Characterisation of technologically proficient wild

Lactococcus lactis strains resistant to phage infection

Carmen Maderaa, Pilar Garcıaa, Thomas Janzenb,Ana Rodrıguezc, Juan E. Suareza,c,*

aArea de Microbiologıa, Universidad de Oviedo, Julian Claverıa s/n, 33006 Oviedo, SpainbDepartment of Genetics, Chr. Hansen A/S, Boege Alle 10, DK-2970 Hoersholm, Denmark

c Instituto de Productos Lacteos de Asturias (IPLA-CSIC), Carretera de Infiesto s/n, 33400 Villaviciosa, Spain

Received 26 February 2002; received in revised form 5 July 2002; accepted 26 December 2002

Abstract

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations

exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in

starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a

collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they

were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates

allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately

proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of

phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although

genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one

mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot

fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely

abolished the process when promoted by a phage-susceptible strain.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Lactococcus lactis; Bacteriophage; Milk fermentation; Starter; Cheese

1. Introduction

Lactic acid bacteria (LAB) is the group of Gram-

positive microorganisms most frequently used in food

and feed fermentations. The role of these starter

bacteria is twofold; on one hand, they contribute to

extend the useful life span of the food, usually through

fermentative degradation of the sugars present in the

raw materials. This causes a lowering of the pH that

makes the medium inhospitable for most spoilage and/

or pathogenic organisms (excellent comprehensive

reviews on the roles of LAB in food fermentation

may be found in de Roissart and Luquet, 1994;

Salminen and von Wright, 1998). Additionally, some

0168-1605/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1605(03)00042-4

* Corresponding author. Area de Microbiologıa, Facultad de

Medicina, Universidad de Oviedo, Julian Claverıa s/n, 33006

Oviedo, Spain. Tel.: +34-985-103559; fax: +34-985-103148.

E-mail address: jsuarez@sauron.quimica.uniovi.es

(J.E. Suarez).

www.elsevier.com/locate/ijfoodmicro

International Journal of Food Microbiology 86 (2003) 213–222

of these strains produce antagonistic peptides towards

other Gram-positive bacteria (Nes et al., 1996; Moll et

al., 1999; Cleveland et al., 2001). On the other hand,

acid accumulation changes the rheological and orga-

noleptical properties of the product, a process that is

complemented by the production and, in some instan-

ces, the secretion of hydrolytic enzymes, mainly

proteinases, peptidases, amino acid hydrolases and,

to a lower extent, lipases and esterases (Kok and De

Vos, 1994; Christensen et al., 1999; Fernandez et al.,

2000).

The development of the starter bacteria may be

affected by a series of environmental factors, such as

the presence of growth inhibitors in the raw material,

disinfectants from inappropriately rinsed equipment

and by contamination with bacteriophages. The pres-

ence of any of these deleterious factors results in

failure of the fermentation or at least in generation

of low quality products, with the consequent eco-

nomic losses (Desmazeaud, 1994; Josephsen and

Neve, 1998). Of all these, phages constitute the most

important source of fermentation failure, especially in

milk fermentations driven by Lactococcus lactis,

probably due to the highly controlled processes devel-

oped by the dairy industry when compared to other

food fermentation processes (Gasson, 1983; Joseph-

sen and Neve, 1998). As a consequence, multiple

phages specific for L. lactis have been isolated and

characterised (for a recent review on the molecular

characteristics of dairy phages, see Brussow, 2001).

They have been grouped in 12 species (Jarvis et al.,

1991), later reduced to 10 (Josephsen and Neve,

1998), out of which 936, P335 and c2 are the most

frequently isolated from dairy fermentations (Moineau

et al., 1996; Labrie and Moineau, 2000).

The phage problem has promoted the adoption of

defensive strategies such as the use of hermetic or

pressurised fermentation vats and media rich in phos-

phates for the growth of the starters, although the

problem is far from being solved (Accolas et al.,

1994). Fortunately, phage infection may be controlled

through appropriate selection of the starter strains.

This selection is based on naturally occurring resist-

ance mechanisms evolved in the dairy environment,

which has allowed the incorporation of bacteriophage-

resistant strains as components of starter cultures,

whenever their presence did not modify the final

product quality. According to their mode of action,

these mechanisms were divided into four groups:

inhibition of phage adsorption, blocking of DNA

ejection, restriction–modification (R/M), and abortive

infection (Daly et al., 1996; Forde and Fitzgerald,

1999; Moineau, 1999). Unfortunately, phages have

the capacity to adopt genetic strategies to circumvent

the resistance mechanisms shown by their hosts

(Moineau et al., 1994; Durmaz and Klaenhammer,

2000). Thus, new strains that show good technolog-

ical properties and are resistant to a significant range

of the phages prevalent in the industry are always

needed. A convenient source for such bacteria might

be the small factories that produce artisan cheeses.

