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Screening of lactic acid bacteria from fermented vegetables

Screening of lactic acid bacteria from fermented vegetables
Screening of lactic acid bacteria from fermented vegetables

Screening of lactic acid bacteria from fermented vegetables by carbohydrate pro?ling and PCR–ELISA

M.Tamminen1,T.Joutsjoki2,M.Sjo¨blom1,M.Joutsen1,A.Palva3,E.-L.Ryha¨nen1

and V.Joutsjoki1

1Food Research and2Plant Protection,MTT Agrifood Research Finland,Jokioinen,Finland,and3Department of Basic Veterinary Sciences,Faculty of Veterinary Medicine,University of Helsinki,Helsinki,Finland

2004/0203:received23February2004,revised22April2004and accepted28July2004

ABSTRACT

M.T A M M I N E N,T.J O U T S J O K I,M.S J O¨B L O M,M.J O U T S E N,A.P A L V A,E.-L.R Y H A¨N E N

A N D V.J O U T S J O K I.2004

Aims:The aim of this study was to identify potential souring agents,isolated from fermented plant material,by API50CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR–ELISA).

Methods and Results:Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API50CHL and PCR–ELISA.Oligonucleotide probes used for hybridization in PCR–ELISA were speci?c for lactobacilli,the Leuconostoc family,Lactobacillus pentosus/plantarum and Lactobacillus brevis.The hybrides were detected by a colour-developing reaction.Bacteria isolated from fermented cucumbers were identi?ed as Lact.plantarum-related(Lact.plantarum and Lact.pentosus)and Leuconostoc species.Most of the strains isolated from sauerkraut were identi?ed as Lact.pentosus/plantarum.

Conclusions:Complementary results were obtained in the identi?cation of bacterial strains,isolated from fermented cucumbers and sauerkraut,by API50CHL and PCR–ELISA.

Signi?cance and Impact of the Study:PCR–ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables.This will be useful for the identi?cation of strains suitable for the design of starter cultures for the fermentation of plant material.

Keywords:fermented vegetables,identi?cation,lactic acid bacteria,PCR–ELISA,starter cultures.

INTRODUCTION

Traditionally,fermented vegetables have been produced by taking advantage of the natural microbiota associated with plant material.However,spontaneous fermentation is unsure and the quality of products varies depending on fermented material and inherent microbiota.Starter cultures with desirable properties and high counts of bacteria are of particular importance for the production of high-quality fermented vegetable products.However,at the moment,there is lack of commercial bacterial cultures suitable for fermentation of vegetables.

In the manufacture of fermented cucumbers,lactic acid bacteria(LAB)starter cultures are known to prevent economic losses due to pickle spoilage such as bloater, softness and off colours.Utilization of starter cultures enables manufacturers to make food products with standard quality in a shorter time(Hala′sz et al.1999).Desai and Sheth(1997)have reported that the use of Lactococcus lactis, Pediococcus pentosaceus,Lactobacillus brevis,Lact.plantarum and Leuconostoc mesenteroides as starters in the fermentation of cucumbers signi?cantly increased acid production,when compared with spontaneously fermented cucumbers.

Correspondence to:Vesa Joutsjoki,MTT Agrifood Research Finland,

Food Research,ET-house,Myllytie1,FIN-31600Jokioinen,Finland

(e-mail:vesa.joutsjoki@mtt.?).

a2004The Society for Applied Microbiology

Letters in Applied Microbiology2004,39,439–444doi:10.1111/j.1472-765X.2004.01607.x

Lactobacillus plantarum prevents bloater formation,but it may cause too acidic?avour(Etchells et al.1975referred by Fleming1991).

