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Characterization of lactobacilli isolated from meats and meat products
International Journal of Food Microbiology, 3 (1986) 19-29
19
Elsevier JFM 00081
Characterization of lactobacilli isolated from meats and meat products Y. Morishita and K. Shiromizu Department of Biomedical Research on Food. National Institute of Health, Kamiosaki 2-10-35, Shinagawa-ku, Tokyo 141, Japan
(Received 30 April 1985; accepted 29 October 1985)
Lactobacillus strains isolated from meats and meat products were biochemically characterized to fall into
9 groups of streptobacteria, 5 groups of thermobacteria, and 4 groups of betabacteria. Of the streptobacterial groups, groups $6 and $7 which were the most predominant resembled Lactobacillus curvatus or L. sake. Groups $4 and $5 which were of secondary importance were distinguished from groups $6 and $7 in that they did not ferment amygdalin and salicin or hydrolysed aesculin, and they were not similar to known species. There were three minor groups which were similar to or identical with L. coryniformis subsp, torquens, L. casei group or L. plantarum. Of the thermobacterial groups, two major groups were identical with L. crispatus and L. salivarius, and another major group was relatively similar to but not identical with L. helveticus. There was a minor group of thermobacteria which produced L-(+ )-lactate and fermented D-ribose, cellobiose, and starch. Four groups of betabacteria were identified as or were similar to L. fermentum, L. viridescens, L. brevis, or L. confusus. L. fermentum and L. viridescens of the betabacteria were found most frequently. All the groups of thermobacteria and L. fermentum were much better isolated at the incubation temperature of 37°C than 25°C. The distribution of each group in meats and meat products was shown. The occurrence of the intestinal type of lactobacilli in meats is discussed in connection with the sanitary conditions typical of meats, especially chicken. Key words: Lactobacilli; Meats; Meat products
Introduction L a c t o b a c i l l i are a n essential c o m p o n e n t of t h e m i c r o b i a l p o p u l a t i o n s in m e a t a n d m e a t p r o d u c t s , h a v i n g i n f l u e n c e s o n their q u a l i t y . A m o n g the s u b g e n e r a of l a c t o b a cilli, s t r e p t o b a c t e r i a a n d b e t a b a c t e r i a h a v e b e e n r e p o r t e d to p r o l i f e r a t e d u r i n g the p r e s e r v a t i o n of m e a t a n d m e a t p r o d u c t s , a n d to b e p r e s e n t in large n u m b e r s ( A l l e n a n d F o s t e r , 1960; G a r d n e r , 1968; K e m p t o n a n d Bobier, 1970; R e u t e r , 1970; M o l et al., 1971; K i t c h e l l a n d Shaw, 1975; R o t h a n d Clark, 1975; S u t h e r l a n d et al., 1975; G e n i g e o g i s , 1976; N e w t o n et al., 1977; P a t t e r s o n a n d G i b b s , 1977; E n f o r s et al., 1979; B l i c k s t a d et al., 1981; V a n d e r z a n t et al., 1982; B l i c k s t a d a n d M o l i n , 1983). Their biochemical characteristics or classifications have been described by many i n v e s t i g a t o r s ( N i v e n a n d E v a n s , 1957, C a v e t t , 1963; R e u t e r , 1970; M o l et al., 1971; K i t c h e l l a n d S h a w , 1975; H i t c h e n e r et al., 1982; H o l z a p f e l a n d G e r b e r , 1983; S h a w a n d H a r d i n g , 1984).
0168-1605/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
20 Most of the Lactobacillus isolates from meat and meat products are psychrotrophic, growing at 15°C. The occurrence of thermophilic lactobacilli in meat and meat products was usually overlooked due to the isolation temperatures of less than 30°C. Some of them might originate in the intestinal tracts and indicate hygienic conditions of meat and meat products. In Japan, lactobacilli in meat and meat products were not characterized before our previous study (Morishita et al., 1985a, b). In the previous study we isolated a wide variety of lactobacilli at incubation temperatures of 15, 25 and 37°C from meats and meat products. Isolates were biochemically characterized to fall into streptobacteria, betabacteria, and thermobacteria which were separated into 10, 4, and 5 groups, respectively. This report describes the biochemical characteristics of the groups of lactobacilli studied further and the distribution of the groups and subgroups in foods such as chicken, pork, beef, bacon, ham, and sausage.
