Characterization of the lactic acid bacteria in artisanal dairy products

Journal of Dairy Research (1997) 64 409–421 Printed in Great Britain 409

Characterization of the lactic acid bacteria in artisanal dairy

products

B TIMOTHY M. COGAN*, MANUELA BARBOSA†, ERIC BEUVIER‡,BRUNA BIANCHI-SALVADORI§, PIER S. COCCONCELLI¶, ISABEL

FERNANDES†, JESUS GOMEZs, ROSARIO GOMEZ**, GEORGEKALANTZOPOULOS††, ANTONIO LEDDA‡‡, MARGARITA MEDINAs,

MARY C. REA* EVA RODRIGUEZs

*National Dairy Products Research Centre, Teagasc, Fermoy, Irish Republic† Instituto Nacional de Engenharia e Tecnologia Industrial, Azinhaga dos Lameiros,

P-1699 Lisbon, Portugal‡ Institut National de la Recherche Agronomique, Station de Recherches en Technologie

et Analyses Laitie[ res, BP 89, F-39801 Poligny, France§Centro Sperimentale del Latte, Strada per Merlino 3, I-20060 Zelo Buon Persico

(Mi), Italia¶ Istituto di Microbiologia, Universita[ Cattolica del Sacro Cuore, Via Emilia

Parmense, 84, I-29100 Piacenza, Italias Instituto Nacional de InvestigacioU n y TecnologıUa AgrarıUa y Alimentaria,

Apartado 8111, E-28080 Madrid, Espanh a** Instituto del Frio, Ciudad Universitaria, E-28040 Madrid, Espanh a

††Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece‡‡ Istituto Zootecnico Caseario per la Sardegna, I-07040 Olmedo (Sassari), Sardinia,

Italia

(Received 20 December 1995 and accepted for publication 20 December 1996 )

S. In all, 4379 isolates from 35 products, including 24 artisanal cheeses, weresurveyed with a view to identifying strains that could be used as starters incommercial dairy fermentations. Of the isolates, 38% were classified as Lactococcus,17% as Enterococcus, 14% as Streptococcus thermophilus, 12% as mesophilicLactobacillus, 10% as Leuconostoc and 9% as thermophilic Lactobacillus. Acidproduction by the isolates varied considerably. Of the 1582 isolates of Lactococcusand 482 isolates of mesophilic Lactobacillus tested, only 8 and 2% respectivelyproduced sufficient acid to lower the pH of milk to ! 5±3 in 6 h at 30 °C. In contrast,53, 32 and 13% of Str. thermophilus, thermophilic Lactobacillus and Enterococcusisolates respectively reduced the pH to 5±3. These isolates were found only in someFrench, Italian and Greek cheeses. Bacteriocins were produced by 11% of the 2257isolates tested and 26 of them produced broad-spectrum bacteriocins which inhibitedat least eight of the ten target strains used, which included lactic acid bacteria,clostridia and Listeria innocua. The most proteolytic of the 2469 isolates tested wereStr. thermophilus from Fontina cheese followed by Enterococcus from Fiore Sardo andToma cheese and thermophilic Lactobacillus from all sources. Exopolysaccharideswere produced by 5±3% of the 2224 isolates tested.

In many Southern European countries cheeses are made from cows’, goats’, ewes’

410 T. M. C

and buffalo milk by farmers and shepherds on a small scale in the farmhouse or dairyusing traditional techniques. In this paper these cheeses will be referred to as‘artisanal ’. Commercial starter cultures are not normally used in making thesecheeses. Instead the cheesemaker relies on the lactic acid bacteria (LAB) naturallypresent in the milk, as adventitious contaminants, to grow and produce the lacticacid required for expulsion of the whey (moisture) from the coagulum duringcheesemaking. Alternatively, different types of natural cultures are used which areproduced by incubating milk or whey from the previous day under definedconditions. With some notable exceptions, e.g. Manchego, Cabrales, Maho! n andMajorero cheese (Nun4 ez & Martı!nez-Moreno, 1976; Nun4 ez, 1978; Sua! rez et al. 1983;Fontecha et al. 1990), little is known about the LAB involved in artisanal cheeses.Manchego, Cabrales, Maho! n and Majorero cheese usually contain high numbers oflactococci, leuconostocs, mesophilic lactobacilli and enterococci.

Proteolytic activity and lactic acid, bacteriocin and exopolysaccharide (EPS)production are important attributes of the starter bacteria used in commercialcheesemaking. Milk is deficient in many of the amino acids required by starterbacteria for growth. Consequently these bacteria must have a proteolytic system tohydrolyse the milk proteins to the amino acids and peptides required for growth.Proteolysis is also important in the development of flavour in cheese. The ability toproduce acid rapidly is probably the most important property of starter bacteria. Ithelps to reduce the pH which in turn increases the expulsion of whey from the curdand reduces the moisture content. The low pH and low moisture are important inreducing the propensity of the cheese for microbial spoilage. Bacteriocin productionby the starter LAB may also be important in this regard. Broad-spectrumbacteriocins produced by GRAS (generally regarded as safe) organisms such as LABhave potentially wide uses in the food industry especially if they are heat and acidstable and active at the neutral or slightly acidic pH values of many foods (for areview see DeVuyst & Vandamme, 1994). EPS production is not an importantproperty of the starters used in cheese manufacture but it is important in those thatare used to make fermented milks where the EPS increases the body and texture ofthe product.