Especially interesting to this purpose might be those

producing cheese without addition of any starters, i.e.

depending on the indigenous microbiota of raw milk

and/or of the environment of the factory. The rationale

is that these strains have probably been subjected to a

strong, albeit unnoticed, selection towards good milk

processing and phage insensitivity. To test this possi-

bility, 100 isolates of L. lactis, originated from a

popular farmhouse cheese produced in Northern

Spain, were analysed with respect to both parameters

and the data obtained are described below.

2. Materials and methods

2.1. Bacterial strains, bacteriophages and media

The L. lactis strains used in this study were isolated

in several local dairies from Afuega’l Pitu, a farm-

house cheese of acidic clotting, over two consecutive

years (Cuesta, 1996). The 34 bacteriophages used

were isolated from failed fermentations occurring in

Europe and the USA (Christian Hansen collection,

Horsholm, Denmark) and belonged to the 936 group

(14), to the c2 group (10) or were taxonomically

undefined (10). Appropriate sensitive indicator strains

for particular phages were from the Chr. Hansen

collection as well. In addition, L. lactis MG1614

was used as a general indicator strain (Gasson, 1983).

The bacterial strains were grown at 30 jC in M17

(Oxoid, Madrid, Spain) broth supplemented with

0.5% lactose or (in the case of MG1614) 0.5%

glucose. For phage propagation, the double-layer

method (Terzaghi and Sandine, 1975) was used on

M17 agar supplemented with 10 mM CaCl2 and 10

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222214

mMMgSO4 (MMC medium). The top layer contained

0.7% agar while in the bottom layer its concentration

was 1.5%.

2.2. Screening for phage sensitivity and adsorption

The host range of the phages was determined by

mixing 0.1-ml aliquots of stationary-phase host cul-

tures with appropriate dilutions of individual phage

suspensions in 3 ml of molten M17 top agar and the

mixture was poured on MMC agar plates. Plates were

incubated at 30 jC overnight and examined for the

appearance of plaques.

The degree of phage adsorption was determined in

0.5-ml aliquots of stationary-phase host cultures, to

which individual phage suspensions were added, to

get a multiplicity of infection of 0.001. The mixture

was incubated at 30 jC for 10 min, diluted with cold

medium and centrifuged (4 jC, 15 min, 7000 rpm).

The supernatants were assayed for non-adsorbed

phages by the double-layer method, and the results

compared with the titre of a control without cells.

2.3. Evaluation of prophage induction

The lysogenic status of the strains was determined

by measuring the induction of prophages with mito-

mycin C (MC) (Cuesta et al., 1995). Exponential

cultures (OD600 = 0.1) were divided into two 10-ml

aliquots, and MC was added to one of them to obtain a

final concentration of 0.6 Ag/ml. Incubation pro-

ceeded for 3 h, and the turbidity was continuously

monitored in a spectrophotometer Uvikon 900 (Kon-

tron Instruments, Madrid, Spain). Both tubes were

centrifuged at 5000 rpm for 10 min, and the super-

natants were filter-sterilised (0.22 Am) before being

tested for the presence of phage by the double-layer

method using six indicator strains (selected for their

susceptibility to a range of phages).

2.4. Biogenic amine production

Production of biogenic amines was determined

according to published methods (Niven et al., 1981)

and their modifications (Joosten and Northolt, 1989;

Bover-Cid and Holzapfel, 1999). Plates of M17 con-

taining 0.06% bromocresol purple and 1% tyrosine,

tryptophan, histidine, ornithine or lysine were inocu-

lated with the strains to be tested and incubated for 48

h at 30 jC. Amino acid decarboxylation was detected

as a purple halo surrounding the colonies, except for

tyramine which developed as a transparent halo in the

cloudy tyrosine containing medium.

2.5. Technological characterisation of L. lactis

The acid-producing ability of the strains was tested

by inoculating stationary cultures of each of them

(1%, v/v) into 11% (w/v) sterile reconstituted

skimmed milk (RSM). Incubation was at 30 jC for

6, 8 and 24 h and the acidity was measured by titration

of the cultures to pH 8.2 with 0.1 M NaOH (Bradley

et al., 1992). The data were expressed as g lactic acid

per 100 ml milk culture.

The determination of the proteolytic activity of the

strains was measured by the method of Citti et al.

(1963). Stationary cultures of each strain were inocu-

lated into RSM and incubated at 30 jC for 24 h.

Afterwards, the concentration of free aromatic amino

acids liberated was measured by a standard method

(Folin and Ciocalteau, 1927).

Diacetyl production was determined according to

King (1948), by mixing 1 ml of milk (inoculated at 1%

and incubated for 24 h at 30 jC) with 0.5 ml of a-

naphthol (1% in 96% ethanol) and 0.5 ml of 16%

KOH. Diacetyl producer strains showed a red ring at

the top of the tube after incubation at 30 jC for 10 min.

