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Classification of Citrate Fermenting Lactobacilli Isolated from Lamb Stomach, Sheep Milk and Pecorino Romano Cheese
Zbl. Bakt. Hyg., 1. Abr. Orig. C 2, 349-356 (1981)
1
2
Istituto di Microbiologia Agraria e Tecnica, Facolta Agraria, Universita Cattolica del Sacro Cuore, 29100-Piacenza, Italia Istituto Zootecnicoe Caseario per la Sardegna, 07040 Olmedo (SS), Italia
Classification of Citrate Fermenting Lactobacilli Isolated from Lamb Stomach, Sheep Milk and Pecorino Romano Cheese FRANCO DELLAGLI01, SEVERINO ARRIZZA2, and ANTONIO LEDDA2
Received May 25, 1981
Summary Three hundred and thirty strains of citrate-fermenting lactobacilli were isolated from lamb stomach, lamb rennet paste, sheep milk and Pecorino Romano cheese. Fifty-two representative strains belonging to four physiological groups were checked for their genetic relationship using the DNA-DNA hybridization test. Most of the heterofermentative strains, isolated from lamb stomach and lamb rennet paste belonged to Lactobacillus reuteri, while the others were found to be related to L. fermentum. The homofermentative citrate-fermenting lactobacilli, isolated mainly from Pecorino Romano cheese, shared high levels of genetic homology with L. plantarum. Only one strain was found to be similar to L. casei.
Key words: Citrate-fermenting lactobacilli - Lactobacillus reuteri - Lactobacillus fermentum - Lactobacillus plantarum - Lactobacillus casei - lamb stomach - Rennet - Pecorino Romano
Introduction
The fermentation of citrate is a fairly general property within the genus Lactobacillus. Both homofermentative and heterofermentative citrate-fermenting lactobacilli have been isolated from silage (Keddie, 1959), faeces (Gasser, 1964), Dutch cheese (de Man, 1956), dairy products (Fryer, 1970), Grana cheese (Battistotti and Bottazzi, 1972) and Pecorino Romano cheese (Pettinau et al., 1972). In some cases the presence of these lactic acid bacteria has been correlated with the "blowing" of the cheeses due to the production of carbon dioxide (Freyer et al., 1970; Bottazzi et al., 1971). Several experiments have shown (Pettinau et al., 1972) that the main source of these bacteria is improperly ripened and salted lamb-rennet which is used in
350
F. Dellaglio, S.Arrizza, and A. Ledda
the production of Pecorino Romano cheese. This paper reports the results of a taxonomic study on citrate-fermenting lactobacilli isolated from fresh lamb abomasum, fresh lamb-rennet paste, sheep milk and Pecorino Romano cheese.
Materials and Methods Organisms and growth conditions The strains L. plantarum ATCC 14917, L. casei ATCC 334, L. fermentum ATCC 14932 and L. reuteri DSM 20016, used as references, were obtained from the American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen (DSM), respectively. Before inoculating the test media the strains were cultured in MRS broth (De Man et a!., 1960). MRS agar with a modified glucose content (5 gil instead of 20 gil) and supplemented with 1 ml of a sterile solution of calcium citrate (100 gil) per plate was used for the isolation of citrate-fermenting lactobacilli. Plates were incubated anaerobically in an atmosphere of 80 Ofo N 2 and 20 010 CO 2 for three days at 37°C. Colonies from MRS agar were inoculated into MRS broth. All cultures were purified by streaking on MRS agar and picking single colonies. The physiological characteristics were determined according to Bergey's Manual (Buchanan and Gibbons, 1974). DNA-DNA hybridization and G+C molOfoof DNA MRS broth containing 14C uracil and 14 C adenine (each 50 {lei/l) was employed to label DNA. Lysis of cells and isolation and purification of DNA were carried out as described by Marmur (1961). The G+C molOfo of DNA was determined by the DNA thermal denaturation curve applying De Ley's equation (De Ley, 1970). DNA-DNA hybridization was performed according to the single point competition technique (Johnson and Ordal, 1968) at a reassociation temperature of 60°C as previously described (Dellaglio et aI, 1973). Results and Discussion Three hundred and thirty citrate-fermenting lactobacilli were isolated from the abomasum of suckling lamb just after slaughtering, from fresh improperly Table 1. Source and distribution of citrate fermenting lactobacilli Isolated from Group
Number of strains
Lamb-abomasum and lamb-rennet paste
A CG FC
33 19
A = homofermentative lactobacilli. B = heterofermentative lactobacilli.