This paper is a report on the identification of a large number of LAB, isolatedfrom artisanal products, to genus level and their characterization in terms of thecommercially important properties outlined above. Such strains should be useful inextending the number of commercially available cultures and in forming the basis ofnew starter cultures for the manufacture of fermented dairy products.

Origin of samples

Altogether 35 different products were used, including 24 cows’, ewes’ or goats’milk cheeses, 6 natural starters, 2 fermented milks and cows’, ewes’ and goats’ milk.The origin and the number of samples tested are shown in Table 1.

Isolation of strains

Thermophilic lactobacilli and streptococci were isolated on milk (100 g}l) agarcontaining 0±2 g bromcresol purple}l. The spread plate technique with anaerobicincubation at 42 °C was used for the lactobacilli and the pour plate technique withaerobic incubation at 42 °C for Streptococcus thermophilus. Mesophilic strains wereisolated on KCA agar (Nickels & Leesment, 1964), KCA agar containing X-Gal

Lactic acid bacteria in artisanal cheeses 411

Table 1. Location, source, age and number of products sampled in this study

Product Type of product Type of milk Source Laboratory

No. oflocationssampled

No. ofsamplestested

Age ofsamples,

d

Kefir Fermented milk Cow Ireland DPC 33 33 3Sa4 o Jorge Cheese Cow Azores DPC 5 5 4–5Sa4 o Joa4 o Cheese Cow Azores DPC 1 4 4–5Fresh Greek Cheese Ewe Greece AUA 1 11 5cheese

Kasseri Cheese Ewe Greece AUA 1 30 9–120Feta Cheese Ewe Greece AUA 2 7 2–365Galotyri Cheese Ewe Greece AUA 1 3 9, 30Scotta innesto Natural starter Ewe Sardinia IZCS 10 10 1Gioddu Yogurt Ewe Sardinia IZCS 4 4 2Fiore Sardo Cheese Ewe Sardinia IZCS 6 8 1, 130Casu axedu Cheese Goat Sardinia IZCS 10 10 2Mozzarella Natural starter Cow Italy CSL 1 2 1–3Caciotta Natural starter Cow Italy CSL 1 1 1–3Grana Natural starter Cow Italy CSL 1 4 1–2Pecorino Natural starter Ewe Italy CSL 1 3 1–3Caciotta Cheese Cow Italy CSL 1 6 1–30Fontina Cheese Cow Italy UCSC 1 11 1–2Toma Cheese Cow Italy UCSC 1 4 1–2Val Itelvi Cheese Cow Italy UCSC 1 2 1–2Serra da Estrela Cheese Ewe Portugal INETI 6 33 30–40Milk Ewe Portugal INETI 24 31 1–2Manchego Cheese Ewe Spain INIA 17 17 1–2Gredos Cheese Goat Spain INIA 12 15 1–2Majorero Cheese Goat Spain IFPL 4 8 2, 30Milk Goat Spain IFPL 2 4 1–2Fresh French Cheese Cow France INRA 25 40 1–8cheese

Munster Cheese Cow France INRA 2 4 1Reblochon-type Cheese Cow France INRA 1 2 1Vacherin-type Cheese Cow France INRA 1 1 1Chevret Cheese Cow France INRA 1 3 1Tomme Cheese Cow, ewe, goat France INRA 18 33 1–100Morbier Cheese Cow France INRA 1 1 120Comte! Cheese Cow France INRA 6 9 1Comte! Natural starter Cow France INRA 10 14 1Milk Cow France INRA 16 16 1

DPC, National Dairy Products Research Centre, Teagasc, Fermoy, Irish Republic ; AUA, AgriculturalUniversity of Athens, Greece; IZCS, Istituto Zootecnico e Caseario per la Sardegna, Olmedo, Sardinia, Italy;CSL, Centro Sperimentale del Latte, Zelo Buon Persico (MI) Italy; UCSC, Universita' Cattolica del Sacro Cuore,Piacenza, Italy; INETI, Instituto Nacional de Engenharia e Tecnologia Industrial, Lisbon, Portugal ; INIA,Instituto Nacional de Investigacio! n y Tecnologia Agraria y Alimentaria, Madrid, Spain; IFPL, Instituto delFrio, Consejo Superior de Investigaciones Cientificas, Ciudad Universitaria, Madrid, Spain; INRA, InstitutNational de la Recherche Agronomique, Poligny, France.

(Vogensen et al. 1987) or LM17 agar (Terzaghi & Sandine, 1975) at 30 °C. All strainswere stored frozen at ®80 °C in the laboratories in which they were isolated.

Identification

Thermophilic strains were grown in MRS broth (De Man et al. 1960) andmesophilic strains in LM17 broth. All strains were tested for their Gram reaction,catalase using 3 g H

#O

#}l, and shape by phase contrast microscopy of overnight

cultures. Growth of the coccal-shaped organisms was examined in LM17 afterincubation at 10 °C for 7 d, at 45 °C for 2 d, and in LM17 containing 20, 40 and65 g NaCl}l after incubation at 30 °C for 2–3 d (mesophiles) and in LM17 containing65 g NaCl}l after incubation at 42 °C for 2 d (thermophiles). Growth of the rod-

412 T. M. C

Table 2. Tests used in presumptive identification of the isolates in this study

Growth in NaCl of Growth at

Shape Heterofermentation 20 g}l 40 g}l 65 g}l 10 °C 15 °C 45 °C Identification

Cocci ® }® ® ND ® LactococcusCocci }® ND ND ® LeuconostocCocci ® ND EnterococcusCocci ND ® ® ® ND Streptococcus thermophilusRods ND ND ND ND ND ® Thermophilic LactobacillusRods }® ND ® Mesophilic Lactobacillus

, Positive; ®, negative; }®, positive or negative; ND, not determined.

shaped organisms was examined in MRS at 15 and 45 °C after 5 and 2 d respectively.All mesophilic strains were tested for heterofermentation or homofermentation ofsugars by the procedure of Abd-el-Malek & Gibson (1948). Based on these results, allGram-positive, catalase-negative strains were presumptively classified to genus levelaccording to the outline shown in Table 2.