2.6. DNA techniques

Plasmid DNAwas isolated from overnight cultures

in M17 broth according to O’Sullivan and Klaenham-

mer (1993). Chromosomal restriction profiles were

obtained by pulsed-field gel electrophoresis (PFGE)

(Hung and Bandziulis, 1990). Essentially, 10 ml of

culture (OD600 = 0.5–1.2) grown in M17 supple-

mented with 20 mM DL-threonine were centrifuged,

resuspended in 50 mM EDTA, mixed with 1% low

melting point agarose (w/v) and poured into 100 Almoulds to produce plugs where the cell lysis and DNA

purification was undertaken. PCR was performed by a

standard procedure using the following conditions: 3

min at 94 jC followed by 30 cycles (1 min at 94 jC, 1min at 50 jC, 1 min at 72 jC) and a final step of 5 min

at 72 jC. Plasmid DNA, restriction fragments and

PCR products were separated by electrophoresis in

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222 215

0.7% agarose gels using Tris–borate buffer (pH 8) as

electrolyte, stained with ethidium bromide and visual-

ised under UV light. Hybridisation experiments were

performed according to Sambrook et al. (1989). The

target DNA was fixed to Hybond membranes (Amer-

sham Biotech, Cerdanyola, Spain). Labelling and

detection of the probes was performed with the non-

radioactive DNA kit ECL (Amersham) in accordance

with the recommendations of the manufacturer.

2.7. Milk fermentation assays

Aliquots of pasteurised milk were inoculated in

parallel with 107 cfu/ml of L. lactis IPLA542 or L.

lactis MG1614, which was used as a phage sensitive

control (in this case, the milk was enriched with 0.5%

glucose and 0.25% yeast extract to compensate the

inability of the strain to ferment lactose and to degrade

casein). Both cultures were divided into two aliquots,

and phage f647 (final titre: 104 pfu/ml) was added to

one of them. Samples were taken every 2 h and their

pH, titratable acidity, starter evolution and phage titre

were determined.

3. Results

3.1. Pattern of phage insensitivity among wild L.

lactis isolates

The 33 L. lactis isolates out of the 100 obtained

from farmhouse dairy products were resistant against

the 34 industrial bacteriophages of our collection.

Another 35 isolates were susceptible to between one

and five phages (Fig. 1). It is noticeable that none of

the isolates were susceptible to more than 20 phages.

Furthermore, when resistant, our isolates appeared to

be completely refractory to infection, no plaques were

detected even when 109 viable phages were used per

lawn of the test organism. On the other hand, no

correlation was found between the sensitivity/resist-

ance phenotype of the isolates and the group to which

the phage belonged, e.g. some of them turned out to be

sensitive to some c2-type phages but resistant to

others. The same was found for the 936-phage group

(data not shown).

To determine whether the phage-resistance was due,

at least in some cases, to a superinfection immunity

mechanism based on the presence of a resident pro-

phage in the genome of any of the isolates, their

induction was attempted with sublethal concentrations

of mitomycin C (0.6 Ag/ml). In most cases, a delay in

the growth of the strains was observed, as expected

from their susceptibility to the antibiotic, but no exten-

sive lysis was noticeable (Fig. 2). Complementarily, no

phages were detected in the supernatants of the treated

cultures. However, in 6 out of the 33 isolates tested,

mitomycin C induced a sharp decrease in the absorb-

ance of the cultures (Fig. 2). In one case, this was

accompanied by the appearance of plaques on lawns of

the test organism L. lactis MG1614, when inoculated

with supernatants of the antibiotic treated cultures.

None of the other five strains produced plaques when

their supernatants were tested against any of the six

strains used for propagation of the phages of the

collection. Despite this, all of them were discarded

because their behaviour might indicate the presence

of defective prophages in their genomes or, alterna-

tively, the absence in our collection of appropriate

susceptible strains.

3.2. Biogenic amine production

Biogenic amines are generated through amino acid

decarboxylation, which is an undesirable trait for food

grade microorganisms. The 27 isolates shown to be

resistant to all phages were tested for production of

tyramine, histamine, cadaverine, putrescine and trypt-

amine. Only five isolates synthesised tyramine from

Fig. 1. Distribution of resistance to 34 bacteriophages among 100

wild L. lactis isolates. The number of phage susceptible bacteria is

plotted against the number of phages able to form plaques on them.

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222216

tyrosine, being the rest negative for any of the amines

tested. Thus, these five were discarded as starter

precursors. Consequently, only 22 out of the 100

starting isolates were selected for further work.

3.3. Technological characterisation of the phage-

resistant L. lactis isolates

All 22 L. lactis isolates selected produced acid from

lactose and 16of them,when inoculated at 1%, rendered

0.4% or higher lactic acid after 6 h in RSM at 30 jC(Table 1). Moreover, 17 reached values in excess of

0.6% lactic acid after 8 h of incubation (data not shown).

