International Journal of Food Microbiology 45 (1998) 85–92

Characterization of lactococci isolated from minimally processed

fresh fruit and vegetables
a, b b
William J. Kelly *, Graham P. Davey , Lawrence J.H. Ward
a
Horticulture and Food Research Institute, Palmerston North Research Centre, Private Bag 11030, Palmerston North, New Zealand
b
New Zealand Dairy Research Institute, Palmerston North, New Zealand

Received 17 August 1998; accepted 28 August 1998

Abstract

Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus
lactis subsp. lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the
16S rRNA gene. Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic
DNA. Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products
during the period they are intended to be eaten. Although these plant strains showed many similarities to strains of L. lactis
used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental
origin. Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose. Most of the
bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin
activity, or that produced a different bacteriocin with a narrow spectrum of activity. One strain produced nisin but was
unable to ferment sucrose, properties that have been generally regarded as linked. These strains may have uses as
biopreservatives for minimally processed plant products.  1998 Elsevier Science B.V. All rights reserved.

Keywords: Lactococcus lactis; PFGE; Bacteriocins; Nisin; Sucrose fermentation; Fruit and vegetable products

1. Introduction Listeria monocytogenes are a food safety concern

with these products and there is interest in providing
Minimally processed fresh fruit and vegetables extra hurdles to control these bacteria. One approach
sold in ready-to-eat or ready-to-use form make up an is to use lactic acid bacteria as protective cultures to
increasing share of the fresh produce market. Diverse provide biological preservation without changing the
products are now available, and most rely on modi- sensory characteristics of the product (Schillinger et
fied atmosphere packaging and refrigeration to main- al., 1996). Trials using lactic acid bacteria for this
tain quality and food safety. Pathogens such as purpose have been reported (Vescovo et al., 1996;
Cai et al., 1997).
*Corresponding author. Tel.: 1 64-6-356-8080; fax: 1 64-6- In previous work we examined a range of ready-
354-6731; e-mail: [email protected] to-eat foods for the presence of bacteriocin-produc-

0168-1605 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved.
PII: S0168-1605( 98 )00135-4
86 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92

