Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced...

Characterization of lactic acid bacteria strains on the basisof neutral volatile compounds produced in whey

G. Mauriello, L. Moio, G. Moschetti, P. Piombino, F. Addeo and S. CoppolaDipartimento di Scienza degli Alimenti, UniversitaÁ degli Studi di Napoli `Federico II', Naples, Italy

617/11/00: received 1 November 2000, revised 2 February 2001 and accepted 7 February 200111

G. MAURIELLO, L . MOIO, G. MOSCHETTI , P . P IOMBINO, F . ADDEO AND S. COPPOLA. 2001.

Aims: Seventy-eight strains of lactic acid bacteria belonging to ®ve genera and showing six

different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and

Cit (citrate degradation) characters were investigated for their main ¯avouring properties with

the aim to detect variability among and within the groups.

Methods and Results: High resolution gas chromatography±mass spectrometry analysis of

neutral volatile compounds produced in whey showed that, considering both neo-formation

compounds and substances quanti®ed in the whey cultures at different concentrations in

comparison to the extract from sterile whey, the groups of lactococci, enterococci,

thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles

than thermophilic lactobacilli and leuconostocs. Applying principal component analysis

(PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci

showed a broad diversity, while lactococci included rather similar strains as well as strains

with special ¯avouring properties. Applying PCA to thermophilic streptococci and

enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to

mesophilic and thermophilic lactobacilli, the strains gathered consistently with their

systematic position.

Conclusions: The study evidenced strains producing some volatile compounds responsible

for food ¯avouring. Flavouring properties were variable among the systematic groups and in

some cases different within the same bacterial group.

Signi®cance and Impact of the Study: The potential of the ®ndings is discussed with

reference to the development of ¯avouring adjuncts for the dairy industry.

INTRODUCTION

Lactic acid bacteria (LAB) differ considerably in morpho-

logical, physiological and functional properties (Wijtzes

et al. 1997). While lactic acid is the major end-product of

their metabolism, diacetyl, acetoin, 2,3-butanediol, acetate,

ethanol, formate, CO2 and many others are also produced

(Cogan 1995). These substances, important in ¯avour

perception and texture of many fermented foods, are

produced in different ratios according to species and/or

strain, with the consequence that the composition of the

LAB community can greatly affect product quality.

Biotyping of LAB isolates, within studies on microbial

biodiversity occurring in fermented foods, is today widely

performed by molecular as well as biochemical proce-

dures, which generally give poor information about

¯avouring abilities and cannot therefore be considered as

exhaustive in de®ning the technological properties of a

strain.

Flavour molecules can be produced during growth by

biosynthesis (de novo synthesis) and bioconversion (precur-

sor biotransformation). Most volatile ¯avour compounds are

produced during the stationary growth phase as secondary

metabolites (terpenes, esters, ketones, lactones, alcohols and

aldehydes) (Belin et al. 1992).

In this paper, 78 LAB strains were studied to focus upon

signi®cant differences among volatile compounds produced

in standard culture conditions. The possibility of using

Correspondence to: Salvatore Coppola, Dipartimento di Scienza degli Alimenti,

Sezione di Microbiologia, UniversitaÁ di Napoli ¢Federico II', 80055 Portici,

Italy (e-mail: [email protected]).

ã 2001 The Society for Applied Microbiology

Journal of Applied Microbiology 2001, 90, 928±942

https://www.researchgate.net/publication/248484851_Microbial_biosynthesis_the_production_of_food_flavours?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/13733894_A_computerised_system_for_identification_of_lactic_acid_bacteria?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2


¯avouring properties as a marker trait of a single strain or

LAB groups was also investigated.