Similarly, all 22 isolates turned out to be proteo-

lytic, although their levels of activity varied broadly.

Thirteen of them rendered between 25 and 50 Ag/ml

tyrosine equivalents after 24 h of incubation in milk at

30 jC, while another two produced concentrations of

tyrosine equivalents close to 60 Ag/ml (Table 1).

Finally, 11 of the 22 bacteriophage-resistant iso-

lates selected in this work produced diacetyl (which is

responsible for the buttery flavour of dairy products),

although 7 of them were weak producers (Table 1).

3.4. Genetic characterisation of the strains

The 100 isolates used in this work were obtained

during a 2-year period and thus a high variability was

expected. However, to know precisely how many

different strains existed among the 22 isolates resistant

to the phage collection that were not biogenic amine

producers, their plasmid profiles and PFGE chromoso-

mal patterns were determined. Ten plasmid arrange-

ments were observed (Fig. 3a). Some were more

common than others and thus a particular profile was

Table 1

Technological parameters of the phage-resistant L. lactis isolates

grown in milka

L. lactis

isolates

Lactic

acid (%)

Proteolysis

(Ag Tyr/ml)

Diacetyl

production

IPLA514 0.30 45.6 �IPLA524 0.46 32.8 +

IPLA542 0.48 57.9 + + +

IPLA549 0.30 31.0 �IPLA551 0.44 25.0 + + +

IPLA555 0.49 15.3 �IPLA560 0.32 27.4 + + +

IPLA578 0.42 33.6 +

IPLA625 0.46 33.5 �IPLA629 0.50 28.1 �IPLA630 0.47 33.1 �IPLA832 0.49 13.9 �IPLA838 0.42 6.4 + + +

IPLA910 0.40 31.1 +

IPLA969 0.36 64.3 +

IPLA974 0.24 50.0 +

IPLA981 0.42 45.3 +

IPLA992 0.42 51.3 �IPLA1066 0.42 51.0 �IPLA1110 0.48 11.7 �IPLA1557 0.50 24.2 +

IPLA1738 0.23 26.1 �a A 1% inoculum was used. Data for lactic acid correspond to 6

h of incubation, while those of proteolysis and diacetyl production

were taken at 24 h.

Fig. 2. Effect of mitomycin C (MC) (0.6 Ag/ml) on the growth of the L. lactis isolates (E). (a) A strain partially susceptible to MC. (b) A strain

in which a prophage became induced. Control without MC (n).

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222 217

shown by eight isolates while another four included

two or three isolates each. Conversely, five patterns

were represented by single isolates.

Eight chromosomal profiles were observed upon

restriction with SmaI and CspI of total DNA from the

22 isolates (Fig. 3b). Some of them were represented

by several isolates, up to eight, while others were

peculiar to one strain. By combining the plasmid and

chromosomal profiles, the 22 isolates could be

reduced to 16 strains (enumerated in Table 2).

3.5. Analysis of the mechanisms of bacteriophage

resistance

In order to find out the mechanisms through which

our strains were refractory to phage infection, all of

them were subjected to specific tests. Lack of adsorp-

tion turned out to be the most abundant cause of

insensitivity. Eleven of the strains did not adsorb

significantly to any of the three phages used, which

belonged to the c2 and 936 species and to the unclas-

sified group (Table 2). Another five showed percen-

tages of adsorption lower or equal to 30%, a value that

although much lower than the 90–95% observed for

the control strain L. lactis MG1614 might indicate the

need for complementary systems of defence. To find

out which might be present in our strains, PCR experi-

ments using primers specific for the genetic determi-

nants of the abortive infection systems abiI, abiK, abiG

and for the restriction–modification system LlaBI

were conducted with the plasmid complement of all

16 strains. Positive amplification was observed when

the abiG and abiI specific primers were used against

the DNA of L. lactis IPLA1110 and L. lactis

IPLA1738, respectively, being the PCR reactions neg-

ative for the rest (Table 2). Complementarily, total

DNA from each strain was hybridised against probes

matching abiP and abiD1, with negative results. How-

ever, the hybridisation with an abiB specific probe

resulted positive in 11 out of the 16 strains (Table 2).

The combined lack of adsorption plus the presumptive

Table 2

Mechanisms of resistance against phage infection present in the 16

selected strains

L. lactis

strains

abiB abiI abiK abiG abiD1 abiP LlaBI %

Adsorptiona

IPLA514 � � � � � � � V 5

IPLA524 + � � � � � � 0

IPLA542 + � � � � � � 0

IPLA549 � � � � � � � 10.5

IPLA551 � � � � � � � 25

IPLA555 + � � � � � � 0

IPLA578 � � � � � � � V 5

IPLA625 + � � � � � � 26.1

IPLA832 + � � � � � � 26.3

IPLA838 + � � � � � � V 5

IPLA910 + � � � � � � 0

IPLA974 + � � � � � � 0

IPLA981 + � � � � � � 0

IPLA1066 + � � � � � � V 5

IPLA1110 � � � + � � � V 5

IPLA1738 + + � � � � � 31.5

a The % of adsorption was initially determined for a phage of the

c2 group. The strains that did not adsorb to it were tested against

one representative phage of the other two groups (936 and

unclassified). The figures indicate the maximum adsorption values

with either of the three phages.