Sheep milk
B
A
B
21
5
7
Pecorino Romano cheese
A
B
17
2
Classification of Citrate Fermenting Lactobacilli
351
prepared lamb rennet paste and from Pecorino Romano cheese. With the exception of G+C mol % and DNA-DNA homology the characters listed in Table 2 were determined in all isolates. According to their sources and physiological properties the strains were divided into 4 groups: CG (an abbreviation of the Italian "caglio" indicating bacteria isolated from abomasum, rennet and sheep milk) and FC (an abbreviation of the Italian "formaggio-citrati" indicating bacteria isolated from Pecorino Romano cheese), homofermentative and heterofermentative. As shown in Table 1, fresh rennet and abomasum yielded exclusively, heterofermentative lactobacilli whereas similar numbers of homo- and heterofermentative lactibacilli were isolated from sheep milk. Of 19 strains 17 were homofermentative, however, in Pecorino Romano cheese. From the 4 groups 52 representative strains were selected to investigate also their genotypical characters. Table 2 shows the physiological and genetical characters of the heterofermentative lactobacilli from the various sources. The G+C mol % of DNA as well as the DNA-DNA homology show that 8 strains belong to 1. fermentum and 22 strains to 1. reuteri. In agreement with the observation of Kandler et al. (1980), arabinose is fermented by all strains of 1. reuteri but only by 25 Ufo of 1. [ermentum. Table 2 shows also that fructose is utilized by all strains of 1. fermentum but only by 2 of 22 strains of 1. reuteri and that mannose is normally fermented by strains of 1. fermentum but not by those of 1. reuteri. The two species were also clearly distinguishable by their ability to grow at 15 DC. While all strains of 1. fermentum grew at 15 DC, none but one of the 1. reuteri strains grew at this temperature. A clear genetical difference between 1. reuteri and other heterofermentative lactobacilli species was previously also found by Vescovo et al. (1979). Sarra et al. (1979) have shown that 1. reuteri is the dominant species of lactic acid bacteria in the faeces of milking calves. Moreover, it has also been demonstrated (Dellaglio, unpublished) that the intestine of chicken, pig and sheep is also natural habitat fo this species. Therefore it is not surprising that 1. reuteri is also present in lamb stomach and thus in rennet. It is very likely that a large percentage of the heterofermentatve lactic acid bacteria isolated by various authors from faeces and intestine of domestic animals in the past and classified on a physiological basis as 1. fermentum, in reality, were strains of 1. reuteri. As shown in Table 3, 21 of the 22 homofermentative lactobacilli isolated from sheep milk and Pecorino Romano cheese have to be allotted to 1. plantarum and 1 strain to 1. casei according to both, the physiological and genetical data. It is particularly interesting that almost all of citrate-fermenting homofermentative lactobacilli belong to 1. plantarum and that of 21 strains 16 were isolated from Pecorino Romano cheese. This study on citrate-fermenting lactobacilli shows that 1. reuteri is the dominating heterofermentative species in the stomach of lamb and in fresh rennet, whereas in Pecorino Romano cheese most lactobacilli are homofermentative and belong to 1. plantarum but not to 1. casei as one might expect. Acknowledgments Acknowledgement. We express our thanks to Prof. V. Bottazzi for his interest and helpful discussion and to Prof. O. Kandler for critical reading of the manuscript.
00
"
S
S
0
N
N
+
+
+
+
+
CG 317A
CG 318B
CG 320B
CG 326B
FC 5B
zoois"
CG 22A
DSM
reuteri.
+
0
+
CG 310A
Laatobaci. l:Ius
+
+
+
+
+
+
+
+
+
+
+
+
CG 244B
CG 309D
u
0
+
8
""
""
...0
...
.....
0
OJ
.,
...>: .,
~
.......
0
., ... ...
..c:: OJ
.,
"0 ....
u
Growth
at
0
u
'"...
'0
....
0
+
+
-
0
+
-
+
-
-
+
+
+
+
+
-
-
+
+
-
+
+
-
+
..."
~
-
Q
-
+
-
-
..'i:
~
~
0
-
-
-
-
+
-
+
+
+
+
+
+
w
-
+
;;:
III
§
0
OJ
.,
-
~
H
"
........
~
+
w
+
+
+
+
+
+
+
+
-
~
........
0
OJ
., .......
-
0
-
-
-
-
-
-
III
... '"
-
w
-
w
-
-
-
-
-
~
....0
-
-
w
+
w
w
w
w
-
w
-
-
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
'"
0
.... '"
0
u
u 00
...
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
'""
:>
..... ....~
III
-
+
+
+
+
+
w
-e
... ...., ""
'C
'"
0
""
u
of 0
Fermentation
.,
+
ATCC 14932 a
fermentwn
Laatobaci. l.Lus
Strains
III
...., ... ....... III
-
-
-
-
-
-
-
w
+
'"'
...0
...
:> ..c:: :>
..c::
.......
s-e
'" .d
Z
.... u
==============- ====-===:;;;:- ================~=====~-~-~-~--~-------------------~----~- ---------
-
-
-
+
+
+
+
+
+
0
u
00 III 0
"
.....III
~
.......0
""0
S
....... .""
----
s-e
39,0
40,9
50,1
51,7
51,9
52,1
53,9
51,0
52,7
51,5
53,4
'"'
+
u
~
....
0
27
77
92
84
87
82
85
88
88
100
ATCC 14932
90
100
0
0
0
0
0
0
0
0
20
DSM 20016
homology with
% DNA-DNA
---~~====~========================
Table 2. Phenotypic and genotypic characters of heterofermentative citrate fermenting lactobacilli isolated from sheep milk and Pecorino Romano cheese
w
~
~
~
0.. 0..