MATERIALS AND METHODS

Strains and culture conditions

Seventy-eight LAB strains were used in this study

(Table 1). Twenty-four lactococci, 14 thermophilic lactoba-

cilli, 10 enterococci, two leuconostocs and two thermophilic

streptococci were from natural whey cultures (NWC) used

as starter for the traditional manufacture of water buffalo

Mozzarella cheese (Coppola et al. 1988); eight mesophilic

lactobacilli and four leuconostocs were from pizza doughs

(PD) (Coppola et al. 1996); four thermophilic streptococci

were from pasteurized milk (PM). Lactococci, enterococci

and thermophilic streptococci were identi®ed by physio-

logical and biochemical techniques (Schleifer et al. 1991;

Teuber et al. 1991; Devriese and Pot 1995) and by

ampli®cation of the 16S±23S rDNA spacer region (Mosch-

etti et al. 1998). They were differentiated at strain level by

DNA ®ngerprinting (Moschetti et al. 1993) and randomly

ampli®ed polymorphic DNA (Moschetti et al. 1998). Lac-

tobacilli and leuconostocs were identi®ed according to

Hammes et al. (199122 ) and Villani et al. (1997), respectively.

Five type strains of leuconostocs and the lactococcal strains

DSM4644 (lactose/citrate plasmid carrier) and DSM4367

(protease plasmid carrier) were from DSM (Deutsche

Table 1 Strains and relative phenotype with respect to Lac, Prt and Cit characters

Phenotype

Microbial strains Lac Prt Cit

Lactococcus lactis subsp. lactis NWC:60, 109, 119, 125, 148, 167, 169, 170, 174, 175, 176, 186, DSM4644 + + +

Lactococcus lactis subsp. lactis BU2-60 ± ± ±

Lactobacillus plantarum PDM41

Lactobacillus brevis PDM207

Lactobacillus delbrueckii subsp. lactis NWC:400, 408, 413

Lactobacillus delbrueckii subsp. delbrueckii NWC475

Lactobacillus helveticus NWC:18, 412,

Lactobacillus acidophilus NWC417

Lactococcus lactis subsp. lactis NWC:0148, 120, 128, 146, 147, 154, 155, 156, 163 + ± +

Lactobacillus plantarum PD:E5, E7

Lactobacillus paracasei PD:T228, G11

Leuconostoc mesenteroides subsp. mesenteroides DSM20343, NWC543

Leuconostoc mesenteroides subsp. dextranicum DSM20484

Leuconostoc citreum DSM20188, PDA29

Leuconostoc carnosum PDLA17

Leuconostoc lactis DSM20202

Enterococcus faecalis NWC:95, 214, 222, 224

Lactococcus lactis subsp. lactis NWC:61, 126, 177 + ± ±

Enterococcus faecalis NWC:70, 76, 93, 94, 211

Lactobacillus paracasei PDE1

Lactobacillus plantarum PDT34

Lactobacillus delbrueckii subsp. delbrueckii NWC:425, 482

Lactobacillus helveticus NWC485,

Lactobacillus acidophilus NWC:447, 470

Lactobacillus delbrueckii subsp. lactis NWC:83, 455

Leuconostoc mesenteroides subsp. mesenteroides NWC532,

Leuconostoc mesenteroides subsp. dextranicun PDA7

Streptococcus thermophilus77 : PM:L1, LP25, NWC286, NCIMBNCDO822

Lactococcus lactis subsp. cremoris DSM4367 + + ±

Enterococcus faecalis NWC92

Streptococcus thermophilus88 PM:LP30, LP45, CNRZ302, NWC8C2

Leuconostoc mesenteroides subsp. cremoris DSM20200

Leuconostoc citreum PDA21 ± ± +

NWC: natural whey culture; PD: pizza dough; PM: pasteurized milk; DSM: Deutsche Sammlung von Mikroorganismen; NCIMB: National

Collection of Industrial and Marine Bacteria; CNRZ: Centre National de Recherches Zootechniques.

NEUTRAL VOLATILE COMPOUNDS PRODUCED BY LACTIC ACID BACTERIA 929

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 928±942

https://www.researchgate.net/publication/13563202_Random_amplified_polymorphic_DNA_RAPD_and_amplified_ribosomal_DNA_spacer_polymorphism_powerful_methods_to_differentiate_Streptococcus_thermophilus_strains?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/14047140_Characterization_of_strains_of_Leuconostoc_mesenteroides_by_analysis_of_soluble_whole-cell_protein_pattern_DNA_fingerprinting_and_restriction_of_ribosomal_DNA_J_Appl_Microbiol?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/45245649_The_microflora_of_natural_whey_cultures_utilized_as_starter_in_the_manufacture_of_Mozzarella_cheese_from_water-buffalo_milk?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/291902576_Characterization_of_leavened_doughs_for_pizza_in_Naples?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/257237915_Revival_of_the_species_Streptococcus_thermophilus_ex_Orla-Jensen_1919_nom_rev_Syst_Appl_Microbiol_14_386-388?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/275890439_The_genus_Lactococcus?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