ing (Bac 1 ) cultures that may be useful in biop- 2.2. Bacterial identification
reservation of minimally processed fresh fruit and
vegetables (Kelly et al., 1996a). Most of the Bac 1 Isolates identified as Gram positive, catalase nega-
isolates from plant products were lactococci and tive cocci were tested for the following properties at
several produced a nisin-like activity and were 288C. Gas production from glucose was determined
capable of inhibiting Listeria monocytogenes strains. in tubes of MRS broth (without citrate) containing
Sprouted seeds were a particularly rich source of inverted Durham tubes. Sensitivity to NaCl was
strains and workers in Canada and Germany (Cai et tested in MRS broth containing 4% or 6.5% (w / v)
al., 1997; Franz et al., 1997) have subsequently NaCl, and sensitivity to vancomycin was tested by
reported the isolation of nisin-producing lactococci streaking cultures on MRS agar plates containing
from bean sprouts implying that lactococci are a vancomycin at 5, 25 or 150 mg ml 21 . Dextran
feature of the normal microflora of these products. production was tested by growth on sucrose agar
This paper reports the further isolation and identi- (Garvie, 1984). The isomer of lactic acid produced
fication of lactococci from minimally processed fruit from growth on glucose was determined using a
and vegetable products, and examines the diversity D,L-lactic acid kit (Boehringer Mannheim, Mann-
of strains present. While these strains show strong heim, Germany). Arginine deamination was ex-
similarities with the well studied dairy starter strains, amined using the method described by Niven et al.
they also have some unusual properties which may (1942). Carbohydrate fermentation patterns were
make them useful for other purposes. determined using API 50CH galleries as specified by
´
the manufacturer (Bio Merieux SA, Marcy-l’Etoile,
France), and also in microtitre plates (Jayne-Wil-
2. Materials and methods liams, 1976) with MRS broth supplemented with 50
mg l 21 chlorophenol red as the basal medium to
2.1. Bacterial cultures and media which sugar solutions were added. Ability to clot
milk was tested by inoculating cultures into 10%
Lactic acid bacteria were isolated from 76 retail reconstituted skim milk and checking for clot forma-
fruit and vegetable products and screened for bac- tion after incubation at 22 and 288C.
teriocin production as previously described (Kelly et
al., 1996a). Lactococci were grown at 288C in MRS 2.3. DNA methodology
broth (De Man et al., 1960; Merck, Darmstadt,
Germany) or M17 broth (Terzaghi and Sandine, The preparation and restriction endonuclease di-
1975; Merck) supplemented with 0.5% (w / v) glu- gestion of genomic DNA in agarose blocks, and its
cose (GM17). Lactose indicator agar (McKay et al., analysis by pulsed-field gel electrophoresis has been
1972) was used to differentiate lactose fermenting described previously (Kelly et al., 1993). DNA was
lactococci, and sucrose indicator agar plates were digested for 16 h with SmaI (New England Biolabs,
prepared in the same way with sucrose substituted Beverly, MA), and gels were run for 21 h at 200 V
for lactose as the carbon source. Listeria monocyto- and 148C with the time pulse ramped from 1 to 30 s
genes was grown in Trypticase Soy broth (Becton using a CHEF DR II pulsed-field gel electrophoresis
Dickinson Microbiology Systems, Cockeysville, apparatus and model 1000 mini chiller (Bio-Rad
MD) with 0.6% yeast extract (Oxoid, Basingstoke, Laboratories, Richmond, CA).
UK) at 378C. Clostridia were grown in Reinforced A fragment of the 16S rRNA gene (corresponding
Clostridial Medium (Difco Laboratories, Detroit, to positions 20 to 362 of the Escherichia coli 16S
MI) at 308C. Other bacteria used as indicators were rRNA gene) was amplified by PCR using primers Y1
grown in either MRS or M17. Solid media were and Y2 (Young et al., 1991). PCR conditions were
prepared by the addition of 1.5% agar (Sigma, St. one cycle: 948C / 3 min, 558C / 45 s, 708C / 1 min; 30
Louis, MO) to broth. Soft agar used for overlays cycles: 948C / 45 s, 558C / 45 s, 708C / 1 min; one
contained 0.8% agar. Stock cultures were maintained cycle: 948C / 45 s, 558C / 45 s, 708C / 5 min. The PCR
at 2 808C in broth supplemented with 20% (v / v) products were passed through Wizard TM PCR prep
glycerol. columns (Promega Corporation, Madison, WI) to
 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92 87