In sauerkraut fermentations,starters such as Lact. alimentarius and P.pentosaceus have been used.Pediococci have been reported to ferment cabbage effectively by decreasing the pH to lower levels than lactobacilli or natural LAB microbiota(Peta¨ja¨and Myllyniemi2000).According to the recent study of Tolonen et al.(2002),Leuc.mesenteroides and Pediococcus dextranicus produced a sauerkraut of good quality.The starter decomposed glucosinolates during fermentation,which led to the formation of many bioactive breakdown products.Strain-dependent reduction of nitrates in the fermentation of carrot–cabbage juices and fresh cabbage by lactobacilli has been reported by Karovicova′et al.(1999). The lowest nitrate concentrations were observed with a Lactobacillus delbrueckii and two Lact.plantarum strains. Comparison of sugar fermentation patterns by the API50 CHL assay has been widely used for species-speci?c identi?cation of LAB.However,several LAB have only minor differencies in these characteristics and some species have a highly variable phenotype,which makes reliable identi?cation complicated.Therefore,the use of a pheno-typic identi?cation method in combination with a genotypic method would be desirable.The polymerase chain reaction (PCR)is currently widely applied as a typing method for microbes.The analysis of PCR-ampli?ed products has been improved by the introduction of solution hybridization and enzyme-linked immunosorbent assay(ELISA).These tech-niques proved to be more sensitive when compared to agarose gel electrophoresis stained with ethidium bromide and more convenient and faster than Southern hybridiza-tion.Solution hybridization reactions are also applicable to automation,which makes them promising for routine screening of large numbers of microbes(Satokari et al. 1998).

In this work,a well-known metabolic differentiation method API50CHL,which has been formerly used for differentiation of LAB from fermentations of plant material (Johansson et al.1995;Nigatu2000),and a genomic method PCR–ELISA(Lawson et al.1999;Antol?′n et al.2001; Logan et al.2001;Laitinen et al.2002),applicable to the differentiation of large numbers of microbes,were used to screen LAB isolated from naturally fermented cucumbers and commercial sauerkraut.

MATERIALS AND METHODS

Fermentation of cucumbers and isolation of bacterial strains from fermented vegetables Fresh pickling cucumbers were washed and rinsed in lukewarm water.Plastic pails were?lled with5kg of cucumbers and5l of brine.The brining solution contained 4?5%NaCl2(rough sea salt).Spontaneous cucumber fermentation occurred in four parallel chambers at+25°C for19days.For the isolation of LAB,samples taken from brine during the course of fermentation(days1–19)and after storage at+4°C(day40)were plated onto MRS agar (Difco Laboratories,Detroit,MI,USA)with0?02%sodium azide as previously described(McDonald et al.1991).Plates were incubated in anaerobic vessels at+30°C for4days.In order to get clones originating from a single cell,colonies selected at random from MRS plates were cultivated in MRS medium and plated onto MRS agar,from which a single colony was picked into MRS medium for cultivation. This procedure was repeated several times.Twenty-six puri?ed cultures from cucumber fermentations were stored at)70°C in15%glycerol.

To obtain single LAB isolates from sauerkraut,a sample was taken from a fresh commercial sauerkraut preperation and plated onto MRS agar.Puri?cation of single LAB isolates was performed as described above for fermented cucumbers.Twenty-seven strains altogether were isolated from sauerkraut.

Identi?cation of LAB by API50CHL assay

For primary species-speci?c identi?cation,bacterial strains isolated from fermented cucumbers were subjected to API 50CHL(bioMe′rieux,l’Etoile,France)assay.Puri?ed strains were cultivated on MRS plates in anaerobic vessels. Grown colonies were cultivated in5ml of MRS medium at +30°C over night,after which the culture was washed and resuspended into1/4strength Ringer’s solution(Merck, Darmstadt,Germany).The turbidity of the suspension was determined by the McFarland method according to the instructions provided by the manufacturer.Cell suspension was applied into API50CH strip wells,which were coated with paraf?n oil.The strips were incubated at+30°C.The result was read after24h and veri?ed after48h.Fermen-tation of carbohydrates in the carbohydrate medium was indicated by a yellow colour except for esculine(dark brown).Colour reactions were scored against a chart provided by the manufacturer.

Preparation of bacterial crude cell extracts Bacterial strains were cultivated in Eppendorf tubes in200l l of MRS broth at+30°C over night.For inoculation,5% inocula of overnight bacterial cultures were used.Cells were collected by centrifugation at16000g for5min and washed with0?05mol l)1HEPES(Sigma Aldrich Chemie Gmbh, Steinheim,Germany).Sterile glass beads(B.Braun Biotech International GmbH,Melsungen,Germany)in the propor-tion of1:3(glass beads to cell culture ratio)and10l l of