930 G. MAURIELLO ET AL .


Sammlung von Mikroorganismen und Zellkulturen GmbH,

Braunschweig, Germany). Moreover, free-plasmid Lacto-coccus lactis subsp. lactis BU2-60 was kindly provided from

Neve et al. (1984), Streptococcus thermophilus NCDO822

and CNRZ302 were from NCIMB (National Collection of

Industrial and Marine Bacteria, Aberdeen, UK) and CNRZ

(Centre National de Recherches Zootechniques, Jouyen-

Josas, France), respectively. Lactobacilli and leuconostocs

were routinely grown in MRS broth (Oxoid Ltd, London,

England); enterococci, streptococci and lactococci in M17

broth (Oxoid).

Whey powder (Lactoserum doux, Unilait International,

Paris, France) was reconstituted with deionized water up to

4% solid content and autoclaved for 5 min at 121°C.

Lac, Prt and Cit phenotype determination

All the strains were tested on Lactic Agar (Elliker et al.1956) containing 0á004% bromocresol purple as pH

indicator, on Transparent Milk Citrate Agar (Brown and

Howe 1922) and on Citrate Agar (Kempler and Mckay

1980) for Lac, Prt and Cit phenotype attribution,

respectively.

Table 2 Volatile compounds isolated in

whey culture extracts of different strains of

lactic acid bacteriaPeak no.

Retention

time (min) Compound Odour*

1 2á391 3-hydroxy-2-butanone buttery

2 2á736 2-methyl-1-butanol fused oil, whiskey

3 3á715 3-methyl butanal malt

4 4á722 furfural sweet, woody, almond,

fragrant, baked bread

5 5á566 ethylbenzene

6 8á724 n.i.

7 8á931 3-hydroxy-methyl butanoate fruity

8 10á714 phenol stable

9 11á945 n.i.

10 12á716 3-hydroxy-4,4-dimethylbutyrolactone nut

11 13á001 2-hydroxy benzaldehyde pungent, phenolic, spicy,

almond, burning taste

12 14á785 n.i.

13 14á963 methyl-2-furoate fruity, mushroom

14 15á054 n.i.

15 15á257 n.i.

16 16á001 n.i.

17 16á449 maltol malt, toasted

18 23á756 n.i.

19 24á115 naphtalene camphoraceous

20 24á959 hydrocarbon



23 27á229 n.i.

24 27á484 n.i.

25 31á228 n.i.

26 34á403 dodecane

27 35á022 2,4-diterbutilphenol

28 35á355 4-ethoxy ethylbenzoate fruity

29 36á246 tetradecane

30 38á601 hexadecane

n.i. not identi®ed.

*Odour described in: Anon. (1998); Arctander (1969).

Fig. 1 Whey extracts chromatograms of representative strains of each

microbial group.(a) Control; (b) Lactococcus lactis subsp. lactis 119; (c)

Enterococcus faecalis 211; (d) Streptococcus thermophilus L1; (e) Leuco-

nostoc mesenteroides subsp. mesenteroides 532; (f) Lactobacillus delbrueckii

subsp. delbrueckii 482; (g) Lactobacillus paracasei T228.IS1: methyl

decanoate as ®rst internal standard; IS2: isopropyl decanoate as second

internal standard

b



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Whey culture preparation

Overnight cultures of each strain were used to prepare 10 ml

of whey subculture; 150 ml of whey were inoculated at 2%

with whey subculture and incubated for 18 h at 30°C and

40°C for mesophilic and thermophilic cultures, respectively.

An uninoculated batch of whey was used as control.

Extraction of neutral volatile compounds

The volatile constituents of whey cultures were isolated as

described by Moio et al. (1993) for milk aqueous distillates.