Materials and Methods
Samples Samples of meats and cooked meat products were obtained from retail stores. Items of samples were as follows: twelve samples of checken (minced, 2; sliced, 1; steak, 9), 17 of pork (minced, 4; sliced, 3; steak, 10), 3 of minced beef, 4 of ham, 5 of sausage, and 8 of bacon. All the samples of meats and meat products, except for one sample for sausage and 3 for bacon, were unpacked. The surface materials scraped from the steak samples using sterile gauze and the homogenized materials for the others were diluted serially 10-fold in phosphate buffer solution and bacteriologically examined as described previously (Morishita et al., 1985a). Media and culture conditions Briggs agar (Lerche and Reuter, 1960; Nissui, Japan), MRS agar (de Man et al., 1960), LBS agar (BBL), and Rogosa SL agar (Difco) were smeared on the agar plates. The plates were incubated at 15°C for 6 days, 25°C for 3 days, and 37°C for 2 days in an atmosphere of CO 2 in an anaerobic jar. After incubation individual isolates were selected on the basis of colony and cell morphology and the Gram reaction from each of the plates, and after purification they were maintained on prereduced agar slants (Mitsuoka, 1969) at 4°C. Physiological and biochemical examination For inoculum preparation, most isolates were grown in MRS broth (Oxoid) at 30°C, and some thermophilic strains at 37°C. The cells were harvested from MRS broth after two subcultures with a 24 h interval. They were resuspended in sterile neutralized saline containing 0.2% cysteine-HC1 to give approximately a doubled concentration.
21 Except where otherwise stated each tube with test medium was inoculated with 0.05 ml of inocula and incubated at 30°C. Inocula were also used for the Gram reaction and morphological examination. Gas production from glucose and gluconate and acid production from carbohydrates were determined by the method of Mitsuoka (1969) with the exception that the basal medium for fermentation with agar and indicator omitted was used for gas production. Tubes were incubated for 10 days at 30°C and observed 3, 6, and 10 days after inoculation. Catalase was tested on cultures grown on Briggs agar and nitrate reduction was examined using tryptic nitrate medium (Difco) according to the description of Cowan (1974). Hydrolysis of arginine was determined using the modified arginine broth containing 2% glucose (Hitchener et al., 1982). Hydrolysis of aesculin was determined by the method of Sharpe et al. (1966). Growth in 7.5% NaC1 was determined in MRS broth containing 7.5% NaC1, and growth at 15, 30, and 45°C examined in MRS broth. Growth on LBS agar was tested in comparison with growth on Briggs agar. The media were streak inoculated and incubated under 100% CO 2 for 3 days. Clotting of milk was determined using Litmus milk (Difco). B.C.P.-Plate count agar (Eiken Chemical Co., Japan) supplemented with 5% sucrose was streak inoculated for dextran production from sucrose and incubated at 25°C for 6 days under 100% CO2. To determine the lactic acid configuration strains were grown at 30°C for most of them but at 37°C for some in T N Y G medium. T N Y G medium had the following composition: tomato juice (Mitsuoka, 1969), 20 ml; neopeptone (Difco), 1 g; yeast extract (BBL), 0.5 g; glucose, 1 g; K 2 H P O 4, 0.3 g; Na-citrate, 0.5 g; Tween 80, 0.1 g; mineral solution B (Mitsuoka, 1969), 0.5 ml; and distilled water, 80 ml (pH 6.8). The cells were removed by centrifugation at 3000 r.p.m, for 20 min after two subcultures with a 24 h interval. L-(+)- and D-( -- )-lactate were estimated in supernatants using enzymic methods. L-(+)-Lactate was assayed using the reaction kits provided by Boehringer-Mannheim, and n - ( - ) - l a c t a t e assayed using D-( -- )-lactate dehydrogenase in place of L-(+)-lactate dehydrogenase in the kits according to the prescription.
Results and Discussion
A total of 690 isolates were examined. 13 of them were leuconostocs because they were cocci or short rods and produced gas and D - ( - )-lactate from glucose. Of the remainder, 440 strains from 45 samples were streptobacteria, 122 from 18 samples were thermobacteria, and 115 from 34 samples were betabacteria. Table I shows the properties of the groups and subgroups of streptobacteria. Groups $6 and $7 were dominant, and the strains were isolated from 32 samples each. Groups $4 and $5 were subordinate, and their isolates were from 20 and 17 samples, respectively. Groups $6 and $7 as well as groups $4 and $5 had mutually similar fermentation properties. However, groups $4 and $6 were separated from
22
TABLE
I
Characteristics of streptobacterial groups from meats and meat products
Growth
, - - , , - - ,
~
~
o
~
f
o
,~ ?.
~o ,-
Z
S1
+ +
x
(+ )
-
t,/oL
--
-
-
+
+
DL
--
--
--
zz
'~
+
+
+
DL
+
--
--
_
_
+
+
+
+
DL
+/--
d
_
_
+
+
+
DE
+
d
-
I
a
+ +
_
_
b
+
+
+
DL
+
--
--
_
_
C
+
+
+
DL
d
d
-
_
_
+
+
+
DI.