Fig. 3. (a) Examples of the plasmid patterns of some of the wild isolates of L. lactis resistant to phage infection; migration of size standards is

indicated and corresponds to the data obtained from lane 8 (supercoiled DNA ladder; Gibco BRL). (b) CspI chromosome restriction pattern of a

sample of the strains as viewed after pulsed-field gel electrophoresis (PFGE); size standards (lane 1) are those contained in the pulsed-field

marker 50–1000 kb (Sigma).

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222218

presence of Abi systems justified the bacteriophage

resistance of most of the strains characterised in this

work. However, two of the five strains that presented

detectable adsorption values (10% and 25%, respec-

tively) were negative in the PCR and hybridisation

tests, suggesting either that the level of adsorption

interference shown was enough to protect them against

infection or that they might carry other resistance

mechanisms.

3.6. Milk fermentation by a resistant strain in the

presence of phage

To find out the real value of our strains as starters

and the stabilities of their phage resistance phenotypes,

pilot milk fermentation assays were performed. The

strain selected was L. lactis IPLA542 because it fer-

ments lactose efficiently, is moderately proteolytic,

produces diacetyl from citrate and, importantly, is

refractory to phage adsorption and harbours an abiB-

like genetic determinant. Besides, it is a representative

of the most common plasmid and chromosome struc-

tural patterns. Parallel fermentations were carried out

with L. lactis MG1614, which was used as a phage-

susceptible control.

The milk inoculated with L. lactis IPLA542 became

coagulated irrespective of the presence or absence of

the phage in it. After 8 h of incubation a firm clot was

generated from both fermentations. On the contrary,

the milk inoculated with L. lactis MG1614 (supple-

mented with 0.5% glucose and 0.25% yeast extract)

plus phage f647 did not coagulate, although it did

when no phage was added to the milk.

These results correlate with the analytical data

obtained from the samples. In the milk inoculated with

L. lactis IPLA542, the pH of the culture became

stabilised after 6 h of incubation at a value of around

4.4, while the lactic acid concentration rose to 0.75%

(Fig. 4a). Conversely, in the milk inoculated with L.

lactis MG1614 and contaminated with phage, neither

pH lowering nor lactic acid accumulation was

observed, while the control without phage showed an

acidification ability similar to the test strain (Fig. 4b).

Viable counts fitted with these physicochemical data.

L. lactis IPLA542 concentration increased during the

six first hours of incubation to reach levels close to 1010

Fig. 4. Physicochemical and microbiological behaviour of the strains L. lactis IPLA542 (a, c) and MG1614 (b, d) when grown in milk in the

absence (o, 5) or presence (., n) of phage f647. Evolution of the bacteriophage populations (E).

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222 219

cfu/ml, regardless of whether the milk was contami-

nated with phage or not (Fig. 4c). Similar results were

obtained for L. lactis MG1614 when grown in the

absence of phage. However, this last strain became

lysed by the phage, which resulted in a lowering of the

viable count by almost 4 logarithmic units during the

first 2 h of incubation (Fig. 4d). Phage concentration

did not change in the milk inoculated with L. lactis

IPLA542 (Fig. 4c), but showed an increase of about 6

logarithmic units in the milk containing L. lactis

MG1614 (Fig. 4d).

On the other hand, growth of L. lactis IPLA542 for a

week in liquid medium (with successive inoculations

into fresh M17 broth every 24 h) followed by challeng-

ing of single colonies to phages revealed that all

conserved the phage-resistant phenotype, indicating

the complete stability of the character.

4. Discussion

To determine whether dairy artisan environments

could be a good source of new strains to be incorpo-

rated into industrial starters, 100 isolates of L. lactis,

collected throughout a period of 2 years, were chal-

lenged to a collection of phages isolated from indus-

trial environments and, later on, assayed for their

technological properties. None of them were suscep-

tible to more than half of a collection of phages isolated

in geographically distant milk environments to max-

imise their diversity. Furthermore, one third were

resistant to the whole collection, although, in some

cases this resistance was due in part to the presence of

resident prophages in their genomes. A further selec-

tion was undertaken among the isolates refractory to

all phages, based on their inability to form biogenic

amines through amino acid decarboxylation. These

compounds are undesirable in dairy products because

they induce systemic pathological effects, probably

because they may act as haptens in the intestine, thus

promoting allergic reactions in its surface (Rice et al.,

1976; Ten Brink et al., 1990; Buckenhuskes, 1993).