Whey cultures were centrifuged for 20 min at 9000 rev

min±1 and 100 ml of supernatant ¯uid were maintained

in ice bath and neutralized with 0á2 mol l±1 NaOH until

pH 9á00 to salify free fatty acid. After adding of

0á1032 ppm of methyl decanoate and 0á1044 ppm of

isopropyl decanoate as internal standards, the whey,

containing free neutral volatile compounds and salts of

fatty acids, were extracted three times with freshly distilled

dichloromethane (50 ml) with vigorous agitation (magnet-

ically stirred). The whey/CH2Cl2 emulsion formed during

stirring was separated from the aqueous layer and frozen

at )30°C (Moio and Etievant 1995). The ¯ask was then

allowed to reach room temperature, and the CH2Cl2solution, progressively separated from the remaining whey,

was dried over anhydrous MgSO4 and reduced to 0á5 ml

under a stream of nitrogen.

High resolution gas chromatography (HRGC)

HRGC analysis was carried out with a Hewlett Packard

series II 5890 gas chromatograph, equipped with a split-

splitless injector, a ¯ame ionization detector, and an HP-5

fused silica capillary column (30 m ´ 0á32 mm ID; ®lm

thickness, 1 lm) (Moio et al. 2000). The operating condi-

tions were as follows: the temperature was programmed to

rise from 40°C to 210°C at a rate of 3°C min±1. The injector

and detector temperatures were set at 250°C and 255°C,

respectively. The hydrogen carrier velocity was 37 cm s±1.

Quantitative measurements of each component were ob-

tained from the peak areas of the components relative to the

internal standards, assuming that the extraction ef®ciency

and the GC response were identical for all compounds

separated.

High resolution gas chromatography±massspectrometry (HRGC±MS)

The electron impact mass spectra were obtained with an

HP 5970 quadrupole mass spectrometer coupled with an

HP 5890 gas chromatograph equipped with a DB-5

Table 5 Concentration (ppm) of neutral volatile compounds produced in whey by Streptococcus thermophilus strains

PeakStreptococcus thermophilus strains

no. 286 L1 LP25 LP30 LP45 8C2 CNRZ308 NCDO822

1 0á221 � 0á044 0á301 � 0á051 0á550 � 0á027 0á031 � 0á027 0á398 � 0á075 0á056 � 0á004 0á459 � 0á004 0á321 � 0á019

2 ± 1á661 � 0á280 ± ± 0á041 � 0á008 ± ± 1á050 � 0á063

3 ± 0á008 � 0á001 0á012 � 0á0 0á007 � 0á0 0á011 � 0á002 0á010 � 0á0 0á011 � 0á0 0á006 � 0á05 0á029 � 0á006 0á037 � 0á006 0á036 � 0á002 ± 0á040 � 0á008 ± 0á028 � 0á0 0á039 � 0á002

7 ± 0á004 � 0á0 0á008 � 0á0 0á004 � 0á0 0á007 � 0á0 0á009 � 0á0 0á007 � 0á0 0á007 � 0á09 0á006 � 0á0 0á006 � 0á0 0á006 � 0á0 0á006 � 0á0 0á006 � 0á0 0á006 � 0á001 0á005 � 0á0 0á005 � 0á0



14 0á010 � 0á001 0á005 � 0á0 0á006 � 0á0 0á005 � 0á0 0á006 � 0á002 0á010 � 0á0 0á005 � 0á0 0á004 � 0á015 ± 0á007 � 0á0 0á011 � 0á0 0á010 � 0á0 0á010 � 0á0 0á012 � 0á0 0á011 � 0á0 0á010 � 0á016 ± 0á005 � 0á0 0á004 � 0á0 0á003 � 0á0 0á004 � 0á0 0á007 � 0á0 0á006 � 0á0 0á004 � 0á018 0á041 � 0á008 0á033 � 0á006 0á024 � 0á001 0á03 � 0á003 0á034 � 0á006 0á029 � 0á002 0á024 � 0á0 0á037 � 0á002


21 ± 0á003 � 0á0 0á004 � 0á0 0á004 � 0á0 0á004 � 0á0 0á004 � 0á0 0á004 � 0á001 0á004 � 0á022 ± 0á005 � 0á0 0á007 � 0á002 0á006 � 0á0 0á006 � 0á001 0á005 � 0á0 0á006 � 0á0 0á005 � 0á023 0á002 � 0á0 0á004 � 0á0 0á008 � 0á0 0á005 � 0á0 0á006 � 0á0 0á006 � 0á0 0á006 � 0á0 0á006 � 0á026 0á004 � 0á0 0á012 � 0á0 0á013 � 0á0 0á013 � 0á0 0á014 � 0á0 0á013 � 0á003 0á012 � 0á002 0á014 � 0á002