+
--
--
+
a
+
+
+
DL
+
--
--
b
+
+
+
DL
+
--
--
+
+
+
DL
d
-
+
a
+
+
+
DL
d
-
+
b
+
+
+
DL
d
c
+
+
+
DL
+
--
+
+
+
+
DL
+
--/+
a
+
+
+
DL
+
b
+
+
+
DL
c
+
+
+
DL
+
+
+
+
+
+
$5
$6
$7
$8 $9
'=
o
$2
$3 $4
=
+
_
+
--
_
+
+
--
_
+
+
--
_
+
_
_
_
+
--
--
--
d-
_
_
+
_
_
_
+
+
+
--
--/+
+
--
+
+
--
_
+
+
+/--
+
+
--
_
+
+
--
+
+
d
+
+
L/DL
+
--
+
_
_
_
+
DL
+
--
+
d
-
d
+
-
+
+
All the groups fermented glucose, mannose and fructose, did not grow at 45°C, produced no a m m o n i a from arginine, and showed no clotting of milk. + , positive for 90% or more strains; - , negative for 90%
groups $5 and $7, respectively, mainly by i-arabinose fermentation. Groups $6 and $7 differed clearly from groups $4 and $5 in aesculin and salicin, and also from groups $8 and $9 in melezitose, D-mannitol, and u-sorbitol. Groups $4, $5, $6, and $7 were separated into a few subgroups each as shown in Table I. Of the predominant groups group $6 was subdivided into 3 subgroups (S6/a, S6/b, and $ 6 / c ) on the basis of D-sucrose and starch fermentation. Subgroup S 6 / a is D-sucrose and melibiose negative and Starch positive; subgroup S 6 / b is D-sucrose and starch positive and variable in melibiose and shows a little weak growth in 7.5% NaCI; subgroup $ 6 / c is D-sucrose positive and starch negative. Biochemical characteristics of group $6 resemble those of group 1 of Hitchener et al. (1982), Lactobacillus sake, or L. curvatus (Rogosa, 1974, Stetter and Stetter, 1980), and is also similar to L. alimentarius with the exception that the latter produces L-(+)-lactate (Reuter, 1983). Subgroup S 6 / a is more similar to L. curvatus except
23
Fermentation A
o
o
.
~a
-
+
-/+
-
-
-
-
+
+
d
-
d
d
-
+
+
+
-
+
+
d
d
+ d
d
+/-
d
d
d
-
-/+
+
+
d +
+
d
-
-
d +
d +
+ .
+ + +
d
+ d + --
-
d
+/-
.
+
+
+
--/+
d
-+
d
+
d
-
-
-
+
-/+
-
d
d
+
+
-
-
-
+
-
-
-
d
+
.
.
.
+/-
d
+
.
.
=
-
.
.
+
.
.
.
+
d
d
+
d
+
d
-/+
-
d
+
+
+
-
+
d
+
-
-
-
+
+
+
+
+
+
+
d
d
d
-
-
+
+
d
+
d
+
+/-
d
-
-
+
d
+
d
-
d
+
d
+
d
d
+ d
+
+
+ d
+ d
+
+
+ +
+
d
-
-
+
d
+ d
+
d
+ -
+
d
-
-
+
d
+
+
+ +
d
+
-
-
+
d
+
+
+
+
+
+
-
-
+
d
d
d
+
+
+
+
+
+
+
+
+
+
+/-
+
+
+/-
+
+
+ d
o r m o r e strains; 21-79%
+/-
o f strains; ( + ) ,
and - / +, positive and negative, respectively
!
for 80% o f strains; d, p o s i t i v e for
w e a k r e a c t i o n . $ 8 is an k casei g r o u p , a n d $ 9 m o s t p r o b a b l y
is L. plantarum.
for amygdalin fermentation. Subgroups. S6/b and $6/c resemble L. sake. Subgroup 6 / c is not distinguished from group $7, especially subgroup S7/a, except for L-arabinose fermentation. No notable difference was found in the distribution of the subgroups of group $6 in foods. Subgroups of group $7 were mutually distinguished by fermentation patterns of rhamnose and trehalose. Subgroup S7/a is rhamnose negative and trehalose positive; subgroup S7/b is negative for both sugars; subgroup $ 7 / c is positive for both. Group $7 resembles group 3 of Hitchener et al. (1982), and is similar to cluster II of Shaw and Harding (1984), or L. bavaricus and L. sake. However, L. bavaricus is an L-(+)-lactate producer and L. sake is L-arabinose negative (Stetter and Stetter, 1980). There is no different distribution of the subgroups of group $7 in foods except that subgroup $ 7 / c was present more frequently in pork than the others were.
24
TABLE
II
Characteristics
of thermobacterial
and
betabacterial
groups
from
meats
and
meat
products
Growth
^
..
~
~
~
8
~
2 ¢-,
,--
"*
t--
.~
,z
~
©
¢~
~
<
<
Therrnobacterium T1
-
+
-
+
DL
.
T2
-
+
-
+
DL
--
. --
.
d
-
T3
-
+
-
+
DL
--
--
d
-
+
T4
-
+
-
+
L
--
-
+ / -
-
d
T5
-
+
-
+
L
.
-
+
DL
+
-
+ +
t)L I)L
+ +
. --
DL
4-
if-//--
and
melezitose
.
.
.
d -
+
.
Betabacterium
B1
-
+
B3
+ +
-
B4
+
-
B2
All the groups as for Table
+ (+ ) -
fermented I. T 3
and
(q-)
glucose and T4
are
were rhamnose
identified
with
the
L. acidophilus
-
.
. ---
negative. group,
+
-
+
--
q.-
q-
.
Reaction
probably
marks
shown
L. crispatus
and
arc L.