(l)
r-
~
0..
;:l
~
N N
::l.
,.,
~
a,
ti
v, N
+
+
CG 176B
CG 195B
+
+
+
+
+
CG 264A
CG 281A FC 3A
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
-
+
+
+
-
-
-
+
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
w
- -
-
-
-
+
+
+
+
+
- -
-
-
w
-
-
-
-
-
-
-
-
-
-
-
-
w
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
-
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
w
w
+
w
+
w
w
w
+
+
+
+
-
-
-
-
-
+
-
+
+
0
0
37 , 8 38,8
37,8
0 0
0
0 0
38 , S
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
38,3
37 ,8
+
38,8
39,S
40,9
38,1
38,8
38,8
39.5
0
0
37,8
38,8
0
0
38,8
37,8
+
+
+
+
0
0
-
-
+
0
+
+
-
-
-
-
-
-
-
0 ..: not investigated; - = negative result; + = positive result; w = weak reaction; a = Type strain. Positive reactions by all strains: galactose, glucose, lactose, maltose, melibi ose, raffinose, ribose, succrose, NH s from arginine and Gram-stain. Negative reacti ons by all strains: cellobiose, glycerol, mannitol, rh amn ose, sorbitol, salicin , hydrolysis of esculin and production of catal ase.
+
+
+
+
CG 248C
CG 257B
+
+
+
+
+
+
+
+
CG 176A
+
+
+
CG 161A
+
+
+
+
CG 151B
CG 233D
+
CG 147D
+
+
+
CG 129A
+
+
+
+
+
CG 231B
+
+
+
CG 122B
CG USB
i-
-
-
+
-
-
+
+
+
+
+
+
+
CG 231A
+
CG 75A
CG 95
+
CG 223E
+
CG 33B
95 93
98
99
96
99
96
80
81
89
87
92
81
79
80
90
78
81
86
89
94
'"
'-" '-"
§;
t->
80-
rv
rt->
(J'l
S'
..,"'T1 3 " ;:;
~
~
o' e s, Q ..,
~
n
s.;
'/> '/>
or
o
+
+
+
+
+
+
+
+
+
+
+
+
+
CG 215B
CG 225D
CG 300B
CG 303B
CG 305e
FC 2A
FC 4A
FC 16A
FC 17B
FC 18A
FC 96B
FC 98A
8
N
"-<
...
a0
u
ATCC 1491 7a
plantal'W71
Lac tobaci. l.Lus
Strains
=•••
X
<1J
<1J U
., ....0 ...0 ...
..-< ::l
..."
......
....0 <1J
<1J
<1J
u
0
u
0
....0 .... ..,. ., .......0 ., '0" ..-< 0 .c .0 ri ..0..... .0...0 ...u .c'" .....'0" .... " 0 0 ...'" H~ ~ l§ ~ '" '" '" '"'"
...... ..... ..."
u
0
Growth at
u co er
0
""...
a
-
-
-
-
-
-
8'
a
...0 "-<
00
0
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
10<
...::l
u
+
~
- -
-
+
+
+
" -e
...
.0
0
-
- - -
w
- - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
<1J
~
..-<
w +
+
+
+
+
+
+
+
+
+
+
+
- - -
- - + - - + + + - + + - - + + - - + + - + + + - - - + + - - + - w + - + + - - + - - -
+
"-< "-<
0
-
+
c
~" :>:"'"
N
<1J
0
OJ
- + - - - + - + + + - + + +
-
- -
w
-
H
" "
.....~
0
0
+
-
+
+
+
w
- -
w
+
w
+
+
+
+
+
+
w +
-
+
+
+
+ +
+
-
+ - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U")
-
'"
...::l
...>>'"
+
+
+
+
+ +
-
+
-
+
+
+
+
+
+
+
+
+
+
+ - - + + + -
+
- - -
U")
"-<
'0
"" ... ...'" ...."
0
::l
+
+
+
+
+
+
+
+
+
-
+
-
+
:I:
~
... -e
....p-, 0
....,
"-< 0
'"
u
......."
_ U== == = = = = == ==_ == _ _ ==
U
..." ....."....
__
::l
<1J
Fe r men t a t i on of
C == = . G= _======= = == _ . _ c_ a == . _ ==== ~ ====_. ~= _ =
0 "-<
<1J
__ .C==========__
=~
...'" '" ......"... ....u
_._ - = =- - = =====- == =
~
45,6
0
0
0
45 , 4
46,6
43 ,9
43,4
43,9
44,6
43,2
43,7
44, 8
+
"
U
""....