27 ± ± 0á002 � 0á0 ± 0á002 � 0á0 0á001 � 0á0 0á002 � 0á0 0á002 � 0á028 0á003 � 0á0 0á007 � 0á0 0á010 � 0á0 0á001 � 0á0 0á011 � 0á0 0á010 � 0á001 0á010 � 0á0 0á010 � 0á001

29 ± 0á006 � 0á0 0á008 � 0á0 0á007 � 0á0 0á009 � 0á001 0á008 � 0á0 0á008 � 0á0 0á008 � 0á0

Values are means of three experiments � standard deviation; ±: absence of peak.



https://www.researchgate.net/publication/12476980_Odour-impact_compounds_of_Gorgonzola_cheese?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

https://www.researchgate.net/publication/284319935_Ethyl_anthranilate_ethyl_cinnamate_23-dihydrocinnamate_and_methyl_anthranilate_Four_important_odorants_identified_in_Pinot_noir_wines_of_Burgundy?el=1_x_8&enrichId=rgreq-39f1b8eb721c36cf91adec11f628af5a-XXX&enrichSource=Y292ZXJQYWdlOzExOTI4MzYyO0FTOjE3MTMyMTcxOTc5NTcxMkAxNDE3ODU3Njg5Njc2

capillary column (30 m ´ 0á32 mm ID; ®lm thickness,

1 lm) connected directly to the ion source of the mass-

spectrometer according to the operating procedure des-

cribed for HRGC. The operating HRGC conditions were

the same as those described for HRGC. The mass spectra

were determined at 70 eV while the ion source and the

interface were held at 150°C and 280°C, respectively (Moio

et al. 2000). Acquisition and processing of mass spectra

were carried out by means of a computer and the

compounds were identi®ed with the aid of mass spectral

data bases (33 Nover de Brauw et al. 1987).

Statistical analysis

Statistical treatment of data, by cluster analysis (CA) and

principal component analysis (PCA), was performed using

Systat software for Macintosh ver. 5.2.1. PCA was per-

formed using Principal Components procedure of Factor

function on data matrix, with two PCs and without

rotational factor. CA was carried out by the Pearson

Correlation with the Corr procedure and then relationships

were established using Single Linkage Method by the

Cluster procedure.

RESULTS AND DISCUSSION

Lac, Prt and Cit phenotype

Glycolysis, proteolysis and citrate degradation are consid-

ered the principal metabolic pathways of LAB involved in

the formation of aroma and ¯avour compounds in

fermented products (Law 1981). On the basis of lactose

fermentation, degradation of caseins and citrate meta-

bolism, the strains were grouped into six phenotypes

(Table 1). Only lactococcal strains (about half of those

tested) showed the Lac+Prt+Cit+ phenotype. The

Lac±Prt±Cit± phenotype was shown by the plasmid-free

strain BU2-60, as expected, and by nine strains belonging

to species of Lactobacillus. As already reported (Cogan

1995), citrate is not metabolized by thermophilic cultures,

so in this study thermophilic lactobacilli and streptococci

showed the Cit± phenotype.

HRGC±MS analysis

On comparing volatile components detected in whey culture

extracts qualitative and quantitative differences could be

evidenced. All microbial groups produced new compounds

and the gas-chromatogram complexity is higher than the

control. Furfural (peak 4), 2-hydroxy benzaldehyde (peak

11) and maltol (peak 17) were already present in the control

chromatogram as shown in Fig. 1 where results are reported

from representative strains.

Tab

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The identity and aroma description of all volatile com-

pounds detected are given in Table 2. Tables 3±8 show, for

each microbial group, the concentration of neutral volatile

compounds isolated from each whey culture, undetected in

the control and/or quanti®ed at different concentration

levels in comparison to the control. The quantity of each

component was calculated with respect to the internal

standards, assuming that the extraction ef®ciency and the

GC response were identical for all compounds separated.