remove unincorporated primers. The products were Lac 2 , Suc 1 , Sm r transconjugants were purified and
sequenced using Y1 and Y2 as primers for cycle tested for the ability to produce nisin.
sequencing (Promega fmol DNA sequencing system)
with direct incorporation of [ 35 S]d-ATP.
Probes for the insertion sequences ISS1 and IS981
3. Results
and the nisin structural gene, were constructed by
amplification of lactococcal DNA with primers al-
3.1. Bacterial isolation and differentiation
ready described (Ward et al., 1993, 1994). From the
published sequence data on lacticin 481 (Piard et al.,
Bac 1 lactic acid bacteria were isolated without
1993) primers were made to the lct structural gene
enrichment from fruit and vegetable products over a
and used in PCR reactions.
period of 2 years. Cell counts on MRS agar ranged
between 10 3 and 10 7 CFU ml 21 , and the proportion
2.4. Bacteriocin activity
of the population that showed inhibition of the
indicator strains Lactobacillus plantarum ATCC
Bacteriocin activity was measured using both the
8014 and Lactococcus lactis subsp. lactis T-21 was
agar spot test, and the agar well diffusion assay as
between 1 and 78%. Isolates with no inhibitory
previously described (Kelly et al., 1996b). To test for
activity (Bac 2) were also selected from samples that
protease sensitivity, neutralized culture supernatant
gave a high proportion ( . 15%) of Bac 1 isolates.
was treated for 1 h at 378C with a-chymotrypsin,
Pulsed-field gel electrophoresis (PFGE) of SmaI
trypsin (Sigma), or proteinase K (Boehringer) at final
digests of genomic DNA was used to differentiate
concentrations of 1 mg ml 21 . To test heat stability
150 Bac 1 and 33 Bac 2 isolates, and 29 different
the supernatant (at pH 2 and pH 6.5) was heated in
Bac 1 strains could be distinguished along with 10
boiling water for 5, 10, and 60 min, or autoclaved at
Bac 2 strains. These are described in Table 1, with
1218C for 15 min. Activity remaining after treatment
their PFGE patterns shown in Fig. 1. All strains were
was measured by agar well diffusion assay.
isolated from sprouted seeds except for KF67 which
Nisin sensitivity was determined by spotting 5 ml
was isolated from unpasteurized grapefruit or orange
of a stationary phase culture onto plates of GM17
juice on three occasions, and KF201 from minimally
agar containing nisin (dissolved in 0.02 M HCl) at
processed sliced mixed vegetables. Sprouted seed
concentrations between 0.1 and 500 mg ml 21 .
products included alfalfa sprouts, alfalfa and onion
sprouts, alfalfa and radish sprouts, Japanese kaiware
2.5. Conjugation experiments
(white radish) shoots, mung bean sprouts, mustard
and cress, and soya sprouts. Up to 11 different
Conjugal matings were set up between four of the
strains (seven Bac 1 and four Bac 2) were isolated
strains which showed the presence of nisin genes and
from a single sample. Four strains were found
the plasmid-free recipient strain L. lactis subsp.
throughout the 2-year sampling period. KF67 was
cremoris MG1614 (Lac 2 , Suc 2 , Bac 2 , Sm r), (Gas-
the only strain isolated from the unpasteurized fruit
son, 1983). Overnight donor and recipient cultures
juices although it showed several variations in
were inoculated into fresh GM17, grown for 4 h,
plasmid profile (Kelly et al. (1996a) and data not
harvested by centrifugation and washed twice in
shown). Of the 17 sprouted seed products from
quarter strength Ringer’s solution (Oxoid). Donor
which Bac 1 strains were isolated, KF4 was found in
and recipient cells were mixed in a 1:1 ratio and 0.2
seven, KF7 in eight and KF5 in 12.
ml was plated onto a nonselective GM17 plate and
incubated at 288C for 2–4 h. Cells were harvested
from mating plates in quarter strength Ringer’s 3.2. Bacterial characteristics
solution, diluted and plated onto sucrose indicator
agar supplemented with 600 mg ml 21 streptomycin All strains were Gram positive, catalase negative
(Sigma). Plates were incubated at 288C for up to 5 cocci, that did not produce gas from glucose or
days. Donor and recipient strains alone were pre- dextran from sucrose. All produced L-lactate from
pared in a similar manner and used as controls. glucose, were able to deaminate arginine, and were
88 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92

Table 1
Lactococci from minimally processed fresh fruit and vegetables
Strain Phenotype a Nisin genes Nisin sensitivity b
1
1. Bac (nisin)
KF4 Raf 1 Mel 1 1 . 500
KF7 1 200
KF24 1 200
KF30 Raf 1 Mel 1 1 . 500
KF67 Lac 2 1 . 500
KF129 1 . 500
KF132 1 . 500
KF134 1 . 500
KF146 Raf 1 Mel 1 1 . 500
KF165 Raf 1 Mel 1 1 . 500
KF178 1 200
KF196 1 . 500
KF197 1 . 500
KF218 1 200
KF221 Suc 2 1 . 500
KF237 Raf 1 Mel 1 1 . 500
KF244 1 200
KF245 Raf 1 Mel 1 1 . 500
KF253 Raf 1 Mel 1 1 . 500
KF282 1 . 500
KF283 1 . 500
KF289 1 . 500
KF292 Raf 1 Mel 1 Lac 2 1 . 500