======
96
87
89
89
88
98
78
97
95
93
82
82
100
ATCC 14917
0
0
0
0
0
0
0
0
0
0
0
0
10
ATCC 334
% DNA-DNA
= _ ~ = = == =
homology with
_._o..__
Tabl e 3. Phenotypic and genotypic characters of homofermentative citrate ferment ing lactobacilli isolated from lamb stomach, lamb -rennet paste, sheep milk and Pecorino Romano cheese w
VI
.,
~
Il>
"00--
r-
:>
0-
::l
N
II>
J'
,,'
~
..,..,>-
9'
'a
a,
tl
-I>-
+
+
+
+
+
FC l37A
FC 148A
FC l5 0A
FC l53A
+
FC lA
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
w
0
-
-
+
+
+
+
+
- + - -
+
+
+
-
w
0
w
+
0
- -
-
v
- - - - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
0
-
-
+
- - -
+
-
- - + - + -
-
- + - - -
- + - - +
+
+
+
+
-
- -
-
+
+
+
-
+
+
+
+
+
+
+
+
+
w
w
+
+
+
+
w
-
-
w
- -
+
-
- + -
+
-
+
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
+
+
+
+
investigated; - == negative result; + = positive result; w = weak reaction; a = Type strain. Positive reaction s by all strains: cellobiose, galactose, glucose , lactose , maltose, ribose, sucrose, salicin, grow th in presence o f 6.5 Nega tive reaction s by all stra ins: NH , from arginine and prod uction of catalase.
o"'" not
+
AlCC 334
casei:
Lac:tobac:i l l us
+
+
FC 107A
FC 109B
+
FC l Ol A
FC 129A
+
FC 99A + +
-
+
+ +
-
+
+
0
+
+
+
+
+
+
+
+
+
43, 9
45, 4
43,9
45,1
44 , 9
45 ,6
45 , 4
0
45 ,6
0
43, 2
72
0
6
78
94
89
92
78
96
90
92
104
100
0
0
0
0
0
0
0
0
0
'I. NaCI, coag ulation o f milk and Gram-stain.
-
-
+
-
+
+
-
-
+
+
+
-
f;l
v, v,
w
§
~
80"
r>
~
r-
Cl'l
~
,..... ,....'T1 ,.3 ::l g .
9...
g"
::l
~
g.
r>
'" S;
'"
o
356
F.Dellaglio, S.Arrizza, and A.Ledda
References
Battistotti, B., Bottazzi, V.: Ricerche sulla microbiologia del formaggio grana. VIII Parte: sulla presenza di lattobacilli citrato-ferrnentanti in formaggio grana. Sci. Teen. latt.-cas. 23, 59-70 (1972) Bottazzi, V., Ledda, A., Arrizza, S.: Bacteries fermentant les citrates et gonflement du fromage "Pecorino Romano". Le lait 505-506,328-331 (1971) Buchanan, R. E., Gibbons, N. E. (eds.): Bergey's Manual of Determinative Bacteriology, 8th edn. Baltimore: The Williams and Wilkins Co. 1974 De Ley, J.: Reexamination of the association between melting point, bouyant density and chemical base composition of deoxyribonucleic acid. J. Bact. 101, 738-754 (1970) Dellaglio, F., Bottazzi, V., Trovatelli, L. D.: Deoxyribonucleic acid homology and base composition in some thermophilic lactobacilli. J. gen. Microbiol. 74, 289-297 (1973) De Man, J. C;; Enige eigenschappen van staafvormige melkzunrbacterien geisoleerd uit Nederlandse Kaas. Ned. melk-eu Zuiveltijdschr. 10, 240-248 (1956) De Man, }. C., Rogosa, M., Sharpe, M. E.: A medium used for the cultivation of lactobacilli. J. Appl. Bacteriol., 23, 130-135 (1960) Fryer, T F.: Utilization of citrate by lactobacilli isolated from dairy products. J. Dairy Res. 37, 9-15 (1970) Fryer, T. F., Sharpe, M. E., Reiter, B.: Utilization of milk citrate by lactic acid bacteria and "blowing" of filmwrappedd cheese. J. Dairy Res. 37, 17-28 (1970) Gasser, F.: Identification des Lactobacillus fecaux. Ann. Inst. Pasteur (Paris) 106, 778 (1964) Johnson, }. L., Ordal, E. 1.: Deoxyribonucleic acid homology in bacterial taxonomy: effect of incubation temperature in reaction specificity. J. Bact. 95, 893-900 (1968) Kandler, 0., Stetter, K. 0., Kohl, R.: Lactobacillus reuteri sp. nov., a new species of heterofermentative lactobacilli. Zbl. Bakt. Hyg., I Abt. Orig. C 1, 264-269 (1980) Keddie, R. M.: The properties and classification of lactobacilli isolated from grass and silage. J. appl. Bact. 22, 403-416 (1959) Marmur, }.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. molec. BioI. 3, 208-218 (1961) Pettinau, M., Ledda, A., Arrizza, S., Bottazzi, V.: I batteri citrato-fermentanti come agenti di gonfiore nel formaggio Pecorino Romano. Sci. Teen. Iarr, cas. 23, 131-146 (1972) Sarra, P. G., Magri, M., Bottazzi, V., Dellaglio, F., Bosi, E.: Frequenza di bacilli lattici eterofermentanti nelle feci di vitelli lattanti. Arch. Vet. Ital. 30, 16-21 (1979) Vescovo, M., Dellaglio, F., Bottazzi, V., Sarra, P. G.: Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Betabacterium Orla-jensen, Microbiologica 2,317-330 (1979) Prof. F. Dellaglio, Istituto di Microbiologia Agraria e Tecnica, Facolta di Agraria, Universita Cattolica del S. Cuore, 29100 - Piacenza, Italia
1
2
Istituto di Microbiologia Agraria e Tecnica, Facolta Agraria, Universita Cattolica del Sacro Cuore, 29100-Piacenza, Italia Istituto Zootecnicoe Caseario per la Sardegna, 07040 Olmedo (SS), Italia
Classification of Citrate Fermenting Lactobacilli Isolated from Lamb Stomach, Sheep Milk and Pecorino Romano Cheese FRANCO DELLAGLI01, SEVERINO ARRIZZA2, and ANTONIO LEDDA2
Received May 25, 1981
Summary Three hundred and thirty strains of citrate-fermenting lactobacilli were isolated from lamb stomach, lamb rennet paste, sheep milk and Pecorino Romano cheese. Fifty-two representative strains belonging to four physiological groups were checked for their genetic relationship using the DNA-DNA hybridization test. Most of the heterofermentative strains, isolated from lamb stomach and lamb rennet paste belonged to Lactobacillus reuteri, while the others were found to be related to L. fermentum. The homofermentative citrate-fermenting lactobacilli, isolated mainly from Pecorino Romano cheese, shared high levels of genetic homology with L. plantarum. Only one strain was found to be similar to L. casei.