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5mmol l)1EDTA were added on the pellet.The cells were disintegrated in a cell mill(Vibrogen Zellmu¨hle,Edmund Bu¨hler,Germany)by vigorous shaking for1min.After cell disintegration the mixture was resuspended into200l l of TE buffer(10mmol l)1Tris-HCl,1mmol l)1EDTA)and centrifuged.The supernatant was transferred into a clean Eppendorf tube.Two microlitres of10mg ml)1RNase (Boehringer,Mannheim Gmbh,Germany)was added into the cell extract,which was incubated at+37°C for20min. Cell extracts were analysed by agarose gel electrophoresis,the sample volume applied on0?8%agarose gel was5–20l l. Oligonucleotide primers and probes

Primers used for the ampli?cation of16S rDNA and23S rDNA regions were as in Ehrmann et al.(1994)or designed by utilizing the sequence data obtained from GenBank.The primers used for the ampli?cation of16S rDNA regions were5¢-AGAGTTTGATNNTGGCTCAG-3¢(forward primer)and5¢-ACGGGCGGTGTGTAC-3¢(reverse pri-mer).The primers used for the ampli?cation of23S rDNA regions were5¢-CCGAATGGGGRAACCC-3¢(forward primer)and5¢-CGACAAGGAATTTCGCTAC-3¢(reverse primer).In the primer sequences N?(ACGT)and R?(AG).The oligonucleotide probes used in this study (Table1)were biotin labelled at the5¢-end.Both published oligonucleotide sequences and sequence data obtained from GenBank were used to design the probes used.

PCR ampli?cation and DIG labelling of the PCR products

PCR reactions were performed in a PTC-100TM Program-mable Thermal Controller(MJ Research Inc.,Waltham,MA, USA).Ampli?cation of bacterial16S and23S rDNA regions was carried out by using the16S and23S rDNA forward and reverse primers described above.PCR products were labelled with the digoxigenin(DIG)DNA-labelling mix(Boehringer) according to the instructions of the manufacturer.

The reaction mixture consisted of the DNA template (0?5l l of the bacterial lysate obtained by disintegration of the cells with glass beads),MgCl2-free1x F-510PCR buffer(Finnzymes,Espoo,Finland),2?0mmol l)1MgCl2, 200l mol l)1of each deoxyribonucleoside triphosphate (including digoxigenin-dUTP),0?2l mol l)1of each primer and0?04U l l)1Dynazyme II DNA polymerase(Finn-zymes).The following PCR protocol was used:a primary denaturation cycle at+95°C for3min,then30cycles of denaturation at+95°C for45s,annealing at+50°C for90s and elongation at+72°C for90s.The?nal extension step (+72°C for5min)was followed by incubation at+4°C. PCR products were visualized on a0?8%agarose gel. Hybridization and hybrid detection

Hybridizations were carried out in commercial streptavidin-coated microtitre plates(Labsystems,Vantaa,Finland).The protocol was essentially as described in Laitinen et al.(2002) with the exceptions that denatured PCR products were hybridized with biotinylated,streptavidin-bound oligonu-cleotide probes for45min at room temperature and reactions with TMB substrate(3,3¢,5,5¢-tetramethylbenzi-dine;Sigma)were terminated with sulphuric acid after 10min incubation.

Speci?city of the probes

To ensure the speci?city of the probes lab448,lab86,mes348 and lab876(Table1),hybridization experiments were performed with strains originating from culture collections: Lactobacillus plantarum DSM20174,Lact.brevis DSM 20054and Leuc.mesenteroides DSM14486.An apparent colour reaction could be detected in test hybridizations of lab448with Lact.plantarum DSM20174,lab86with Lact. brevis DSM20054and mes348with Leuc.mesenteroides DSM14486.The genus-speci?c probe lab876hybridized with the lactobacilli Lact.plantarum DSM20174and Lact. brevis DSM20053.For speci?c identi?cations,a cut-off value for each probe was determined as3·the mean value (absorbance at450nm)of all negative controls(rDNA from LAB species,for which the probe was not speci?c).The cut-off values for the probes lab448,lab86,mes348and lab876 were0?098,0?094,0?121and0?134,respectively. RESULTS

Twenty-six bacterial strains isolated from different phases of the fermentation of cucumbers were characterized by API50