Acid production

This was determined in heat-treated (5 min at 121 °C) reconstituted skim milk(RSM, 100 g}l) from each laboratory or in Nilac skim milk, (NIZO, NL-6710 Ede,The Netherlands). A 10 ml}l inoculum was used with incubation for 6 h at 30 °C formesophiles and 42 °C for the thermophiles. In this regard, enterococci wereconsidered to be thermophiles.

Proteolysis

This was measured by the o-phthaldialdehyde method (Church et al. 1983) afterincubation in RSM at 30 °C (mesophiles) or 42 °C (thermophiles) for 24 h. The resultswere calculated as the increase in A

$%!over the uninoculated control.

Bacteriocin production

This was measured by the overlay (O) and agar diffusion (AD) techniques. In theO technique, spots (10 µl) of log phase cells were spotted on the appropriate agar(MRS or LM17) and, after overnight growth at the appropriate temperature,overlaid with 5 ml of the target strain (C 10& cfu}ml) in sloppy agar (7±5 g}l). Theplates were incubated at the temperature appropriate for the target strain for 2–3 dand the diameter of the zone of inhibition measured. For the AD technique, the teststrain, grown in the appropriate broth, was centrifuged and the supernatantadjusted to pH 6±5 before filtration (Millex GV; Millipore, Bedford, MA 01730, USA).The target organism was seeded into the appropriate medium (C 10& cfu}ml). Wells(diam. 6 mm) were cut in the solidified agar and filled with 50 µl of test solution. Theplates were held at 4 °C for 2 h to allow diffusion of the bacteriocin into the agarbefore incubating them at the optimum temperature of the target strain for 1–2 d.

The target (indicator) organisms used were Lactobacillus casei ATCC 334, Lb.helveticus ATCC 15009, Lb. reuteri DSM 20016, Pediococcus pentosaceus FBB 63,Lactococcus lactis subsp. cremoris CNRZ 117, Listeria innocua BL 86}26, Str.thermophilus ST20, Enterococcus faecalis EF1, Clostridium sporogenes C22}10, and Cl.tyrobutyricum NCDO 1754. All of these were provided to each author by Dr P. S.Cocconcelli, Istituto di Microbiologia, Universita' Cattolica del Sacro Cuore, I-29100Piacenza, Italy. These strains have also been used to screen bacteriocin productionin the FLAIR and BRIDGE programmes of the EU. Str. thermophilus ST112 was


used instead of Str. thermophilus ST20 in some laboratories while Ln. mesenteroidesDB 1275 and Lc. lactis subsp. lactis IFPL 186 were also used in the IstitutoZootecnico e Caseario per la Sardegna and the Instituto del Frio respectively. Cl.sporogenes NCFB 1755 and Cl. tyrobutyricum NCFB 1791 were used instead of thetarget clostridia at the National Dairy Products Research Centre.

Polysaccharide formation

EPS production from lactose was determined by qualitatively measuring thedegree of ‘stringiness’ of cultures grown in RSM at 30 °C (mesophiles) or 42 °C(thermophiles) for 18 h. A positive result was recorded if the coagulated culture couldbe teased into a string with an inoculating loop. Strains that failed to coagulate milkin 18 h were rechecked in RSM containing yeast extract (3 g}l).

Characterization

Handling large numbers of isolates meant that relatively few tests would be doneon each one. The tests outlined in Table 2 can discriminate adequately between mostof the common genera of starter bacteria but they do not unequivocally distinguishbetween Enterococcus and Lactococcus since Ec. dispar and Ec. sulfureus do not growat 45 °C (Devriese et al. 1993) and some Lactococcus strains can grow in 65 g NaCl}l(Facklam & Collins, 1989; Pot et al. 1994, 1996); thus Lactococcus and Enterococcuscould be confused. However, it is likely that Ec. faecalis, Ec. faecium, Lc. lactis andLc. raffinolactis would be the commonest species of enterococci and lactococci foundin milk and cheese. These enterococci and lactococci would be distinguished fromeach other at the genus level by their growth responses at 45 °C and in 65 g NaCl}l(Table 2).

A total of 4379 isolates from 35 different sources were classified (Table 3). Mostof the cheeses examined were relatively young and would, therefore, be dominatedby the bacteria responsible for acid production. Most strains were identified asLactococcus (38%) followed in order by Enterococcus (17%), Str. thermophilus (14%),mesophilic Lactobacillus (12%), Leuconostoc (10%) and thermophilic Lactobacillus(9%). Only 26 (! 0±6%) of the strains could not be characterized by the tests usedand were considered to be atypical. For the purposes of this paper, Enterococcusstrains are considered to be thermophilic.