Key words: Citrate-fermenting lactobacilli - Lactobacillus reuteri - Lactobacillus fermentum - Lactobacillus plantarum - Lactobacillus casei - lamb stomach - Rennet - Pecorino Romano
Introduction
The fermentation of citrate is a fairly general property within the genus Lactobacillus. Both homofermentative and heterofermentative citrate-fermenting lactobacilli have been isolated from silage (Keddie, 1959), faeces (Gasser, 1964), Dutch cheese (de Man, 1956), dairy products (Fryer, 1970), Grana cheese (Battistotti and Bottazzi, 1972) and Pecorino Romano cheese (Pettinau et al., 1972). In some cases the presence of these lactic acid bacteria has been correlated with the "blowing" of the cheeses due to the production of carbon dioxide (Freyer et al., 1970; Bottazzi et al., 1971). Several experiments have shown (Pettinau et al., 1972) that the main source of these bacteria is improperly ripened and salted lamb-rennet which is used in
350
F. Dellaglio, S.Arrizza, and A. Ledda
the production of Pecorino Romano cheese. This paper reports the results of a taxonomic study on citrate-fermenting lactobacilli isolated from fresh lamb abomasum, fresh lamb-rennet paste, sheep milk and Pecorino Romano cheese.
Materials and Methods Organisms and growth conditions The strains L. plantarum ATCC 14917, L. casei ATCC 334, L. fermentum ATCC 14932 and L. reuteri DSM 20016, used as references, were obtained from the American Type Culture Collection (ATCC) and Deutsche Sammlung von Mikroorganismen (DSM), respectively. Before inoculating the test media the strains were cultured in MRS broth (De Man et a!., 1960). MRS agar with a modified glucose content (5 gil instead of 20 gil) and supplemented with 1 ml of a sterile solution of calcium citrate (100 gil) per plate was used for the isolation of citrate-fermenting lactobacilli. Plates were incubated anaerobically in an atmosphere of 80 Ofo N 2 and 20 010 CO 2 for three days at 37°C. Colonies from MRS agar were inoculated into MRS broth. All cultures were purified by streaking on MRS agar and picking single colonies. The physiological characteristics were determined according to Bergey's Manual (Buchanan and Gibbons, 1974). DNA-DNA hybridization and G+C molOfoof DNA MRS broth containing 14C uracil and 14 C adenine (each 50 {lei/l) was employed to label DNA. Lysis of cells and isolation and purification of DNA were carried out as described by Marmur (1961). The G+C molOfo of DNA was determined by the DNA thermal denaturation curve applying De Ley's equation (De Ley, 1970). DNA-DNA hybridization was performed according to the single point competition technique (Johnson and Ordal, 1968) at a reassociation temperature of 60°C as previously described (Dellaglio et aI, 1973). Results and Discussion Three hundred and thirty citrate-fermenting lactobacilli were isolated from the abomasum of suckling lamb just after slaughtering, from fresh improperly Table 1. Source and distribution of citrate fermenting lactobacilli Isolated from Group
Number of strains
Lamb-abomasum and lamb-rennet paste
A CG FC
33 19
A = homofermentative lactobacilli. B = heterofermentative lactobacilli.