Considering all the substances referable to the strains of

each group, lactococci, enterococci, thermophilic strepto-

cocci and mesophilic lactobacilli showed 12, 20, 20 and 20

compounds, respectively. Thermophilic lactobacilli and

leuconostocs produced a small number of new substances

(eight and six volatile compounds, respectively). Only two

compounds, 3-hydroxy-2-butanone (acetoin, peak 1) and

the unidenti®ed peak 18 were found as produced by all

bacterial groups. The ®rst, together with diacetyl, plays an

important role in the aroma of dairy products and its level

is strain-dependent. Only six strains produced more than

1 ppm of 3-hydroxy-2-butanone. A high level (12á75 ppm)

was produced by the strain Lactococcus lactis subsp. lactis4644 according to its plasmid content. The strain Lacto-bacillus paracasei T228 produced 2á74 ppm of 3-hydroxy-

2-butanone, while lactococcal strains 154, 155 and 156

produced 4á61, 4á81 and 4á24 ppm, respectively. Finally,

the leuconostocs produced low concentrations of acetoin

probably due to the low acidity reached in the whey

cultures of these microorganisms. The citrate transport

system is known to be induced by natural acidi®cation of

the medium during growth and by a shift to media

buffered at acidic pHs (Garcia-Quintans et al. 1998).

Therefore low acid-producing microorganisms are respon-

sible for low acetoin production. However, it is well known

that leuconostocs, cocultured with acidifying microrgan-

isms, are able to produce high quantities of acetoin during

their metabolism (Levata-Jovanovic and Sandine 1996a,

1997)44 . Acetoin contributes, together with diacetyl, to the

butter note (Cogan 1995). Due to the procedure carried

out, we were unable to evaluate the diacetyl level since it

was coeluted with dichloromethane used as extraction

solvent.

Naphthalene (peak 19) and the unidenti®ed compound

relative to peak 24 seem to be produced only by lactococci.

Another unidenti®ed compound (peak 25) was found only in

leuconostoc whey cultures.

Thermophilic streptococci and leuconostocs produced

2-methyl-1-butanol (peak 2) and phenol (peak 8), respect-

ively, while lactococcal strains produced both substances.

2-Methyl-1-butanol generally arises from deamination

and subsequent decarboxylation of isoleucine according to

Ehrlich's pathway (Anon. 1995). The isoleucine is probably

already present in the whey since there is no apparent

correlation between 2-methyl-1-butanol production and

Table 8 Concentration (ppm) of neutral volatile compounds produced by mesophilic lactobacilli strains

PeakMesophilic lactobacilli strains

no. G11 E1 E5 E7 M41 M207 T34 T228


3 0á007 � 0á0 0á007 � 0á0 0á005 � 0á0 0á007 � 0á0 0á006 � 0á0 0á005 � 0á0 0á006 � 0á0 0á005 � 0á05 0á023 � 0á003 0á028 � 0á003 0á025 � 0á004 0á024 � 0á001 0á024 � 0á0 ± 0á021 � 0á001 0á031 � 0á002


9 0á006 � 0á0 0á007 � 0á001 0á006 � 0á001 0á006 � 0á0 0á007 � 0á0 0á005 � 0á0 0á006 � 0á0 0á006 � 0á010 ± 0á007 � 0á0 ± ± ± ± ± ±





16 0á009 � 0á0 0á009 � 0á0 0á009 � 0á0 0á008 � 0á0 0á006 � 0á0 0á003 � 0á0 0á009 � 0á0 0á008 � 0á018 0á035 � 0á005 0á030 � 0á004 0á041 � 0á007 0á034 � 0á0 0á035 � 0á001 0á027 � 0á003 0á030 � 0á001 0á032 � 0á003