Strains from each of the above groups of LAB, except Leuconostoc, were found inKasseri, Feta and fresh cheese from Greece, in Tomme cheese from France, and inFontina and Toma cheese from Italy. Manchego, Gredos and Majorero cheese fromSpain and Serra da Estrela cheese from Portugal contained Lactococcus, Leuconostoc,mesophilic Lactobacillus and Enterococcus, which confirms previous results (Nun4 ez &Martı!nez-Moreno, 1976; Nun4 ez, 1978; Sua! rez et al. 1983; Fontecha et al. 1990).

Lactococcus strains were isolated from kefir, natural Mozzarella starter and all 24cheeses, except Galotyri, Caciotta, Morbier and Comte! (Table 3). Many of these alsocontained thermophilic LAB. The presence of mesophilic and thermophilic strains inthe same product is not unusual in artisanal cheeses, which are made withoutcommercial starters.

Mesophilic lactobacilli were isolated from 19 of the 35 products but in smallnumbers from most of them. However, they were a major component of themicroflora of Kasseri, Feta, Serra da Estrela, Gredos and Majorero cheese. Except forKasseri, large numbers of lactococci were also isolated from these cheeses. Cheese is

414 T. M. C

Table

3.Sou

rce

and

iden

tifica

tion

ofth

eis

olate

sfr

omart

isanaldair

ypro

duct

sto

genus

orsp

ecie

sle

vel

Pro

duct

Lact

ococ

cus

Leu

conos

toc

Ente

roco

ccus

Str

epto

cocc

us

ther

mop

hilus

Aty

pic

al

cocc

iM

esophilic

Lact

obaci

llus

Ther

mophilic

Lact

obaci

llus

Aty

pic

al

rods

Tota

ls

Kefi

r361

41

402

Sa4 o

Jorg

ech

eese

238

47

1286

Sa4 o

Joa4 o

chee

se11

11

Fre

shG

reek

chee

se17

36

55

310

121

Kass

erich

eese

590

90

47

60

292

Fet

ach

eese

100

28

324

4159

Galo

tyri

chee

se3

3Sco

tta

innes

to2

93

54

149

Gio

ddu

32

55

87

Fio

reSard

och

eese

17

30

47

Casu

axed

uch

eese

73

11

13

3100

Mozz

are

lla,natu

ralst

art

er57

11

41

8108

Caci

ott

a,natu

ralst

art

er30

30

Gra

na,natu

ralst

art

er73

73

Pec

ori

no,natu

ralst

art

er12

61

73

Caci

ott

a,ch

eese

106

106

Fonti

na

chee

se9

67

45

19

40

180

Tom

ach

eese

534

93

17

68

ValIt

elvich

eese

4152

77

10

243

Ser

rada

Est

rela

chee

se24

85

172

182

Ser

rada

Est

rela

milk

35

60

83

178

Manch

ego

chee

se169

101

59

31

360

Gre

dos

chee

se186

58

74

55

373

Majo

rero

chee

se47

11

74

10

107

249

Majo

rero

milk

48

20

32

100

Fre

shF

rench

chee

se105

19

12

61

2145

Munst

erch

eese

96

11

21

20

Reb

loch

on-t

ype

chee

se8

8V

ach

erin

-type

chee

se5

21

8Chev

ret

chee

se12

31

117

Tom

me

chee

se72

10

17

25

19

2127

Morb

ier

chee

se4

4Com

te! c

hee

se5

49

Com

te! n

atu

ralst

art

er11

57

932

Com

te! m

ilk

21

15

229

Tota

l1649

442

724

608

24

527

403

24379


an ideal medium for the growth of mesophilic lactobacilli (Laleye et al. 1990;Peterson & Marshall, 1990; McSweeney et al. 1993, 1994), but whether they areinvolved in ripening is not clear.

Significant numbers of enterococci were found in fresh Greek, Kasseri, Feta,Fontina, Fiore Sardo, Toma, Val Itelvi, Manchego, Gredos and Majorero cheese.Enterococci were more prevalent in the Sardinian cheeses, Fiore Sardo and Casuaxedu, which had mainly a mesophilic flora, than in the other Sardinian products,Scotta innesto and Gioddu, which had a thermophilic flora. Scotta innesto is anatural starter and Gioddu is a ewes’ milk yogurt.

The presence of enterococci in cheese has been questioned since some strains cancause serious health problems in humans, especially endocarditis and urinary tractinfections. However, they have a positive effect on flavour development in many ofthese cheeses. Their isolation in such large numbers is not surprising since they arenormal inhabitants of the intestine from where they contaminate the udder and themilk. They have been found previously in high numbers in artisanal cheeses (Nun4 ez& Martı!nez-Moreno, 1976; Nun4 ez, 1978; Sua! rez et al. 1983; Fontecha et al. 1990) andsome workers have shown their usefulness in flavour formation in Cheddar cheese(Jensen et al. 1975). Enterococci have several properties (e.g. heat and salt tolerance)which make them ideal starter cultures and many of them are also good acidproducers. Their role in cheese ripening should be re-evaluated.