Based on this, five isolates were discarded due to their

ability to produce tyramine from tyrosine.

The 22 out of the 100 isolates that were refractory

to all 34 phages of the collection, non-lysogenic and

unable to produce biogenic amines turned out to be

able to ferment lactose, proteolytic and, in some cases,

to produce diacetyl, three parameters crucial to their

utility as dairy starters. Lactic acid production from

lactose is the main role played by L. lactis in milk.

According to Timmons et al. (1988), one strain may be

considered useful when, after inoculation at 3% in

milk, it is able to generate 0.4% lactic acid upon

incubation at 30 jC for 6 h. Most of our strains (16

out of 22) overcame this value even when inoculated

at 1% (Table 1). Proteolysis is important for dairy L.

lactis strains because they tend to be auxotrophic for

several amino acids and, since their concentration in

milk can only support about 20% of the needs of a

typical starter, the rest has to be obtained through

hydrolysis of milk proteins, especially casein. In this

respect, all the 22 isolates were proteolytic, although

their activities varied broadly, but 15 of them gener-

ated in excess of 25 Ag/ml tyrosine equivalents after

24 h of incubation in milk, thus fulfilling the require-

ments to be considered useful as proteolytic starters.

Diacetyl, which confers the butter flavour and aroma

to dairy products, is generated from pyruvate through

a minor pathway of recuperation of NAD+ (Hugen-

holtz, 1993). Its main precursor in milk is citric acid,

which is internalised by L. lactis var. diacetylactis,

thanks to a plasmid-encoded permease (David et al.,

1990). Four of the isolates were good diacetyl pro-

ducers, to the point that they could be considered as

aromatic strains. Another seven might act as adjuvant

strains with respect to this parameter.

The chromosomal analysis of the 22 L. lactis

isolates revealed eight restriction patterns, which

indicates that there exists a substantial variability of

genotypes in the microbial community under study.

However, one pattern, represented by eight isolates,

was prominent, possibly indicating that this was

better adapted than the others, especially those repre-

sented by two isolates (two cases) or just one (three

cases). The variability among the isolates was further

increased by the plasmid complement harboured by

the strains. Ten extrachromosomal DNA patterns

were observed, although one of them, which was

shared by eight isolates, was more frequent than the

rest. Concordance was observed between the chro-

mosomal and plasmid pattern of some isolates; how-

ever, this was not complete, finally rendering the 22

isolates into 16 strains. Some of the selective pres-

sures that might have made some genotypes more

frequent than others may be suggested. For example,

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222220

all the eight isolates belonging to the most common

chromosomal pattern presented high lactose fermen-

tation and proteolysis capacities. Conversely, two out

of the three patterns represented by single isolates

showed almost negligible proteolytic values. Interest-

ingly, only three of the eight isolates belonging to the

most frequent genotype were good diacetyl pro-

ducers, possibly indicating that the citrate permease

gene is plasmid-encoded, as is generally the case for

the aromatic strains of L. lactis used in the dairy

industry.

Adsorption interference appears to be the main

cause of phage infection blockage shown by the wild

strains of L. lactis studied. The advantages of having

this mechanism of infection interference might be: (i) it

is more efficient to impede infection than to stop it once

it has started, and (ii) there is not loss of cell viability,

while other systems of phage resistance such as abor-

tive infection (Abi) usually induce death of the infected

cells (Allison and Klaenhammer, 1998; Forde and

Fitzgerald, 1999). However, the presence in most of

our isolates of at least two mechanisms of resistance

that act successively may have a synergistic effect in

the defence against phage attack, especially for the

strains that adsorb phage moderately. The first mech-

anism (reduced adsorption) would restrict the number

and frequency of phages reaching the cytoplasm, thus

facilitating the interference with their development

exerted by any of the Abi mechanisms found in

individual strains. The high frequency of abiB deter-

minants among our isolates is noticeable. This might

reflect the selection pressure exerted by the phages and,

indirectly, indicate that AbiB is more proficient in

aborting phage development than the other Abi sys-

tems detected in the population (AbiG and AbiI). This

effect might rely on the fact that AbiB induces degra-

dation of phage transcripts early after the beginning of

infection (Parreira et al., 1996), while AbiG inhibits

only late transcription (O’Connor et al., 1996), when

high concentrations of template are present in the cell,

thus hindering their neutralization. However, the wide

distribution of abiB might also be, at least in part, the

consequence of the location of its genetic determinant

in a plasmid, in the vicinity of an ISS1 element that

contains the promoter from which abiB is transcribed

(Cluzel et al., 1991), suggesting that this insertion

sequence might somehow promote transfer of the

resistance determinant.

The data reported indicate that the strains selected

in this work fulfil the requirements to be considered as

potential components of starter formulations and also

that the dairy environments in which farmhouse

cheeses are being produced may be a source of

valuable strains with good combinations of properties

which, upon optimization, might help to improve the

competitiveness of the dairy industry.