Sheep milk
B
A
B
21
5
7
Pecorino Romano cheese
A
B
17
2
Classification of Citrate Fermenting Lactobacilli
351
prepared lamb rennet paste and from Pecorino Romano cheese. With the exception of G+C mol % and DNA-DNA homology the characters listed in Table 2 were determined in all isolates. According to their sources and physiological properties the strains were divided into 4 groups: CG (an abbreviation of the Italian "caglio" indicating bacteria isolated from abomasum, rennet and sheep milk) and FC (an abbreviation of the Italian "formaggio-citrati" indicating bacteria isolated from Pecorino Romano cheese), homofermentative and heterofermentative. As shown in Table 1, fresh rennet and abomasum yielded exclusively, heterofermentative lactobacilli whereas similar numbers of homo- and heterofermentative lactibacilli were isolated from sheep milk. Of 19 strains 17 were homofermentative, however, in Pecorino Romano cheese. From the 4 groups 52 representative strains were selected to investigate also their genotypical characters. Table 2 shows the physiological and genetical characters of the heterofermentative lactobacilli from the various sources. The G+C mol % of DNA as well as the DNA-DNA homology show that 8 strains belong to 1. fermentum and 22 strains to 1. reuteri. In agreement with the observation of Kandler et al. (1980), arabinose is fermented by all strains of 1. reuteri but only by 25 Ufo of 1. [ermentum. Table 2 shows also that fructose is utilized by all strains of 1. fermentum but only by 2 of 22 strains of 1. reuteri and that mannose is normally fermented by strains of 1. fermentum but not by those of 1. reuteri. The two species were also clearly distinguishable by their ability to grow at 15 DC. While all strains of 1. fermentum grew at 15 DC, none but one of the 1. reuteri strains grew at this temperature. A clear genetical difference between 1. reuteri and other heterofermentative lactobacilli species was previously also found by Vescovo et al. (1979). Sarra et al. (1979) have shown that 1. reuteri is the dominant species of lactic acid bacteria in the faeces of milking calves. Moreover, it has also been demonstrated (Dellaglio, unpublished) that the intestine of chicken, pig and sheep is also natural habitat fo this species. Therefore it is not surprising that 1. reuteri is also present in lamb stomach and thus in rennet. It is very likely that a large percentage of the heterofermentatve lactic acid bacteria isolated by various authors from faeces and intestine of domestic animals in the past and classified on a physiological basis as 1. fermentum, in reality, were strains of 1. reuteri. As shown in Table 3, 21 of the 22 homofermentative lactobacilli isolated from sheep milk and Pecorino Romano cheese have to be allotted to 1. plantarum and 1 strain to 1. casei according to both, the physiological and genetical data. It is particularly interesting that almost all of citrate-fermenting homofermentative lactobacilli belong to 1. plantarum and that of 21 strains 16 were isolated from Pecorino Romano cheese. This study on citrate-fermenting lactobacilli shows that 1. reuteri is the dominating heterofermentative species in the stomach of lamb and in fresh rennet, whereas in Pecorino Romano cheese most lactobacilli are homofermentative and belong to 1. plantarum but not to 1. casei as one might expect. Acknowledgments Acknowledgement. We express our thanks to Prof. V. Bottazzi for his interest and helpful discussion and to Prof. O. Kandler for critical reading of the manuscript.
00
"
S
S
0
N
N
+
+
+
+
+
CG 317A
CG 318B
CG 320B
CG 326B
FC 5B
zoois"
CG 22A
DSM
reuteri.
+
0
+
CG 310A
Laatobaci. l:Ius
+
+
+
+
+
+
+
+
+
+
+
+
CG 244B
CG 309D
u
0
+
8
""
""
...0
...
.....
0
OJ
.,
...>: .,
~
.......
0
., ... ...
..c:: OJ
.,
"0 ....
u
Growth
at
0
u
'"...
'0
....
0
+
+
-
0
+
-
+
-
-
+
+
+
+
+
-
-
+
+
-
+
+
-
+
..."
~
-
Q
-
+
-
-
..'i:
~
~
0
-
-
-
-
+
-
+
+
+
+
+
+
w
-
+
;;:
III
§
0
OJ
.,
-
~
H
"
........
~
+
w
+
+
+
+
+
+
+
+
-
~
........
0
OJ
., .......
-
0
-
-
-
-
-
-
III
... '"
-
w
-
w
-
-
-
-
-
~
....0
-
-
w
+
w
w
w
w
-
w
-
-
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
'"
0
.... '"
0
u
u 00
...
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
'""
:>
..... ....~
III
-
+
+
+
+
+
w
-e
... ...., ""
'C
'"
0
""
u
of 0
Fermentation
.,
+
ATCC 14932 a
fermentwn
Laatobaci. l.Lus
Strains
III
...., ... ....... III
-
-
-
-
-
-
-
w
+
'"'
...0
...
:> ..c:: :>
..c::
.......
s-e
'" .d
Z
.... u
==============- ====-===:;;;:- ================~=====~-~-~-~--~-------------------~----~- ---------
-
-
-
+
+
+
+
+
+
0
u
00 III 0
"
.....III
~
.......0
""0
S
....... .""
----
s-e
39,0
40,9
50,1
51,7
51,9
52,1
53,9
51,0
52,7
51,5
53,4
'"'
+
u
~
....
0
27
77
92
84
87
82
85
88
88
100
ATCC 14932
90
100
0
0
0
0
0
0
0
0
20
DSM 20016
homology with
% DNA-DNA
---~~====~========================
Table 2. Phenotypic and genotypic characters of heterofermentative citrate fermenting lactobacilli isolated from sheep milk and Pecorino Romano cheese
w
~
~
~
0.. 0..