Groups $6 and $7 were difficult to distinguish morphologically, their strains grow singly or in short chains in MRS broth, being straight, or curved, occasionally horseshoe-like. Their surface colonies on Briggs agar are about 1 to 2 m m in diameter, circular, convex, and greyish-white. Groups $6 and $7 were separated only by L-arabinose fermentation in this report, so further studies may be needed to justify this separation. In chicken, Group $6 was found more frequently than Group $7, while there was no difference in occurrence in the others (Table III). At any rate, the two groups have the common and important characteristics that D-ribose and salicin fermentation and aesculin hydrolysis are positive and melezitose and D-mannitol fermentation are negative, and that they were present at remarkably high frequency in meat and meat products (Table III). Such properties are shared by described species and groups such as L. curvatus (Rogosa, 1974), L. sake, L. bavaricus, and L. alimentarius (Rogosa, 1974; Stetter and Stetter, 1980; Reuter, 1983), and atypical streptobacteria of Cavett (1963), biotype A 1 / 2 of Reuter (1970), groups 1 and 3 of Hitchener et al. (1982), and cluster II of Shaw and Harding (1984). In addition, these streptobacteria are most likely to be important components of the lactobacilli in meat and meat products (Cavett, 1963, Reuter, 1970; Bickstad et al., 1981; Hitchener et al., 1982; Vanderzant et al., 1982; Shaw and Harding, 1984; Morishita et al., 1985b). Of the subgroups of streptobacteria, group $4 was separated into 3 subgroups on the basis of the fermentation of D-sucrose, trehalose, melibiose, and starch. Subgroup S 4 / a is a main subgroup which is positive for D-sucrose, trehalose, and
25
Fermentation A.,
•-
~
.g
~
~
~
~
2
~
~
o
o '-
.~
~
z
~
~.
~
~
,~
~
d +
d +
-/+ -
-
d
+
d
d
+
d
-
d
d
+ +
+ +
+ +
+ +
+ +
d +
+ +
-
d +/-
+
+
-
+
+
+
+
+
-
+
+/-
+
+
-
+
+
+
+
+
+
+
+
+
d
+
+
+
--
+
-
+
+
+ -
+
+
d
d
d
d +
+
d +
+ -
+ +
+
+
+
d
+
+
+
+
+
-
+
+
+
+
salivarius, r e s p e c t i v e l y ; confusus, r e s p e c t i v e l y .
BI,
<
d
-
-
+
d
d
d
-
d
+
+
-
+
+
+
B2, B3, and
B4 are
L. fermentum, L. viridescens, L. brevis,
and
probably
L.
melibiose and negative for starch; subgroup S 4 / b is trehalose positive and negative in the other three sugars mentioned; subgroup $ 4 / c is positive for D-sucrose and starch, and negative for the others. Subgroup S 4 / a is physiologically similar to group 2 of Hitchener et al. (1982). Subgroup S 4 / a was distributed widely in meat and meat products, whereas the other subgroups were found mainly in meat. Group $5 was separated into 2 subgroups by fermentation of melibiose. Both subgroups show o-sucrose negative and trehalose positive reactions, and appear physiologically similar except for melibiose. They are unlikely to be identical to known species. There was a difference in distribution in meat and meat products: subgroup S 5 / a was widely distributed in meat and meat products, and subgroup S 5 / b was present only in meat, especially chicken and pork. Separation of $4 from $5 remains to be studied further because the main key to this separation was only L-arabinose fermentation. Of the minor groups of streptobacteria, group S1 showed o-galactose and o-ribose negative reactions, and no growth on LBS agar. The colony forms of the strains of this group were different from those of the other groups of streptobacteria and were rather similar to that of L. viridescens. This group is a combined group of S1 and $2 from our previous report (Morishita et al., 1985b,). Six strains, which belonged to a previous group $2, produced o - ( - ) - l a c t a t e from glucose, and slightly resemble L. homohiochi (Rogosa, 1974). Group S1 was present almost exclusively in meat products (Table III). Group $2 is negative for D-ribose, cellobiose, lactose, trehalose, and positive for