2. Bac 1 (broad spectrum)

KF201 2 20

3. Bac 1 (narrow spectrum)

KF5 Raf 1 Mel 1 1 50
KF31 Raf 1 Mel 1 1 50
KF164 Raf 1 Mel 1 1 50
KF241 Raf 1 Mel 1 1 50
KF280 1 50

4. Bac 2
KF138 2 5
KF140 Raf 1 Mel 1 2 50
KF147 Raf 1 Mel 1 1 50
KF152 Raf 1 Mel 1 1 50
KF169 Raf 1 Mel 1 Lac 2 2 100
KF181 Raf 1 Mel 1 2 1
KF186 Raf 1 Mel 1 2 50
KF225 Raf 1 Mel 1 2 50
KF257 2 5
KF269 2 1
a
Lac, lactose; Mel, melibiose; Raf, raffinose; Suc, sucrose.
Highest concentration of nisin (mg ml 21 ) at which growth occurred.
b

sensitive to vancomycin at 5 mg ml 21 . Growth was ribose, salicin and trehalose, and did not ferment
recorded in 4% NaCl, but not in 6.5%. melezitose or sorbitol. Most strains fermented lactose
All strains fermented amygdalin, cellobiose, es- (except KF67, KF292 and KF169), mannitol (except
culin, fructose, galactose, glucose, maltose, mannose, KF292), sucrose (except KF221) and xylose (except
 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92 89

metabolism is a major distinguishing characteristic

separating L. lactis from L. raffinolactis, but ribose
and sucrose metabolism are not characteristics com-
mon in the latter species. Thus molecular techniques
were used to further identify some of these isolates.

3.3. Molecular taxonomy

PCR was used to amplify a fragment from the 16S

rRNA gene from five of the plant strains, KF4, KF5,
KF138, KF146 and KF201. These fragments were
sequenced and in four cases (KF4, KF5, KF138 and
KF146) were found to be identical to the published
sequence for Lactococcus lactis subsp. lactis (Col-
lins et al., 1989). Strain KF201 differed from the
published sequence by one base change, A for G at
position 87.
Strains were also probed using the insertion
elements ISS1, and IS981 which occur commonly in
dairy strains of Lactococcus lactis (Polzin et al.,
1993). Representative patterns are shown in Fig. 2.
Fig. 1. Pulsed-field gel electrophoresis patterns of genomic DNA ISS1 was present in most strains, and in many cases
from Lactococcus strains. (A) Bac 1 . Lane 1, KF4; 2, KF237; 3,
KF245; 4, KF5; 5, KF31; 6, KF241; 7, KF7; 8, KF218; 9, KF24;
occurred in multiple copies. Fewer strains harboured
10, KF244; 11, KF30; 12, KF67; 13, KF129; 14, KF283; 15, copies of IS981, and the copy number was lower, but
KF132; 16, KF134; 17, KF146; 18, KF196; 19, KF164; 20, this element could still be detected in many of the
KF165. (B) Lane 1, KF178; 2, KF197; 3, KF201; 4, KF221; 5, strains. Therefore, it was concluded that the plant
KF253; 6, KF280; 7, KF282; 8, KF289; 9, KF292; Bac 2 . 10, isolates are strains of Lactoccus lactis subsp. lactis.
MG1363; 11, KF138; 12, KF140; 13, KF147; 14, KF152; 15,
KF169; 16, KF181; 17, KF269; 18, KF186; 19, KF225; 20,
KF257. 3.4. Bacteriocin characterization