Acknowledgements

This work was supported by grants PB-EXP01-23

from FICYT (Principado de Asturias) and BIO2001-

3621 from CICYT (Spanish Ministry of Researh and

Technology). C.M. is the recipient of a PRI fellowship

from FICYT.

References

Accolas, J.P., Peigney, C., Limsowtin, G.K.Y., Cluzel, P.J., Se-

chaud, L., 1994. Lutte contre les bacteriophages dans l’industrie

laitiere. In: de Roissart, H., Luquet, F.M. (Eds.), Bacteries Lac-

tiques. Lorica, Uriage, France, pp. 473–492.

Allison, G.E., Klaenhammer, T.R., 1998. Phage resistance mecha-

nisms in lactic acid bacteria. International Dairy Journal 8,

207–226.

Bover-Cid, S., Holzapfel, W.H., 1999. Improved screening proce-

dure for biogenic amine production by lactic acid bacteria. In-

ternational Journal of Food Microbiology 53, 33–41.

Bradley, R.L., Arnold, E., Barbano, D.M., Semerad, R.G., Smith,

D.E., Vines, B.K., 1992. Chemical and physical methods. In:

Marshall, R.T. (Ed.), Standard Methods for the Examination of

Dairy Products. American Public Health Association, Washing-

ton, DC, pp. 433–531.

Brussow, H., 2001. Phages of dairy bacteria. Annual Review of

Microbiology 55, 283–303.

Buckenhuskes, H.J., 1993. Selection criteria for lactic acid bacteria

to be used as starter cultures for various commodities. FEMS

Microbiology Reviews 12, 253–272.

Christensen, J.E., Dudley, E.G., Pedersen, J.A., Steele, J.L., 1999.

Peptidases and amino acid catabolism in lactic acid bacteria.

Antonie van Leeuwenhoek 76, 217–246.

Citti, J.E., Sandine, W.E., Elliker, P.R., 1963. Some observation on

the Hull method for measurement of proteolysis in milk. Journal

of Dairy Science 46, 337.

Cleveland, J., Montville, T.J., Nes, I.F., Chikindas, M.L., 2001.

Bacteriocins: safe, natural antimicrobials for food preservation.

International Journal of Food Microbiology 71, 1–20.

Cluzel, P.-J., Chopin, A., Ehrlich, S.D., Chopin, M.C., 1991. Phage

abortive infection mechanism from Lactococcus lactis subsp.

lactis, expression of which is mediated by an Iso-ISS1 element.

Applied and Environmental Microbiology 57, 3547–3551.

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222 221

Cuesta, P., 1996. Desarrollo de un cultivo iniciador para el queso

Afuega’l Pitu. PhD thesis, University of Oviedo, Oviedo, Spain.

Cuesta, P., Suarez, J.E., Rodrıguez, A., 1995. Incidence of lysog-

eny in wild lactococcal strains. Journal of Dairy Science 78,

998–1003.

Daly, C., Fiztgerald, G., Davies, R., 1996. Biotechnology of lactic

acid bacteria with special reference to bacteriophage resistance.

Antonie van Leeuwenhoek 70, 99–110.

David, S., Van Der Rest, M.E., Driessen, A.J.M., Simons, G., De

Vos, W.M., 1990. Nucleotide sequence and expression in Es-

cherichia coli of the Lactococcus lactis citrate permease gene.

Journal of Bacteriology 172, 5789–5794.

de Roissart, H., Luquet, F.M. (Eds.), 1994. Bacteries Lactiques.

Lorica, Uriage, France.

Desmazeaud, M.J., 1994. Le lait milieu de culture. In: de Roissart,

H., Luquet, F.M. (Eds.), Bacteries Lactiques. Lorica, Uriage,

France, pp. 25–36.

Durmaz, E., Klaenhammer, T.R., 2000. Genetic analysis of chromo-

somal regions ofLactococcus lactis acquired by recombinant lytic

phages. Applied and Environmental Microbiology 66, 895–903.

Fernandez, L., Beerthuyzen, M.M., Brown, J., Siezen, R.J., Cool-

bear, T., Holland, R., Kuipers, O.P., 2000. Cloning, character-

ization, controlled overexpression and inactivation of the major

tributyrin esterase gene of Lactococcus lactis. Applied and En-

vironmental Microbiology 66, 1360–1368.

Folin, O., Ciocalteau, V., 1927. On tyrosine and tryptophan deter-

minations in proteins. Journal of Biological Chemistry 73,

1150–1157.

Forde, A., Fitzgerald, G.F., 1999. Bacteriophage defence in lactic

acid bacteria. Antonie van Leeuwenhoek 76, 89–113.

Gasson, M.J., 1983. Plasmid complements of Streptococcus lactis

NCDO712 and other streptococci after protoplast-induced cur-

ing. Journal of Bacteriology 154, 1–9.