26 D-mannitol, resembling L. coryniformis subsp, torquens (Rogosa, 1974), but this group is composed of DL-lactate producers, whereas the corresponding species is D - ( - ) - l a c t a t e producer. This group was present in meats. Group $3 is positive for rhamnose and D-ribose and produces gas from gluconate. These points differentiate this group from group $2. Group $3 was present in meats. Groups $8 is not distinguished from the L. casei group. Two of the three isolates were identical to L. casei subsp, casei, and one is subsp, pseudoplantarum. The three strains were isolated from meat. Group $9 shows morphological and biochemical properties identical to that of L. plantarum. It was found mainly in meat. L. casei and L. plantarum were reported to be present in meat and meat products by many workers (Cavett, 1963; Reuter, 1970; Mol et al., 1971; Enfors et al., 1979; Blickstad et al., 1981; Vanderzant et al., 1982; Blickstad and Molin, 1983). Thermobacterium isolates, which were all isolated from meat, were separated into 5 groups as shown in Table II. Groups T2, T3, and T4 were dominant of thermobacteria from meats. All of the thermobacterial strains were isolated from chicken and pork, but not from beef and meat products (Table III). Chicken was the main source of these isolates. Group T1 is negative for lactose fermentation and is heterogeneous. Group T2 is close in physiology to L. helveticus. This group, however, is positive for D-sucrose, raffinose, and starch. Group T2 was found more frequently in pork than the others were, Group T3 belongs to the L. acidophilus group which consists of L. acidophilus, L. crispatus, L. gasseri, and some unnamed groups (Johnson et al., 1980; Lauer et al., 1980; Lauer and Kandler, 1980; Cato et al., 1983). Since it fermented raffinose and starch but not trehalose, group T3 is most likely to be L. crispatus, although the L. acidophilus group was reported not to be distinguished by sugar fermentation (Johnson et al., 1980; Lauer et al., 1980; Kuroshima and Kodaira, 1983). G r o u p T3 was found most frequently in chicken. Group T4 is identical with L. saliuarius. This group was also found mainly in chicken. Group T4 as well as group T3 are intestinal bacteria, and were isolated with remarkable frequency from chicken in this study. This suggests that the sanitory quality of chicken in the Japanese market should be improved. Group T5 produces L-(+)-lactate and is positive for D-ribose, cellobiose, starch and amygdalin but not for D-mannitol. This group, therefore, apparently differs from L. saliuarius. Of the 122 thermobacteria strains, 104 were isolated at 37°C incubation temperature. This indicates that 37°C incubation is necessary to recover thermobacteria from meat. Betabacterium isolates were separated into 4 groups (Table III). Group B1 is most likely to be L. fermentum since this group grew at 45°C but not at 15°C and showed biochemical characteristics identical to those of L. fermentum. All strains of group B1 were isolated from meat at an incubation temperature of 37°C. The occurrence of this group in meat, as mentioned above for groups T3 and T4, indicates that the meat was under poor sanitary conditions. L. fermentum was reported to occur in meat and meat products (Reuter, 1970; Blickstad et al., 1981).
27 TABLE III Distribution of groups of lactobacilli on meats and meat products Bacterial
Food
group
Chicken
Pork
Beef
Ham
Sausage
Bacon
Streptobacterium
104 (11) a 0 (0) 5 (4) 0 (0) 9 (6) 25 (6) 36 (9) 23 (5) 1 (1) 2 (2)
171 (17) 1 (1) I (1) 3 (1) 8 (7) 21 (7) 70(15) 54 (15) 2 (2) 11 (5)
74 (3) 0 (0) 0 (0) 3 (1) 2 (1) 0 (0) 30(3) 37 (3) 0(0) 2 (2)
25 (4) 7 (3) 0 (0) 0 (0) 3 (1) 2 (2) 4(1) 4 (2) 0(0) 5 (1)
20 (4) 2(1) 0 (0) 0 (0) 2 (2) 2 (1) 3(1) 5 (2) 0(0) 0 (0)
46 (7) 3 (1) 0 (0) 0 (0) 17 (2) 3 (1) 10(3) 13 (5) 0(0) 0 (0)
440 (45) 13 (6) 6 (5) 6 (2) 47 (20) 53 (17) 156(32) 136 (32) 3 (3) 20 (10)
(7) (2) (5) (2) (3) (1)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0(0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
122 (18) 6 (6) 24(11) 60 (12) 25 (11) 7 (4)
(8) (8) (0) (1) (1)
23 (10) 12 (2) 2 (2) 7 (3) 2 (2)
10 (3)
23 (3)
9 (3)
22 (7)
115 (34)
1 (1)
0 (0)
0 (0)
0 (0)
39 (11)
1 (1) 8 (2) 0 (0)
22 (3) 1 (1) 0(0)
2 (2) 0(0) 7 (2)
22 (7) 0(0) 0 (0)
49 (15) 17 (7) 10 (5)
235 (12)
213 (17)
84 (3)
48 (4)
29 (5)
68 (8)
677 (49)
SI $2 $3 $4 $5 $6 $7 $8 $9
Thermobacteriurn T1 T2 T3 T4 T5
Betabacterium B1 B2 B3 B4 Total
Total
103 (11) 4 (4) 15 (6) 57 (10) 22 (8) 5 (3) 28 26 0 1 1
19 2 9 3 3 2
a No. of strains (No. of positive samples).