Acid production

A good acid-producing starter culture will reduce the pH of milk from its normalvalue of C 6±6 to 5±3 in 6 h at 30 °C (mesophiles) or 42 °C (thermophiles) using aninoculum of 10 ml}l. Of the 442 Leuconostoc isolates tested for acid production onlysix reduced the pH of RSM below 5±9, five (one each from Sa4 o Jorge, Majorero andTomme cheese and two from ewes’ milk) to pH 5±5 and one (from ewes’ milk) to 5±4.Despite this leuconostocs are considered to play an important role in ripening of Serrada Estrela cheese. The acid-producing abilities of a further 3475 isolates aresummarized in Table 4. In interpreting these results and those in the rest of thisstudy it should be remembered that some isolates from the same source could beidentical.

The vast majority of Lactococcus isolates were poor acid producers: only 8±3% ofthe 1582 isolates tested reduced the pH of RSM to ! 5±3 (Table 4). This may bebecause lactococci that ferment lactose slowly contain both β-galactosidase (EC3.2.1.23) and phospho-β-galactosidase (EC 3.2.1.85), while those that ferment itrapidly contain only phospho-β-galactosidase (Farrow, 1980). Rapid acid-producingLactococcus were more prevalent in Mozzarella natural starter and Comte! milk andFeta, Fiore Sardo, Casu axedu, Fontina, Majorero and Tomme cheese than in theother cheeses. Some of these cheeses also contained thermophilic LAB. Within themesophilic Lactobacillus, only 2±1% of the isolates reduced the pH to ! 5±3 and mostof these were from Fontina cheese.

Rapid acid producers were much more prevalent in thermophilic than inmesophilic LAB, and were more common in some cheeses than in others. Forexample, large numbers of good, acid-producing Enterococcus were found particularlyin Fontina and Val Itelvi cheese while good acid-producing thermophilic Lactobacilluswere found in Scotta innesto, Gioddu and Fontina cheese and in natural Granastarters. In contrast, slow acid-producing thermophilic Lactobacillus were found inKasseri and Toma cheese and Mozzarella and Pecorino natural starters. Good acid-producing Str. thermophilus were common in all cheeses in which they were found.

416 T. M. C

Table

4.N

um

ber

ofis

olate

sfr

omart

isanaldair

ypro

duct

sin

each

grou

pte

sted

and

the

num

ber

thatre

duce

dth

epH

ofm

ilk

to!

5±3

,!

5±1

,!

4±9

and

!4±7

in6

hat30

°C(m

esop

hiles

)or

42

°C(ther

mop

hiles

)

Lact

ococ

cus

Ente

roco

ccus

Str

epto

cocc

us

ther

mop

hilus

Tota

lno.

No.re

duci

ng

the

pH

toT

ota

lno.

No.re

duci

ng

the

pH

toT

ota

lno.

No.re

duci

ng

the

pH

to

Pro

duct

test

ed!

5±3

!5±1

!4±9

!4±7

test

ed!

5±3

!5±1

!4±9

!4±7

test

ed!

5±3

!5±1

!4±9

!4±7

Kefi

r358

20

00

Sa4 o

Jorg

ech

eese

220

10

00

Sa4 o

Joa4 o

chee

se11

00

00

Fre

shG

reek

chee

se17

00

00

36

64

10

55

15

10

0K

ass

erich

eese

50

00

090

42

10

90

23

12

2F

eta

chee

se96

22

15

83

26

22

21

32

00

Galo

tyri

chee

se7

00

00

Sco

tta

innes

to93

67

57

55

48

Gio

ddu

32

29

18

11

11

Fio

reSard

och

eese

17

12

74

130

00

00

Casu

axed

uch

eese

73

19

70

011

20

00

14

62

00

Mozz

are

lla,natu

ralst

art

er55

12

20

040

15

97

6Caci

ott

a,natu

ralst

art

er30

24

24

24

24

Gra

na,natu

ralst

art

erP

ecori

no,natu

ralst

art

er12

20

00

Caci

ott

a,ch

eese

106

73

73

69

69

Fonti

na

chee

se6

55

21

67

33

27

16

427

96

30

Tom

ach

eese

51

00

034

86

60

10

40

00

ValIt

elvich

eese

44

00

010

81

10

11

00

0Ser

rada

Est

rela

chee

se24

00

00

10

00

0Ser

rada

Est

rela

milk

28

00

00

Manch

ego

chee

se169

10

00

59

00

00

Gre

dos

chee

se186

00

00

74

00

00

Majo

rero

chee

se49

16

14

14

13

Majo

rero

milk

90

00

0F

resh

Fre

nch

chee

se105

10

30

012

10

00

Munst

erch

eese

93

10

01

11

00

10

00

0R

eblo

chon-t

ype

chee

se8

10

00

Vach

erin

-type

chee

se5

00

00

10

00

0Chev

ret

chee

se12

00

00

30

00

0T

om

me

chee

se72

13

72

017

11

00

22

22

1M

orb

ier

chee

seCom

te! c

hee

se5

41

00

Com

te! n

atu

ralst

art

er11

00

00

50

00

07

31

00

Com

te! m

ilk

21

93

10

51

00

0T

ota

l1582

131

64

31

18

482

67

44

27

5528

279

215

173

159


Ther

mophilic

Lact

obaci

llus

Mes

ophilic

Lact

obaci

llus

Tota

lno.

No.re

duci

ng

the

pH

toT

ota

lno.

No.re

duci

ng

the

pH

to

Pro

duct

test

ed!

5±3

!5±1

!4±9

!4±7

test

ed!