Hugenholtz, J., 1993. Citrate metabolism in lactic acid bacteria.

FEMS Microbiology Reviews 12, 253–272.

Hung, L., Bandziulis, R., 1990. Promega Notes No. 24, p. 1.

Jarvis, A.W., Fitzgerald, G.F., Mata, M., Mercenier, A., Neve, H.,

Powell, I.B., Ronda, C., Saxelin, M., Teuber, M., 1991. Species

and type phages of lactococcal bacteriophages. Intervirology 32,

2–9.

Joosten, H.M.L.J., Northolt, M.D., 1989. Detection, growth and

amine-producing capacity of lactobacilli in cheese. Applied

and Environmental Microbiology 55, 2356–2359.

Josephsen, J., Neve, H., 1998. Bacteriophages and lactic acid bac-

teria. In: Salminen, S., von Wright, S.A. (Eds.), Lactic Acid

Bacteria. Microbiology and Functional Aspects. Marcel Dekker,

New York, pp. 385–436.

King, N., 1948. Modification of the Voges–Prokauer test for rapid

colorimetric determination of acetylmethyl carbinol plus diace-

tyl in butter cultures. Dairy Industries 13, 860–866.

Kok, J., De Vos, V.M., 1994. The proteolytic system of lactic

acid bacteria. In: Gasson, M.J., De Vos, W.M. (Eds.), Genetics

and Biotechnology of Lactic Acid Bacteria. Blackie, London,

pp. 169–210.

Labrie, S., Moineau, S., 2000. Multiplex PCR for detection and

identification of lactococcal bacteriophages. Applied and Envi-

ronmental Microbiology 66, 987–994.

Moineau, S., 1999. Applications of phage resistance in lactic acid

bacteria. Antonie van Leeuwenhoek 76, 377–382.

Moineau, S., Pandian, S., Klaenhammer, T.R., 1994. Evolution of a

lytic bacteriophage via DNA acquisition from the Lactococcus

lactis chromosome. Applied and Environmental Microbiology

60, 1832–1841.

Moineau, S., Borkaev, M., Holler, B.J., Walker, S.A., Kondo, J.K.,

Vedamuthu, E.R., Vanderbergh, P.A., 1996. Isolation and char-

acterization of lactococcal bacteriophages from cultured butter-

milk plants in the United States. Journal of Dairy Science 79,

2104–2111.

Moll, G.N., Konings, W.N., Driessen, A.J.M., 1999. Bacteriocins:

mechanism of membrane insertion and pore formation. Antonie

van Leeuwenhoek 76, 185–198.

Nes, I.F., Diep, D.B., Havarstein, L.S., Brurberg, M.B., 1996. Bio-

synthesis of bacteriocins in lactic acid bacteria. Antonie van

Leeuwenhoek 70, 113–128.

Niven, C.F., Jeffrey, M.B., Corlee, D.A., 1981. Differential plating

medium for quantitative detection of histamine-producing bac-

teria. Applied and Environmental Microbiology 41, 321–322.

O’Connor, L., Coffey, L.A., Daly, C., Fitzgerald, G.F., 1996. Abi G, a

genotypically novel abortive infection mechanisms encoded by a

plasmid pCI750 of Lactococcus lactis subsp. cremoris UC653.

Applied and Environmental Microbiology 62, 3075–3082.

O’Sullivan, D.J., Klaenhammer, T.R., 1993. Rapid mini-prep iso-

lation of high-quality plasmid DNA from Lactococcus and Lac-

tobacillus spp. Applied and Environmental Microbiology 59,

2730–2733.

Parreira, R., Ehrlich, S.D., Chopin, M.C., 1996. Dramatic decay of

phage transcripts in lactococcal cells carrying the abortive infec-

tion determinant AbiB. Molecular Microbiology 19, 221–230.

Rice, S.I., Eitenmiller, R.R., Koehler, P.E., 1976. Biologically ac-

tive amines in food: a review. Journal of Milk and Food Tech-

nology 39, 353–358.

Salminen, S., von Wright, S.A. (Eds.), 1998. Lactic Acid Bacteria.

Microbiology and Functional Aspects. Marcel Dekker, New

York.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning:

A Laboratory Manual. Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, NY.

Ten Brink, B., Damink, C., Joosten, H.M.L.J., Huis in’t Veld,

J.H.J., 1990. Occurrence and formation of biogenically active

amines in foods. International Journal of Food Microbiology 11,

73–84.

Terzaghi, B.E., Sandine, W.E., 1975. Improved medium for lactic

streptococci and their bacteriophages. Applied and Environmen-

tal Microbiology 29, 807–813.

Timmons, P., Hurley, M., Drinan, F., Daly, C., Cogan, T.M., 1988.

Development and use of a defined strain starter system for

Cheddar cheese. Journal of the Society of Dairy Technology

41, 49–53.

C. Madera et al. / International Journal of Food Microbiology 86 (2003) 213–222222