G r o u p B2 is i d e n t i c a l to L. viridescens. T h i s g r o u p grew in 7.5% NaC1 a n d p r o d u c e d n o a m m o n i a f r o m a r g i n i n e . S t r a i n s of it were i s o l a t e d f r o m 12 s a m p l e s of m e a t p r o d u c t s a n d f r o m o n l y 3 s a m p l e s of meat. Since N i v e n a n d E v a n s (1957) d e s c r i b e d L. viridescens, the p r e s e n c e of this species in m e a t a n d m e a t p r o d u c t s was r e p o r t e d b y m a n y w o r k e r s ( A l l e n a n d Foster, 1960; C a v e t t , 1963; R e u t e r , 1970; M o l et al., 1971; E n f o r s et al., 1979; V a n d e r z a n t et al., 1982; Blickstad a n d M o l i n , 1983). G r o u p B3 is likely to b e L. brevis. T h i s g r o u p was f o u n d m a i n l y in p o r k a n d beef, a n d grew w e a k l y i n 7.5% NaC1. T h e o c c u r r e n c e of L. brevis i n m e a t a n d m e a t p r o d u c t s was r e p o r t e d b e f o r e (Cavett, 1963; R e u t e r , 1970; E n f o r s et al., 1979). G r o u p B4 shows b i o c h e m i c a l a n d m o r p h o l o g i c a l s i m i l a r i t y to L. confusus ( S h a r p e et al., 1972; H o l z a p f e l a n d v a n W y k , 1982), b u t a n a l y s i s of the cell wall is r e q u i r e d for full j u s t i f i c a t i o n of the d e s i g n a t i o n of this g r o u p ( H o l z a p f e l a n d v a n W y k , 1982). T h e m a j o r i t y of the s t r a i n s of this g r o u p p r o d u c e d d e x t r a n f r o m sucrose. W e d i d n o t isolate L-( + ) - l a c t a t e - p r o d u c i n g b e t a b a c t e r i a , w h i c h were r e p o r t e d b y H i t c h e n e r et al. (1982) a n d H o l z a p f e l a n d G e r b e r (1983), f r o m either m e a t or m e a t products.
28
As shown in Table III thermobacteria were found in chicken in approximately the same frequency as streptobacteria, while in pork and beef streptobacteria were isolated at a much higher frequency than thermobacteria. Such a peculiar composition of the lactobacillus populations in chicken suggests that chicken is exposed to heavy contamination by bacteria originating from the chicken itself, especially the intestines. In meat products streptobacteria and betabacteria were found at a similar frequency but no thermobacteria. These bacteria probably proliferated during the processing or preservation at low temperature after they had survived the curing or heating (Kempton and Bobier, 1970; Mol et al., 1971; Kitchell and Shaw, 1975).
References Allen, J.R. and E.M. Foster, 1960. Spoilage of vacuum-packed slices processed meats during refrigerated storage. Food Res. 25, 19-25. Blickstad, E. S., S.-O. Enfors and G. Molin, 1981. Effect of hyperbaric dioxide pressure on the microbial flora of pork stored at 4°C or 14°C. J. Appl, Bacteriol. 50, 493-504. Blickstad, E. and G. Molin, 1983. The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4°C. J. Appl. Bacteriol. 54, 45-56. Cato, E., W.E.C. Moore and J.L. Johnson, 1983. Synonymy of strains of "Lactobacillus acidophilus" group A2 (Johnson et al. 1980) with the type strain of Lactobacillus crispatus (Brygoo and Aladame, 1953) Moore and Holdeman, 1970. Int. J. Syst. Bacteriol. 33, 426-428. Cavett, J.J., 1963. A diagnostic key for identifying the lactic acid bacteria of vacuum-packed bacon. J. Appl. Bacteriol. 26, 453-470. Cowan, S.T., 1974. Cowan and Steel's Mant,al for the identification of medical bacteria. Cambridge University Press, Cambridge, 238 pp. De Man, J.C., M. Rogosa and M.E. Sharpe, 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135. Enfors, S.-O., G. Molin and A. Ternstrom, 1979. Effect of packaging under carbon dioxide, nitrogen or air on the microbial flora of pork stored at 4°C. J. Appl. Bacteriol. 47, 197-208. Gardner, G.A., 1968. Effects of pasteurization or added sulphite on the microbiology of stored vacuum package bacon burgers. J. Appl. Bacteriol. 31,462-478, Genigeorgis, C.A., 1976. Quality control for fermented meats. J. Am. Vet. Assoc. 169, 1220 1228. Hitchener, B.J., A.F. Egan and P.J. Rogers, 1982. Characteristics of lactic acid bacteria isolated from vacuum-packaged beef. J. Appl. Bacteriol. 52, 31-37. Holzapfel, W.H. and E.S. Gerber, 1983. Lactobacillus divergens sp. nov., a new heterofermentative Lactobacillus species producing L-( + )-lactate. Syst. Appl. Microbiol. 4, 522-534. Holzapfel, W.H. and E. van Wyk, 1982. Lactobacillus kandeleri sp. nov., a new species of the subgenus Betabacterium with glycine in the peptidoglycan. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig, C3, 495-502. Johnson, J.L., C.F. Phelps, C.S. Cummins, J. London and F. Gasser, 1980 Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30, 53-68. Kempton, A.G. and S.R. Bobier, 1970. Bacterial growth in refrigerated, vacuum-packed luncheon meats. Can. J. Microbiol. 16, 287 297. Kitchell, A.G. and B.G. Shaw, 1975. Lactic acid bacteria in fresh and cured meat. In: Lactic acid bacteria in beverages and food, edited by J.G. Carr, C,V. Cutting and G.C. Whiting, Academic Press, London, pp. 209-220. Kuroshima, T. and S. Kodaira, 1983. Differentiation between Lactobacillus acidophilus and L. gasseri. Japan. J. Bacteriol. 38, 809-816. Lauer, E., C. Helming and O. Kandler, 1980. Heterogeneity of the species Lactobacillus acidophilus (Moro) Hansen and Moquot as revealed by biochemical characteristics and D N A - D N A hybridisation. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. C1, 150-168.