5±3

!5±1

!4±9

!4±7

Kefi

rSa4 o

Jorg

ech

eese

Sa4 o

Joa4 o

chee

seF

resh

Gre

ekch

eese

10

43

13

00

00

Kass

erich

eese

60

21

047

00

00

Fet

ach

eese

41

10

11

00

00

Galo

tyri

chee

seSco

tta

innes

to54

40

34

34

33

Gio

ddu

55

45

21

10

7F

iore

Sard

och

eese

Casu

axed

uch

eese

32

22

213

00

00

Mozz

are

lla,natu

ralst

art

er8

00

00

Caci

ott

a,natu

ralst

art

erG

rana,natu

ralst

art

er73

17

80

0P

ecori

no,natu

ralst

art

er61

00

00

Caci

ott

a,ch

eese

Fonti

na

chee

se41

15

10

71

17

55

30

Tom

ach

eese

17

00

00

31

10

0V

alIt

elvich

eese

11

00

0Ser

rada

Est

rela

chee

se72

00

00

Ser

rada

Est

rela

milk

83

00

00

Manch

ego

chee

se31

00

00

Gre

dos

chee

se55

00

00

Majo

rero

chee

se108

31

10

Majo

rero

milk

10

00

00

Fre

shF

rench

chee

se1

00

00

Munst

erch

eese

10

00

0R

eblo

chon-t

ype

chee

seV

ach

erin

-type

chee

seChev

ret

chee

se1

00

00

Tom

me

chee

se1

10

00

19

00

00

Morb

ier

chee

se1

00

00

40

00

0Com

te! c

hee

se4

11

00

Com

te! n

atu

ralst

art

er9

10

00

Com

te! m

ilk

20

00

0T

ota

l401

129

81

54

43

482

10

74

0

418 T. M. C

The reason that good acid producers were not isolated uniformly from all cheeses isnot clear, but the results suggest that each cheese is a unique ecosystem.

Bacteriocin production

The O and AD methods were used to monitor bacteriocin production. The Otechnique was used in the National Dairy Products Research Centre, CentroSperimentale del Latte and INRA Poligny, and the AD technique in the Universita'Cattolica del Sacro Cuoro, the Agricultural University of Athens and the IstitutoZootecnico e Caseario per la Sardegna. Both techniques were used at the Instituto delFrio, Instituto Nacional de Engenharia e Tecnologia Industrial and InstitutoNacional de Investigacio! n y Tecnologia Agraria y Alimentaria. The O method iseasier to use where large numbers of strains have to be screened, but H

#O

#and lactic

acid can give false positives. In the AD method, neutralized supernatants were used,which overcame the problems due to lactic acid. A preliminary trial at InstitutoNacional de Investigacio! n y Tecnologia Agraria y Alimentaria and Instituto del Frioshowed that the number of bacteriocin-producing strains was reduced from 579 bythe O technique to 12 when the AD technique was used. Thus the latter techniqueis better in determining bacteriocin production despite the fact that it is morelaborious. These results suggest that few strains are genuine bacteriocin producersunder the conditions of the assay and that the numbers of bacteriocin-producingstrains checked by the O method may be overestimated. In the O technique used inthe National Dairy Products Research Centre, 10 µl proteinase K was spottedalongside the producer organism to eliminate nonspecific interference. Only strainsthat produced inhibitory zones that were inactivated by proteinase K (EC 3.4.21.14)were considered to be bacteriocin-producing.

Overall, 239 (11%) of the 2257 strains tested produced bacteriocins, most ofwhich were narrow-spectrum, inhibiting only one or two of the target strains, ormedium-spectrum, inhibiting three to seven of the target strains. A total of 26 broad-spectrum bacteriocins, which inhibited eight to ten of the target strains, wereidentified. Fifteen isolates (Lactococcus strains DPC 3147, DPC 3153, DPC 3178, DPC3205, DPC 3216, DPC 3244, DPC 3254, INRA 224, INRA 226 and INIA 515 andthermophilic Lactobacillus strains UCSC 20613, UCSC 3051, UCSC 3052, UCSC 3117and UCSC 3119) inhibited all ten target strains. The remaining eleven isolates haddifferent activities (Table 5). Broad-spectrum bacteriocins were found in all groupsof LAB except Str. thermophilus.

Little is known about the properties of the bacteriocins identified in the presentstudy but several are being actively studied. At least three different bacteriocinswere produced by the lactococcal isolates from kefir; a narrow-spectrum one thatinhibited only lactococci, a medium-spectrum one that inhibited Lb. casei, Lb.helveticus and Pd. pentosaceus and a broad-spectrum one that inhibited the ten targetstrains. That produced by DPC 3147 inhibited the ten target strains and numerousother strains of Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Str. thermophilusand Staphylococcus aureus (Ryan et al. 1996). This was not nisin, since it inhibited anisin producer. Production of the bacteriocin was plasmid encoded. It wasparticularly inhibitory to the non-starter LAB found in Cheddar cheese and is beingused as a tool to study the effect of non-starter LAB on cheese flavour. A novel phageresistance gene is also encoded on the same plasmid (Coakley et al. 1997). Strain IFPL105 (Table 5) has been identified as Lb. curvatus. The bacteriocin produced by thisstrain is heat-stable (partial inactivation occurs at 121 °C in 15 min), has a