Table1Biotinylated probes used in PCR–ELISA

Probe Sequence Speci?city Origin

lab4485¢-ATCTAGTGGTAACAGTTG-3¢23S rDNA of Lact.pentosus/plantarum Hertel et al.1991 lab865¢-GTGCTTGCACTGATTTCAAC-3¢16S rDNA of Lact.brevis Laitinen et al.2002 mes3485¢-GCATCATGATTTACATTTGAGTG-3¢16S rDNA of the Leuconostoc family This study

lab8765¢-GTGTTGGAGGGTTTCCG-3¢16S rDNA of lactobacilli This study

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CHL and PCR–ELISA tests.In PCR–ELISA,all strains were tested(hybridized)with the probes lab876,lab448, lab86and mes348(Table1).Seventeen strains were desig-nated as Lact.pentosus/plantarum and?ve as Leuconostoc sp. by API50CHL and PCR–ELISA.Two isolates were characterized as Lact.pentosus by API50CHL,but could not be identi?ed by PCR–ELISA because of weak hybrid-ization with the probes used.One strain was designated as a Lactobacillus species by PCR–ELISA with the probe lab876, but could not be identi?ed at species level by API50CHL (Table2).

In order to get a view of the rate of lactobacilli in the microbiota of sauerkraut,27bacterial strains isolated from commercial sauerkraut were analysed by PCR–ELISA. Eighteen strains showed strong hybridization with the Lactobacillus-speci?c probe lab876.Of these,17hybridized strongly with the Lact.pentosus/plantarum-speci?c probe lab448and one with the Lact.brevis-speci?c probe lab86 (data not shown).DISCUSSION

Because of the problems encountered with the traditional fermentation method of vegetables,relying on the natural microbiota associated with plants,there is an increasing demand for speci?c starter cultures designed of well-characterized LAB strains.However,a clear identi?cation of species is often rather complicated.Simple phenotypic methods,such as sugar fermentation patterns,may be ambiguous,as several LAB species have only minor differences in these characteristics.Additionally,some species have a highly variable phenotype,which leads to very dif?cult identi?cation:e.g.strains of Lact.delbrueckii and Lactobacillus acidophilus are so heterogeneous in respect to sugar fermentation,that the API50CHL similarity level within species can be as low as50%(Paludan-Mu¨ller et al. 1999).It seems obvious that for a reliable identi?cation of speci?c LAB groups,like https://www.doczj.com/doc/4118790114.html,ctobacilli,a genomic method should be used in reference with a phenotypic assay

Table2Characterization of bacterial strains,

isolated during the course of fermentation and

storage of spontaneously fermented cucum-

bers,by API50CHL and PCR–ELISA.The

percentage value shows the similarity of the

tested strain with a database strain.The

probes used in PCR–ELISA for identi?ca-

tions:lab876(lactobacilli),lab448(Lactoba-

cillus pentosus/plantarum),lab86(Lact.brevis)

and mes348(Leuconostoc sp.)

Strain Fermentation/

storage time

API50CHL PCR–ELISA

10-day fermentation Lact.plantarum(99?3%)Lact.pentosus/plantarum

20-day fermentation Leuc.mesenteroides ssp.

mesenteroides/dextranicum(99?8%)

Leuconostoc sp.

31-day fermentation Lact.pentosus(57?2%)/

Lact.plantarum(47?2%)

Lact.pentosus/plantarum

41-day fermentation Leuc.mesenteroides ssp.

mesenteroides/dextranicum(99?3%)

Leuconostoc sp.

51-day fermentation Lact.plantarum(99?2%)Lact.pentosus/plantarum

61-day fermentation Lact.plantarum(98?9%)Lact.pentosus/plantarum

79-day fermentation Lact.pentosus(98?2%)No valid identi?cation

89-day fermentation Lact.plantarum(99?0%)Lact.pentosus/plantarum

99-day fermentation No valid identi?cation Lactobacillus sp.

109-day fermentation Leuc.mesenteroides ssp.

mesenteroides/dextranicum(99?4%)

Leuconostoc sp.