21 0á006 � 0á0 0á006 � 0á0 0á006 � 0á001 0á005 � 0á0 0á006 � 0á001 0á006 � 0á0 0á006 � 0á001 0á005 � 0á022 0á007 � 0á001 0á008 � 0á0 0á008 � 0á001 0á006 � 0á0 0á008 � 0á0 0á007 � 0á0 0á008 � 0á002 0á006 � 0á023 0á012 � 0á001 0á009 � 0á0 0á009 � 0á0 0á009 � 0á0 0á009 � 0á001 0á008 � 0á0 0á010 � 0á0 0á009 � 0á026 0á017 � 0á002 0á016 � 0á0 0á015 � 0á002 0á015 � 0á003 0á015 � 0á001 0á015 � 0á001 0á016 � 0á003 0á015 � 0á027 0á003 � 0á0 0á003 � 0á0 0á003 � 0á0 0á003 � 0á0 0á004 � 0á001 0á003 � 0á0 0á003 � 0á0 0á004 � 0á028 0á013 � 0á002 0á012 � 0á002 0á012 � 0á001 0á011 � 0á0 0á013 � 0á002 0á012 � 0á002 0á012 � 0á001 0á011 � 0á029 0á011 � 0á0 0á009 � 0á0 0á010 � 0á001 0á009 � 0á0 0á010 � 0á0 0á009 � 0á0 0á010 � 0á0 0á009 � 0á0

Values are means of three experiments � standard deviation; ±: absence of peak.



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Prt+ phenotype. Phenol, often responsible for important off-

¯avours due to its unpleasant stable odour, is produced only

by few lactococcal strains. Only whey cultures of thermo-

philic lactobacilli and of the mesophilic strain Lactobacillusparacasei E1 contain 3-hydroxy-4,4-dimethyl-butyrolactone

(pantolacton), a compound characterized by a typical nut

odour.

3-Hydroxy-methyl butanoate (peak 7), methyl-2-furoate

(peak 13), several hydrocarbons (peaks 20, 21 and 22),

dodecane (peak 26), 4-ethoxy-ethyl benzoate (peak 28),

tetradecane (peak 29) and ®ve unidenti®ed compounds

(peaks 6, 12, 14, 16 and 23) were produced by lactobacilli,

both mesophilic and thermophilic, as well as by enterococci.

Among these, 3-hydroxy-methyl butanoate, methyl-2-furo-

ate and 4-ethoxy-ethyl benzoate are odour-active com-

pounds and responsible for a fruit-like aroma (Arctander

1969).

Lactococci, enterococci, thermophilic streptococci and

mesophilic lactobacilli produced 3-methyl butanale, an

odourant belonging to the aldehyde class. This aldehyde

Fig. 2 Principal component analysis of neutral volatile compounds produced in whey from lactococci (a), enterococci (b), thermophilic streptococci

(c) and mesophilic lactobacilli (d)



was identi®ed in water buffalo Mozzarella cheese as

responsible for a typical odour of malt (Moio et al. 1993).

Finally, the concentration of furfural (peak 4), an

odourant characterized by a typical baker bread aroma and

arising from carbohydrate dehydration and cyclization

(Anon. 1995), seems to change only as a consequence of

the growth of the LA17 leuconostoc strain. However, the

presence of this odourant is unlikely to be linked to

microbial metabolism but it develops during whey powder

preparation.

Principal component analysis (PCA)

PCA was performed by comparing strains of single

microbial groups (Fig. 2) and strains of different microbial

groups (Fig. 3). As shown in Fig. 2, only for lactococci,

Fig. 3 Principal component analysis of neutral volatile compounds produced in whey from strains of different microbial groups. (a) s, Thermophilic

streptococci; d, enterococci; (b) s, lactococci; d, enterococci; (c) s, lactococci; d, thermophilic streptococci; (d) s, thermophilic lactobacilli; d,

mesophilic lactobacilli



enterococci, thermophilic streptococci and mesophilic lac-

tobacilli was it possible to obtain high percentages of total

variance on two components. In fact, leuconostocs and

thermophilic lactobacilli accounted for 40% and 44% of

total variance, respectively (data not shown), probably due

to the small number of substances produced by these

microorganisms.

The score plot of volatile compounds produced by

lactococci is shown in Fig. 2a; the total variance accounted

for was 62á6% with low discrimination among the strains.

