDO 788 wasadequateto dominatethe background LABspecies

of vegetables, thereby ensuring rapid acidification and thus

microbiologically safe end-products.However, yeastgrowthcould

not be suppressed, perhaps because of sucrose addition, which

makes the additionof small amounts of glucose instead of sucrose,

if any, and the introduction of, preferably, a heterofermentative

LAB strain to the starter culture advisable for future experiments,

to limit residual carbohydrate concentrations and to obtain end-

products without yeasts.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the Research

Council fromthe VrijeUniversiteit Brussel, the Romanian Academy

and the Research Foundation Flanders (FWO). DW is the recipient

of a post-graduate grant from the Agency for Innovation by

Science and Technology (IWT) in Flanders. Part of this work was

supported by the post-doctoral research project PD33 of the

Romanian National Research Plan (PNII-RU) and by the project

RO1567-IBB05/2012 of the Institute of Biology Bucharest of theRomanian Academy.

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