119-day fermentation Lact.plantarum(99?3%)Lact.pentosus/plantarum

129-day fermentation Lact.plantarum(99?8%)Lact.pentosus/plantarum

1319-day fermentation Lact.plantarum(98?9%)Lact.pentosus/plantarum

1419-day fermentation Lact.plantarum(98?9%)Lact.pentosus/plantarum

1519-day fermentation Lact.plantarum(98?8%)Lact.pentosus/plantarum

1619-day fermentation Lact.plantarum(99?2%)Lact.pentosus/plantarum

1719-day fermentation Lact.plantarum(99?2%)Lact.pentosus/plantarum

1819-day fermentation Lact.plantarum(98?9%)Lact.pentosus/plantarum

1919-day fermentation Leuc.mesenteroides ssp.

mesenteroides/dextranicum(99?8%)

Leuconostoc sp.

2019-day fermentation Leuc.mesenteroides ssp.

mesenteroides/dextranicum(99?8%)

Leuconostoc sp.

2119-day fermentation Lact.plantarum(98?3%)Lact.pentosus/plantarum

2219-day fermentation Lact.plantarum(99?1%)Lact.pentosus/plantarum

2340-day storage Lact.plantarum(99?2%)Lact.pentosus/plantarum

2440-day storage Lact.plantarum(98?8%)Lact.pentosus/plantarum

2540-day storage Lact.pentosus(97?8%)No valid identi?cation

2640-day storage Lact.plantarum(99?2%)Lact.pentosus/plantarum

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(Johansson et al.1993;Jacobsen et al.1999).Species-speci?c16S and23S rDNA-targeted oligonucleotide probes have become widely adopted for the detection of LAB species from various sources(reviewed in Charteris et al. 1997).The use of oligonucleotide probes and a highly sensitive colorimetric detection assay in combination has made possible the ef?cient identi?cation of spoilage micro-organisms(Satokari et al.1998),and a further developed PCR–ELISA protocol was proven to be applicable for the characterization of intestinal lactobacilli and bi?dobacteria (Laitinen et al.2002;Malinen et al.2002).

The results obtained with API50CHL and PCR–ELISA in this work were mostly consistent.Two bacterial isolates from fermented cucumbers,assigned as Lact.pentosus by API50CHL,did not hybridize with the Lact.pentosus/ plantarum-speci?c probe lab448used in PCR–ELISA.One strain could not be characterized by PCR–ELISA,but hybridized strongly with the Lactobacillus-speci?c probe lab876.The LAB species identi?ed in fermented cucum-bers,including Lact.plantarum and Leuconostoc sp.,are typical for spontaneous fermentation of vegetables(Pederson and Albury,1950referred by Etchells et al.1975).As expected,Lact.plantarum became the dominating species in natural cucumber fermentation(Pederson and Albury,1961 referred by Etchells et al.1975)and could be detected in all phases of fermentation,as well as in the storage period.In all,the results suggest that the use of API50CHL and PCR–ELISA in a complementary fashion enables reliable identi?cation of the majority of LAB isolates.Both Lact. brevis and Lact.plantarum-related lactobacilli,detected in the commercial sauerkraut,are typical species which follow L.mesenteroides in white cabbage https://www.doczj.com/doc/4118790114.html,ctobacillus plantarum forms large quantities of lactic acid from remain-ing carbohydrates,further lowering pH in the?nal stage of fermentation(Pederson and Albury,1969referred by Harris 1998).As with fermented cucumbers,Lact.plantarum-related lactobacilli eventually became the most prevalent group of LAB in sauerkraut(Pederson and Albury,1950 referred by Etchells et al.1975).

Microbiological methods currently used for the detection and enumeration of food microbes are time consuming,and it often takes several days to carry out the procedure (Antol?′n et al.2001).Carbohydrate fermentation for API50 CHL assay is completed in48h,whereas the genomic PCR–ELISA method can be performed in1day after cultivation of bacterial strains in culture medium.As a rapid and easy method to perform,PCR–ELISA can be consid-ered applicable for the screening of large numbers of bacteria.In the current work,a rapid screening of lactoba-cilli from sauerkraut was achieved by PCR–ELISA with the Lactobacillus-speci?c probe lab876and a further species-speci?c differentiation was accomplished with the species-speci?c probes lab448(Lact.pentosus/plantarum)and lab86(Lact.brevis).This proved to be a convenient way to select a representative group of bacterial isolates,which belong to the same species,from fermented plant material.For the design of starter cultures consisting of well-characterized LAB strains,selected isolates can be subjected to more sensitive molecular methods for differentiation at the subspecies and strain level. ACKNOWLEDGEMENTS

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