(l)
r-
~
0..
;:l
~
N N
::l.
,.,
~
a,
ti
v, N
+
+
CG 176B
CG 195B
+
+
+
+
+
CG 264A
CG 281A FC 3A
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
-
+
+
+
-
-
-
+
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
w
- -
-
-
-
+
+
+
+
+
- -
-
-
w
-
-
-
-
-
-
-
-
-
-
-
-
w
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
-
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
w
w
+
w
+
w
w
w
+
+
+
+
-
-
-
-
-
+
-
+
+
0
0
37 , 8 38,8
37,8
0 0
0
0 0
38 , S
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
38,3
37 ,8
+
38,8
39,S
40,9
38,1
38,8
38,8
39.5
0
0
37,8
38,8
0
0
38,8
37,8
+
+
+
+
0
0
-
-
+
0
+
+
-
-
-
-
-
-
-
0 ..: not investigated; - = negative result; + = positive result; w = weak reaction; a = Type strain. Positive reactions by all strains: galactose, glucose, lactose, maltose, melibi ose, raffinose, ribose, succrose, NH s from arginine and Gram-stain. Negative reacti ons by all strains: cellobiose, glycerol, mannitol, rh amn ose, sorbitol, salicin , hydrolysis of esculin and production of catal ase.
+
+
+
+
CG 248C
CG 257B
+
+
+
+
+
+
+
+
CG 176A
+
+
+
CG 161A
+
+
+
+
CG 151B
CG 233D
+
CG 147D
+
+
+
CG 129A
+
+
+
+
+
CG 231B
+
+
+
CG 122B
CG USB
i-
-
-
+
-
-
+
+
+
+
+
+
+
CG 231A
+
CG 75A
CG 95
+
CG 223E
+
CG 33B
95 93
98
99
96
99
96
80
81
89
87
92
81
79
80
90
78
81
86
89
94
'"
'-" '-"
§;
t->
80-
rv
rt->
(J'l
S'
..,"'T1 3 " ;:;
~
~
o' e s, Q ..,
~
n
s.;
'/> '/>
or
o
+
+
+
+
+
+
+
+
+
+
+
+
+
CG 215B
CG 225D
CG 300B
CG 303B
CG 305e
FC 2A
FC 4A
FC 16A
FC 17B
FC 18A
FC 96B
FC 98A
8
N
"-<
...
a0
u
ATCC 1491 7a
plantal'W71
Lac tobaci. l.Lus
Strains
=•••
X
<1J
<1J U
., ....0 ...0 ...
..-< ::l
..."
......
....0 <1J
<1J
<1J
u
0
u
0
....0 .... ..,. ., .......0 ., '0" ..-< 0 .c .0 ri ..0..... .0...0 ...u .c'" .....'0" .... " 0 0 ...'" H~ ~ l§ ~ '" '" '" '"'"
...... ..... ..."
u
0
Growth at
u co er
0
""...
a
-
-
-
-
-
-
8'
a
...0 "-<
00
0
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
10<
...::l
u
+
~
- -
-
+
+
+
" -e
...
.0
0
-
- - -
w
- - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
<1J
~
..-<
w +
+
+
+
+
+
+
+
+
+
+
+
- - -
- - + - - + + + - + + - - + + - - + + - + + + - - - + + - - + - w + - + + - - + - - -
+
"-< "-<
0
-
+
c
~" :>:"'"
N
<1J
0
OJ
- + - - - + - + + + - + + +
-
- -
w
-
H
" "
.....~
0
0
+
-
+
+
+
w
- -
w
+
w
+
+
+
+
+
+
w +
-
+
+
+
+ +
+
-
+ - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
U")
-
'"
...::l
...>>'"
+
+
+
+
+ +
-
+
-
+
+
+
+
+
+
+
+
+
+
+ - - + + + -
+
- - -
U")
"-<
'0
"" ... ...'" ...."
0
::l
+
+
+
+
+
+
+
+
+
-
+
-
+
:I:
~
... -e
....p-, 0
....,
"-< 0
'"
u
......."
_ U== == = = = = == ==_ == _ _ ==
U
..." ....."....
__
::l
<1J
Fe r men t a t i on of
C == = . G= _======= = == _ . _ c_ a == . _ ==== ~ ====_. ~= _ =
0 "-<
<1J
__ .C==========__
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...'" '" ......"... ....u
_._ - = =- - = =====- == =
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45,6
0
0
0
45 , 4
46,6
43 ,9
43,4
43,9
44,6
43,2
43,7
44, 8
+
"
U
""....