The Bac 1 strains could be divided into three

KF7, KF218 and KF140). One strain fermented groups on the basis of their spectrum of inhibitory
inulin (KF165). More variability was noted in the activity with the Bac 2 strains constituting a fourth
fermentation of arabinose (31 strains positive), group (Table 2). Group 1 strains did not inhibit, nor
gluconate (32 strains positive), melibiose and raffin- were inhibited by, the nisin producing strain L. lactis
ose (see Table 1). All strains, except the three that NCFB 1404, and their bacteriocin activity was stable
did not ferment lactose, were able to produce a firm after boiling for 5 min at pH 2.0, resistant to trypsin
clot in milk within 24 h at 288C. One strain (KF4), digestion and sensitive to a-chymotrypsin. These
also clotted milk at 228C. characteristics are typical of nisin, and in addition all
While these strains were identified as lactococci, the Bac 1 strains except KF221, fermented sucrose, a
in many cases the fermentation patterns did not fit property which has been invariably linked to nisin
well with published species descriptions (Schleifer et biosynthesis (Rauch et al., 1994). The large size of
al., 1985; Teuber et al., 1992; Bounaix et al., 1996; inhibition zones produced (15–19 mm) when com-
Pot et al., 1996). Lactococcus lactis is probably the pared with those from L. lactis NCFB1404 (13 mm)
best fit for these strains, especially since many other suggest that these strains are nisin Z producers as
plant isolates have been identified as this species. this variant has better diffusion properties than nisin
However, sucrose and xylose fermentation are un- A (De Vos et al., 1993). KF201 (group 2) also gave a
common properties in this species and melibiose and broad spectrum of activity, but was distinct from
raffinose fermentation is not recorded. Raffinose nisin, and was able to inhibit the nisin producer L.
90 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92

KF5 but differ slightly in their PFGE pattern (Fig.

1A, lanes 4–6). KF152 (Fig. 1B, lane 14) has the
same PFGE pattern as KF5 but lacks any bacteriocin
activity, and is particularly sensitive to the activity
produced by these strains.

3.5. Occurrence and transfer of nisin genes

To confirm nisin production, all strains were

probed for the presence of the structural genes for
nisin, and another lactococcal lantibiotic, lacticin
481. All of the Bac 1 strains except KF201 gave a
positive result for nisin (Table 1). The nisin structur-
al gene was also detected in two strains, KF147 and
KF152, that showed no bacteriocin activity. There
was no reaction with the lacticin 481 probe. Nisin
producing strains showed the highest levels of nisin
resistance, and other strains differed in their sen-
sitivity (Table 1).
Since sucrose fermentation and nisin production
are reported to be genetically linked on a conjugative
transposon, four of the nisin producers were screened
for the ability to transfer the sucrose fermentation
phenotype to strain MG1614. Two nisin producers
(KF165 and KF292) transferred the capacity to
ferment sucrose and nisin production at frequencies
between 2 3 10 26 and 5 3 10 27 CFU per donor cell,
while a further two (KF7 and KF67) did not show
transfer.

Fig. 2. Southern blots of EcoRI digested total genomic DNA from 4. Discussion
Lactococcus lactis strains. (A) Hybridized with ISS1. (B) Hybrid-
ized with IS981. Lane 1, KF4; 2, KF7; 3, KF30; 4, KF67; 5,
The aim of this study was to examine lactococci
KF132; 6, KF146; 7, KF178; 8, KF196; 9, KF31.
isolated from a range of retail fruit and vegetable
products and to determine bacteriocin activity and
lactis NCFB 1404. Activity was lost after digestion strain diversity. Lactococci could be readily isolated,
with a-chymotrypsin or proteinase K, but only especially from products containing sprouted legume
slightly affected by trypsin. Boiling had no effect on and brassica seeds. As Bac 2 lactococci were also
the inhibitory activity and 25% of activity was isolated, we conclude that Lactococcus strains are a
retained after autoclaving. major component of the microflora found on these
Group 3 strains gave a narrow spectrum of products during the period they are intended to be
inhibitory activity which was resistant to trypsin consumed, and that the common occurrence of
digestion, but sensitive to proteinase K and a- bacteriocin-producers aids the dominance of these
chymotrypsin. Activity was lost after autoclaving but strains. The origin of these bacteria is not known but
stable to boiling. KF31 and KF241 have the same it is probable that they are present on the seeds prior
phenotype, plasmid profile and bacteriocin activity as to sprouting, and are thus able to colonise the
 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92 91