Table

5.Sum

mary

ofth

eba

cter

ioci

n-p

roduci

ng

stra

ins

from

art

isanaldair

ypro

duct

sth

atin

hib

ited

eigh

tor

nin

eof

the

targ

etst

rain

s

Bact

erio

cin-p

roduci

ng

stra

in

Leu

conos

toc

Lact

obaci

llus

Ente

roco

ccus

Lact

ococ

cus

stra

ins

stra

ins

stra

ins

stra

ins

Targ

etst

rain

INR

A39

INR

A73

INR

A172

INR

A222

INR

A244

INR

A259

Ther

mophilic

CSL

9±4

Mes

ophilic

IFP

L105

INR

A296

INR

A307

INR

A309

Lact

obaci

llus

case

iA

TCC

334

®

®

®

Lb.

hel

veticu

sA

TCC

15009

Lb.

reute

riD

SM

20016

®

®

®

P

edio

cocc

us

pen

tosa

ceus

FB

B63

®

Lact

ococ

cus

lact

issu

bsp

.cr

emor

isCN

RZ

117

Lis

teri

ain

noc

ua

BL

86}2

6

®

Str

epto

cocc

us

ther

mop

hilus

ST

20

ND

ND

ND

ND

ND

ND

ND

ND

ND

Str

.th

erm

ophilus

ST

112

ND

ND

®

®E

nte

roco

ccus

faec

alis

EF

1

®

®

Clo

stri

diu

msp

orog

enes

C22}1

0

Cl.

tyro

buty

ricu

mN

CD

O1754

®

,In

hib

itio

n;®

,no

inhib

itio

n;N

D,not

det

erm

ined

.

Table

6.E

xop

olysa

cchari

de

pro

duct

ion

bydiff

eren

tis

olate

sof

lact

icaci

dba

cter

iafr

omart

isanalpro

duct

s

Lact

ococ

cus

Leu

conos

toc

Str

epto

cocc

us

ther

mop

hilus

Ther

mophilic

Lact

obaci

llus

Mes

ophilic

Lact

obaci

llus

Ente

roco

ccus

Labora

tory

Tota

lno.

test

edN

o.

posi

tive

Tota

lno.

test

edN

o.

posi

tive

Tota

lno.

test

edN

o.

posi

tive

Tota

lno.

test

edN

o.

posi

tive

Tota

lno.

test

edN

o.

posi

tive

Tota

lno.

test

edN

o.

posi

tive

INIA

355

16

159

086

0133

1A

UA

126

23

155

41

78

8157

10

CSL

57

11

0189

1142

01

0U

CSC

50

61

12

15

010

2D

PC

487

12

47

01

0IN

ET

I12

0T

ota

l1030

52

219

0350

43

232

991

0302

13

INIA

,In

stit

uto

Naci

onaldeIn

ves

tigaci

o! ny

Tec

nolo

gia

Agra

ria

yA

lim

enta

ria,M

adri

d,Spain

;A

UA

,A

gri

cult

ura

lU

niv

ersi

tyofA

then

s,G

reec

e;CSL

,Cen

tro

Sper

imen

tale

del

Latt

e,Zel

oB

uon

Per

sico

(MI)

,It

aly

;U

CSC,U

niv

ersi

ta'Catt

olica

del

Sacr

oCuore

,P

iace

nza

,It

aly

;D

PC,N

ati

onalD

air

yP

roduct

sR

esea

rch

Cen

tre,

Tea

gasc

,F

erm

oy,

Iris

hR

epublic;IN

ET

I,In

stit

uto

Naci

onalde

Engen

hari

ae

Tec

nolo

gia

Indust

rial,

Lis

bon,P

ort

ugal.

420 T. M. C

bacteriolytic mode of action, is plasmid encoded and inhibits several lactobacilli,enterococci, lactococci, pediococci, listeria, clostridia and bacilli (Casla et al. 1996).

Proteolysis

Another technologically important property of starter cultures is possession of aproteinase system. Proteinase activity is necessary for good growth of starterbacteria in milk and in hydrolysing casein during the ripening of cheese. Theproteolytic activity of 2469 strains was measured. Altogether 35 sets of correlationsbetween acid production and proteolysis were calculated for the different groups ofLAB from different products. Within the same group, the coefficients ofdetermination (r#) varied widely, e.g. from 0±00 to 0±49 for the lactococci, from 0±02to 0±59 for Str. thermophilus, from 0±00 to 0±06 for the thermophilic Lactobacillus andfrom 0±01 to 0±55 for Enterococcus (results not shown).

The most proteolytic organisms were Str. thermophilus strains from Fontinacheese followed by Enterococcus isolates from Fiore Sardo and Toma cheese andthermophilic lactobacilli from Grana, Gioddu, Toma, Scotta innesto and Fontina(results not shown). In addition, there was considerable variation in the proteolyticactivity within some bacterial groups from the same cheese, e.g. Str. thermophilusand the enterococci isolated from Toma and Fiore Sardo showed similar extremes.In contrast, there was relatively little variation in the proteolytic activity of thelactococci or the mesophilic or thermophilic lactobacilli, with the possible exceptionof the lactococcal isolates from Serra da Estrela (results not shown). The reason forthese contrasting results are not clear. However, they do suggest that strains of thesame genus or species isolated from the same source were different.

Exopolysaccharide production

Only 117 (5%) of the 2224 strains tested produced EPS (Table 6). This was notsurprising since EPS production, at least in lactococci, is normally plasmidassociated and is therefore easily lost on subculture.