Strains 109, 119, 120, 125 and 146 were well separated in

the right half of plane. In particular, strains 109, 119 and

125, placed in the lower part, produced the higher number

of volatile compounds. Strains 120 and 146, placed in the

upper part, showed a similar GC-pattern, but the latter

produced high quantities of 3-hydroxy-methyl butanoate

(lacking the GC-pattern of strain 120) and 2-methyl-

1-butanol. The citrate plasmid bearing strain DSM4644 was

well separated in the left lower part, where a single group

containing 14 lactococcal strains was also placed. However,

in this part all high-level acetoin-producing strains

(DSM4644, 148, 154, 155, 156 and 0148) were found. In

the left upper part seven strains were found, among which

the protease plasmid carrying strain DSM4367.

Enterococci, as well as mesophilic lactobacilli, showed

similar GC-patterns but the quantitative analysis allowed

reliable discrimination among the strains (Fig. 2b, d).

Thermophilic streptococci (Fig. 2c) were well separated

into eight single-strain groups according to their different

GC-patterns.

PCA results comparing strains of different groups are

reported in Fig. 3. In all the cases considered, there was

good separation between the groups, allowing each strain to

be properly ascribed to the appropriate group.

Cluster analysis (CA)

Results of HRGC were used for hierarchical cluster analysis.

As well as for PCA of the substances produced by strains

of single groups, in this analysis leuconostocs were not

included. For strain clustering purposes, concentrations

of peak ³ 0á01 ppm were coded as 1, concentrations

< 0á01 ppm were coded as 0.

Figure 4 depicts the dendrogram showing the relation-

ships among lactococci, enterococci, thermophilic strepto-

cocci, thermophilic and mesophilic lactobacilli. It was

possible to differentiate almost all the strains tested,

except for a few strains which clustered at the 100%

similarity level (data not shown). At the 71% similarity

level 15 clusters were differentiated: four main multistrain

clusters and 11 single-strain clusters. Two of the main

clusters included 23 lactococcal strains (three in cluster A

and 20 in cluster B). Cluster C included all the

enterococci, all the mesophilic lactobacilli and seven of

the eight studied thermophilic streptococci. However, in

this cluster we can discriminate a subcluster (cluster C1)

at 71% similarity level including six thermophilic strep-

tococci and a subcluster (cluster C2) at 90% similarity

level including enterococci, mesophilic lactobacilli and the

LP30 Streptococcus thermophilus strain. Cluster D included

eight strains of thermophilic lactobacilli; 12 of the 14

studied strains of this group clustered at about 50%

similarity level.

CONCLUSION

By studying Lac, Prt and Cit phenotype characterization of

78 strains of LAB, partially expected information is

achieved about their metabolic properties, lacking direct

evidence for ability to produce ¯avouring compounds.

However, the occurrence in the same habitat of strains

belonging to the same species with different phenotypes

could be observed.

By identifying the neutral volatile compounds occurring

in the whey cultures by HRGC±MS analysis, the

¯avouring potential of each strain could be de®ned,

evidencing that thermophilic streptococci, enterococci and

mesophilic lactobacilli produce a higher number of sub-

stances than the other LAB groups. Quantitative analysis

of results performed by PCA revealed that enterococci,

mesophilic lactobacilli and thermophilic streptococci are

characterized by a certain diversity, while the group of

lactococci include rather similar strains as well as strains

with special properties. Interestingly, by applying PCA to

groups such as thermophilic streptococci and enterococci

as well as mesophilic and thermophilic lactobacilli, the

strains clustered consistently with their systematic posi-

tion.

The production of neutral volatile compounds in

standard cultural conditions has therefore good potential

for characterizing LAB, allowing also preliminary screen-

ing of strains on the basis of ¯avouring capability.

However, the development of cultures to employ as

¯avouring adjuncts, undoubtedly requires experiments and

analyses at the dairy scale with reference to speci®c target

products.

Fig. 4 Dendrogram showing relationships among lactococci, entero-

cocci, thermophilic streptococci, thermophilic and mesophilic lacto-

bacilli on the basis of quantitative analysis of neutral volatile

compounds produced in whey. NWC: natural whey culture; PD: pizza

dough; PM: pasteurized milk; DSM: Deutsche Sammlung von

Mikroorganismen; NCIMB: National Collection of Industrial

and Marine Bacteria; CNRZ: Centre National de Recherches

Zootechniques

b



ACKNOWLEDGEMENTS

This work was supported by a grant of MURST, Rome.

Authors thank Stefania Pesacane for HRGC analysis.

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