29 Lauer, E. and O. Kandler, 1980. Lactobacillus gasseri sp. nov., a new species of the subgenus Thermobacterium. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. C1, 77-78. Lerche, M. and G. Reuter, 1960. Beitrag zur Methodik der Isolierung und Differenzierung von aerob wachsenden "Laktobazillen" (Genus "'Lactobacillus Beijerinck"). Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. 179, 354-370. Mitsuoka, T., 1969. Vergleichende Untersuchungen i~ber die Laktobazillen aus den Faeces yon Menschen, Schweinen und Hi)hnern. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 1. Abt. Orig. 210, 32-51. Mol, J.H.H., J.E.A. Hietbrink, H.W.M. Molin and J. van Tienteren, 1971. Observations on the microflora of vacuum packed sliced cooked meat products. J. Appl. Bacteriol. 34, 377 397. Morishita, Y., K. Shiromizu and T. Hori, 1985a. Lactobacillus flora of meats and meat products from retail stores. J. Food Hyg. Soc. Japan 26, 331-336 (in Japanese). Morishita, Y., K. Shiromizu and T. Hori, 1985b. Characteristics and distribution of lactobacilli in meat and meat products. J. Food Hyg. Soc. Japan 26, 365-370 (in Japanese). Newton, K.G., J.C.L. Harrison and K.M. Smith, 1977. The effect of storage in various gaseous atmospheres on the microflora of lamb chops held at - I°C. J. Appl. Bacteriol. 43, 53-59. Niven, C.F. and J.B. Evans, 1957. Lactobacillus viridescens nov. spec., a heterofermentative species that produces a green discoloration of cure meat pigments, J. Bacteriol. 73, 758-759. Patterson, J.T. and P.A. Gibbs, 1977. Incidence and spoilage potential of isolates from vacuum-packaged meat of high pH value. J. Appl. Bacteriol. 43, 25-38. Reuter, G., 1970. Laktobazillen und eng verwandte Mikroorganismen in Fleisch and Fleischwaren 2. Mitteilung: Die Charakterisierung der isolierten Laktobazillenstamme. Fleischwirtschaft 50, 954-962. Reuter, G., 1983. Lactobacillus alimentarius sp. nov., nom. rev. and Lactobacillusfarciminis sp. nov., nora. rev. Syst. Appl. Microbiol. 4, 277-279. Rogosa, M., 1974. Genus Lactobacillus. In: Bergey's Manual of Determinative Bacteriology, 8th edition, edited by R.E. Buchanan and N.E. Gibbons, Williams & Wilkins, Baltimore, pp. 576 593. Roth, L.A. and D.S. Clark, 1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21,629-632. Sharpe, M.E., T.F. Fryer and D.G. Smith, 1966. Identification of the lactic acid bacteria. In: Identification methods for microbiologists, edited by B.M. Gibbs and F.A. Skinner, Academic Press, London, pp. 65-79. Sharpe, M.E., E.I. Garvie and R.H. Tilbury, 1972. Some slime-forming heterofermentative species of the genus Lactobacillus. Appl. Microbiol. 23, 389-397. Shaw, B.G. and C.D. Harding, 1984. A numerical taxonomic study of lactic acid bacteria from vacuum-packed beef, pork, lamb and bacon. J. Appl. Bacteriol. 56, 25 40. Stetter, H. and K.O. Stetter, 1980. Lactobacillus bavaricus sp. nov. a new species of the subgenus Streptobacterium. Zentralbl. Bakteriol. Parasitenkd. Infektionstr. Hyg. 1. Abt. Orig. C1.70 74. Sutherland, J.P., J.T. Patterson and J.G. Murray, 1975. Changes in the microbiology of vacuum-packaged beef. J. Appl. Bacteriol. 39, 227-237. Vanderzant, C., M.O. Hanna, J.G. Ehlers, J.W. Savell, G.C. Smith, D.B. Griffin, R.N. Terrell, K.D. Lind and D.E. Galloway, 1982. Centralized packaging of beef loin steaks with different oxygen-barrier films: microbiological characteristics. J. Food Sci. 47, 1070-1079.