Conclusions

Considerable variation was found in the types of LAB present in 35 Europeanartisanal products and in their ability to produce acid, EPS, bacteriocins andproteinases, suggesting that each cheese is a unique ecosystem. Some of these isolatesare potentially useful new starters for fermented dairy products and evaluation oftheir cheesemaking characteristics is currently being undertaken. In addition, theisolates producing broad spectrum bacteriocins are being studied to determine ifthese are novel bacteriocins and if they are of value in controlling the growth ofspoilage and pathogenic bacteria in foods.

The European Union is thanked for partly financing this project under ECLAIRcontract CT-91-0064. A data base of 3719 strains on FileMaker Pro for the PC isavailable from Dr P. S. Cocconcelli, Istituto de Microbiologia, Universita' CattolicaSacro Cuoro, I-29100 Piacenza, Italy.

A--M, Y. & G, T. 1948 Studies in the bacteriology of milk. I. The streptococci of milk. Journalof Dairy Research 15 233–248

C, D., R, T. & G! , R. 1996 Antimicrobial activity of lactic acid bacteria isolated from goat’smilk and artisanal cheeses : characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL 105.Journal of Applied Bacteriology 81 35–41


C, F. C., S, H. E., P, D. H. & C, G. L. 1983 Spectrophotometric assay usingo-phthalaldehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science66 1219–1227

C, M., F, G. F. & R, R. P. 1997 Application and evaluation of the phage resistance- andbacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures. Applied andEnvironmental Microbiology 63 1434–1440

D M, J. C., R, M. & S, M. E. 1960 A medium for the cultivation of lactobacilli. Journal ofApplied Bacteriology 23 130–135

D, L. A., P, B. & C, M. D. 1993 Phenotypic identification of the genus Enterococcus anddifferentiation of phylogenetically distinct enterococcal species and species groups. Journal of AppliedBacteriology 75 399–408

DV, L. & V, E. J. 1994 Bacteriocins of Lactic Acid Bacteria: microbiology, genetics andapplications. London: Blackie

F, R. R. & C, M. D. 1989 Identification of Enterococcus species isolated from human infectionsby a conventional test scheme. Journal of Clinical Microbiology 27 731–734

F, J. A. E. 1980 Lactose hydrolysing enzymes in Streptococcus lactis and Streptococcus cremoris and alsoin some other species of streptococci. Journal of Applied Bacteriology 49 493–503

F, J., P! , C., J! , M., R, T., G! , C. & R, M. 1990 Biochemical andmicrobiological characteristics of artisanal hard goat’s cheese. Journal of Dairy Science 73 1150–1157

J, J. P., R, G. W., W, C. J. & V, E. R. 1975 Role of enterococci in Cheddarcheese: organoleptic considerations. Journal of Milk and Food Technology 38 142–145

L, L. C. S, R. E., L, B. H. & H, R. A. 1990 Quality attributes of Cheddar cheesecontaining added lactobacilli. Journal of Food Science 55 114–118

MS, P. L. H., F, P. F., L, J. A., J, K. N. & C, T. M. 1993 Contribution of theindigenous microflora to the maturation of Cheddar cheese. International Dairy Journal 3 613–634

MS, P. L. H., W, E. M., F, P. F., C, T. M., D, F. D. & C-G, M. 1994A procedure for the manufacture of Cheddar cheese under controlled bacteriological conditions and the effectof adjunct lactobacilli on cheese quality. Irish Journal of Agricultural and Food Research 33 183–192

N, C. & L, H. 1964 [Method for the differentiation and quantitative determination of starterbacteria.] Milchwissenschaft 19 374–378

N4 , M. 1978 Microflora of Cabrales cheese: changes during maturation. Journal of Dairy Research 45501–508

N4 , M. & M!-M, J. L. 1976 [Microbial flora of Manchego cheese. 1. Behaviour of themicrobial flora in artisanal Manchego cheese.] Anales del Instituto Nacional de Investigaciones Agrarias, SerieGeneral 4 11–31

P, S. D. & M, R. T. 1990 Non starter lactobacilli in Cheddar cheese. A review. Journal ofDairy Science 73 1395–1410

P, B., D, L. A., U, D., V, P., H, F. & K, K. 1996 Phenotypicidentification and differentiation of Lactococcus strains from animals. Systematic and Applied Microbiology 19213–222

P, B., L, W., K, K. & S, K.-H. 1994 Taxonomy of lactic acid bacteria. InBacteriocins of Lactic Acid Bacteria, pp. 13–90 (Eds L. deVuyst and E. J. Vandamme). London: Blackie

R, M. P., R, M. C., H, C. & R, R. P. 1996 An application in Cheddar cheese manufacture for astrain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Applied andEnvironmental Microbiology 62 612–619

S! , J. A., B, R. & I4 , B. 1983 Contribution to study of Maho! n cheese. III. Lactic acid bacteriaand enterococci. Chemie Mikrobiologie Technologie der Lebensmittel 8 52–56

T, B. E. & S, W. E. 1975 Improved medium for lactic streptococci and their bacteriophages.Applied Microbiology 29 807–813

V, F. K., K, T., L, J. J., K, B., E, D. & W N, E. W.1987 Improved direct differentiation between Leuconostoc cremoris, Streptococcus lactis subsp. diacetylactis,and Streptococcus cremoris}Streptococcus lactis on agar. Milchwissenschaft 42 646–648

Characterization of the lactic acid bacteria in artisanal dairy products

Documents