======
96
87
89
89
88
98
78
97
95
93
82
82
100
ATCC 14917
0
0
0
0
0
0
0
0
0
0
0
0
10
ATCC 334
% DNA-DNA
= _ ~ = = == =
homology with
_._o..__
Tabl e 3. Phenotypic and genotypic characters of homofermentative citrate ferment ing lactobacilli isolated from lamb stomach, lamb -rennet paste, sheep milk and Pecorino Romano cheese w
VI
.,
~
Il>
"00--
r-
:>
0-
::l
N
II>
J'
,,'
~
..,..,>-
9'
'a
a,
tl
-I>-
+
+
+
+
+
FC l37A
FC 148A
FC l5 0A
FC l53A
+
FC lA
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
w
0
-
-
+
+
+
+
+
- + - -
+
+
+
-
w
0
w
+
0
- -
-
v
- - - - - -
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
0
-
-
+
- - -
+
-
- - + - + -
-
- + - - -
- + - - +
+
+
+
+
-
- -
-
+
+
+
-
+
+
+
+
+
+
+
+
+
w
w
+
+
+
+
w
-
-
w
- -
+
-
- + -
+
-
+
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
+
+
+
+
investigated; - == negative result; + = positive result; w = weak reaction; a = Type strain. Positive reaction s by all strains: cellobiose, galactose, glucose , lactose , maltose, ribose, sucrose, salicin, grow th in presence o f 6.5 Nega tive reaction s by all stra ins: NH , from arginine and prod uction of catalase.
o"'" not
+
AlCC 334
casei:
Lac:tobac:i l l us
+
+
FC 107A
FC 109B
+
FC l Ol A
FC 129A
+
FC 99A + +
-
+
+ +
-
+
+
0
+
+
+
+
+
+
+
+
+
43, 9
45, 4
43,9
45,1
44 , 9
45 ,6
45 , 4
0
45 ,6
0
43, 2
72
0
6
78
94
89
92
78
96
90
92
104
100
0
0
0
0
0
0
0
0
0
'I. NaCI, coag ulation o f milk and Gram-stain.
-
-
+
-
+
+
-
-
+
+
+
-
f;l
v, v,
w
§
~
80"
r>
~
r-
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References
Battistotti, B., Bottazzi, V.: Ricerche sulla microbiologia del formaggio grana. VIII Parte: sulla presenza di lattobacilli citrato-ferrnentanti in formaggio grana. Sci. Teen. latt.-cas. 23, 59-70 (1972) Bottazzi, V., Ledda, A., Arrizza, S.: Bacteries fermentant les citrates et gonflement du fromage "Pecorino Romano". Le lait 505-506,328-331 (1971) Buchanan, R. E., Gibbons, N. E. (eds.): Bergey's Manual of Determinative Bacteriology, 8th edn. Baltimore: The Williams and Wilkins Co. 1974 De Ley, J.: Reexamination of the association between melting point, bouyant density and chemical base composition of deoxyribonucleic acid. J. Bact. 101, 738-754 (1970) Dellaglio, F., Bottazzi, V., Trovatelli, L. D.: Deoxyribonucleic acid homology and base composition in some thermophilic lactobacilli. J. gen. Microbiol. 74, 289-297 (1973) De Man, J. C;; Enige eigenschappen van staafvormige melkzunrbacterien geisoleerd uit Nederlandse Kaas. Ned. melk-eu Zuiveltijdschr. 10, 240-248 (1956) De Man, }. C., Rogosa, M., Sharpe, M. E.: A medium used for the cultivation of lactobacilli. J. Appl. Bacteriol., 23, 130-135 (1960) Fryer, T F.: Utilization of citrate by lactobacilli isolated from dairy products. J. Dairy Res. 37, 9-15 (1970) Fryer, T. F., Sharpe, M. E., Reiter, B.: Utilization of milk citrate by lactic acid bacteria and "blowing" of filmwrappedd cheese. J. Dairy Res. 37, 17-28 (1970) Gasser, F.: Identification des Lactobacillus fecaux. Ann. Inst. Pasteur (Paris) 106, 778 (1964) Johnson, }. L., Ordal, E. 1.: Deoxyribonucleic acid homology in bacterial taxonomy: effect of incubation temperature in reaction specificity. J. Bact. 95, 893-900 (1968) Kandler, 0., Stetter, K. 0., Kohl, R.: Lactobacillus reuteri sp. nov., a new species of heterofermentative lactobacilli. Zbl. Bakt. Hyg., I Abt. Orig. C 1, 264-269 (1980) Keddie, R. M.: The properties and classification of lactobacilli isolated from grass and silage. J. appl. Bact. 22, 403-416 (1959) Marmur, }.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. molec. BioI. 3, 208-218 (1961) Pettinau, M., Ledda, A., Arrizza, S., Bottazzi, V.: I batteri citrato-fermentanti come agenti di gonfiore nel formaggio Pecorino Romano. Sci. Teen. Iarr, cas. 23, 131-146 (1972) Sarra, P. G., Magri, M., Bottazzi, V., Dellaglio, F., Bosi, E.: Frequenza di bacilli lattici eterofermentanti nelle feci di vitelli lattanti. Arch. Vet. Ital. 30, 16-21 (1979) Vescovo, M., Dellaglio, F., Bottazzi, V., Sarra, P. G.: Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Betabacterium Orla-jensen, Microbiologica 2,317-330 (1979) Prof. F. Dellaglio, Istituto di Microbiologia Agraria e Tecnica, Facolta di Agraria, Universita Cattolica del S. Cuore, 29100 - Piacenza, Italia