Table 2
Inhibitory activity spectra of L. lactis strains
Indicator Bac 1 Bac 2
Gp1 Gp2 Gp3
Bacillus cereus NCTC 8035 1 2 2 2
Clostridium butyricum BZ2 1 1 2 2
C. perfringens NCTC 8237 1 1 2 2
C. sporogenes NCTC 532 1 1 2 2
Enterococcus faecalis NCTC 775 1 1 2 2
Lactobacillus casei NCFB 161 1 1 2 2
Lb. delbrueckii subsp. lactis ATCC 4797 1 1 2 2
Lb. plantarum ATCC 8014 1 1 1 2
Lactococcus lactis subsp. lactis T-21 1 1 1 2
L. lactis subsp. lactis NCFB 1404 2 1 2 2
L. lactis subsp. cremoris MG1614 1 1 1 2
Leuconostoc lactis NCFB 956 1 1 1 2
Leuc. mesenteroides subsp. cremoris NCFB 2035 1 1 2 2
Leuc. mesenteroides subsp. dextranicum NCFB 529 1 1 2 2
Listeria monocytogenes ATCC 49594 1 1 2 2
Pediococcus pentosaceus ATCC 10791 1 1 2 2
Pseudomonas fluorescens NCTC 10038 2 2 2 2
Staphylococcus aureus ATCC 25923 1 1 2 2
S. epidermidis ATCC 14990 1 1 2 2
Weissella viridescens ATCC 12706 1 1 2 2

developing seedling. The ability of many of these that chromosomal rearrangements occur frequently in
strains to utilise raffinose and sucrose would assist these lactococci.
this colonization. It came as no surprise that most of the Bac 1
The carbohydrate fermentation patterns of many of isolates were producing nisin since this bacteriocin is
the isolates from sprouted seeds did not correspond produced by many strains of L. lactis including those
with published species descriptions, but showed isolated from non-dairy environments. However we
characteristics in common with both L. lactis and L. were surprised by the prevalence and diversity of
raffinolactis. This was resolved when a fragment of nisin producing strains, and by the number of strains
the 16S rRNA gene was sequenced from five strains which hybridized with the nisin probe but did not
and found to be identical to the sequence reported for show nisin activity. This suggests that strains which
L. lactis subsp. lactis, and clearly distinct from that possess the nisin genes (or possibly the sucrose
for L. raffinolactis (Klijn et al., 1995). The common fermentation genes with which they are associated)
presence of the insertion sequences ISS1 and IS981, have a selective advantage in the plant environment.
and the production of nisin are further evidence that In support of this it was shown the nisin production
these plant isolates are strains of Lactococcus lactis and sucrose fermentation phenotypes were transfer-
subsp. lactis. Other studies have also recently re- able properties at least in some strains. It has been
ported the isolation of nisin-producing lactococci stated that nisin production and sucrose metabolism
from bean sprouts (Cai et al., 1997; Franz et al., are invariably linked (De Vuyst, 1994; Rauch et al.,
1997), and these show similar characteristics to the 1994), but one of the strains isolated (KF221)
strains described here. produced nisin but was unable to ferment sucrose
Differentiation of the isolates by pulsed-field gel suggesting that either the sucrose utilizing genes
electrophoresis highlighted the diversity of the popu- have been disrupted or that the structure of the
lation, as most products contained several different nisin–sucrose transposon has changed in this strain.
strains. Some strains were found in several samples, The use of these plant lactococci to control Listeria
while others were isolated only once. Minor changes in refrigerated minimally processed fresh fruit and
in PFGE patterns (see Fig. 1A, lanes 1–10) suggest vegetable products is being further investigated.
92 W. J. Kelly et al. / International Journal of Food Microbiology 45 (1998) 85 – 92

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The authors wish to thank Marie Timmins for McKay, L.L., Baldwin, K.A., Zottola, E.A., 1972. Loss of lactose
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