19
Elsevier JFM 00081
Characterization of lactobacilli isolated from meats and meat products Y. Morishita and K. Shiromizu Department of Biomedical Research on Food. National Institute of Health, Kamiosaki 2-10-35, Shinagawa-ku, Tokyo 141, Japan
(Received 30 April 1985; accepted 29 October 1985)
Lactobacillus strains isolated from meats and meat products were biochemically characterized to fall into
9 groups of streptobacteria, 5 groups of thermobacteria, and 4 groups of betabacteria. Of the streptobacterial groups, groups $6 and $7 which were the most predominant resembled Lactobacillus curvatus or L. sake. Groups $4 and $5 which were of secondary importance were distinguished from groups $6 and $7 in that they did not ferment amygdalin and salicin or hydrolysed aesculin, and they were not similar to known species. There were three minor groups which were similar to or identical with L. coryniformis subsp, torquens, L. casei group or L. plantarum. Of the thermobacterial groups, two major groups were identical with L. crispatus and L. salivarius, and another major group was relatively similar to but not identical with L. helveticus. There was a minor group of thermobacteria which produced L-(+ )-lactate and fermented D-ribose, cellobiose, and starch. Four groups of betabacteria were identified as or were similar to L. fermentum, L. viridescens, L. brevis, or L. confusus. L. fermentum and L. viridescens of the betabacteria were found most frequently. All the groups of thermobacteria and L. fermentum were much better isolated at the incubation temperature of 37°C than 25°C. The distribution of each group in meats and meat products was shown. The occurrence of the intestinal type of lactobacilli in meats is discussed in connection with the sanitary conditions typical of meats, especially chicken. Key words: Lactobacilli; Meats; Meat products
Introduction L a c t o b a c i l l i are a n essential c o m p o n e n t of t h e m i c r o b i a l p o p u l a t i o n s in m e a t a n d m e a t p r o d u c t s , h a v i n g i n f l u e n c e s o n their q u a l i t y . A m o n g the s u b g e n e r a of l a c t o b a cilli, s t r e p t o b a c t e r i a a n d b e t a b a c t e r i a h a v e b e e n r e p o r t e d to p r o l i f e r a t e d u r i n g the p r e s e r v a t i o n of m e a t a n d m e a t p r o d u c t s , a n d to b e p r e s e n t in large n u m b e r s ( A l l e n a n d F o s t e r , 1960; G a r d n e r , 1968; K e m p t o n a n d Bobier, 1970; R e u t e r , 1970; M o l et al., 1971; K i t c h e l l a n d Shaw, 1975; R o t h a n d Clark, 1975; S u t h e r l a n d et al., 1975; G e n i g e o g i s , 1976; N e w t o n et al., 1977; P a t t e r s o n a n d G i b b s , 1977; E n f o r s et al., 1979; B l i c k s t a d et al., 1981; V a n d e r z a n t et al., 1982; B l i c k s t a d a n d M o l i n , 1983). Their biochemical characteristics or classifications have been described by many i n v e s t i g a t o r s ( N i v e n a n d E v a n s , 1957, C a v e t t , 1963; R e u t e r , 1970; M o l et al., 1971; K i t c h e l l a n d S h a w , 1975; H i t c h e n e r et al., 1982; H o l z a p f e l a n d G e r b e r , 1983; S h a w a n d H a r d i n g , 1984).
0168-1605/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
20 Most of the Lactobacillus isolates from meat and meat products are psychrotrophic, growing at 15°C. The occurrence of thermophilic lactobacilli in meat and meat products was usually overlooked due to the isolation temperatures of less than 30°C. Some of them might originate in the intestinal tracts and indicate hygienic conditions of meat and meat products. In Japan, lactobacilli in meat and meat products were not characterized before our previous study (Morishita et al., 1985a, b). In the previous study we isolated a wide variety of lactobacilli at incubation temperatures of 15, 25 and 37°C from meats and meat products. Isolates were biochemically characterized to fall into streptobacteria, betabacteria, and thermobacteria which were separated into 10, 4, and 5 groups, respectively. This report describes the biochemical characteristics of the groups of lactobacilli studied further and the distribution of the groups and subgroups in foods such as chicken, pork, beef, bacon, ham, and sausage.
Materials and Methods
Samples Samples of meats and cooked meat products were obtained from retail stores. Items of samples were as follows: twelve samples of checken (minced, 2; sliced, 1; steak, 9), 17 of pork (minced, 4; sliced, 3; steak, 10), 3 of minced beef, 4 of ham, 5 of sausage, and 8 of bacon. All the samples of meats and meat products, except for one sample for sausage and 3 for bacon, were unpacked. The surface materials scraped from the steak samples using sterile gauze and the homogenized materials for the others were diluted serially 10-fold in phosphate buffer solution and bacteriologically examined as described previously (Morishita et al., 1985a). Media and culture conditions Briggs agar (Lerche and Reuter, 1960; Nissui, Japan), MRS agar (de Man et al., 1960), LBS agar (BBL), and Rogosa SL agar (Difco) were smeared on the agar plates. The plates were incubated at 15°C for 6 days, 25°C for 3 days, and 37°C for 2 days in an atmosphere of CO 2 in an anaerobic jar. After incubation individual isolates were selected on the basis of colony and cell morphology and the Gram reaction from each of the plates, and after purification they were maintained on prereduced agar slants (Mitsuoka, 1969) at 4°C. Physiological and biochemical examination For inoculum preparation, most isolates were grown in MRS broth (Oxoid) at 30°C, and some thermophilic strains at 37°C. The cells were harvested from MRS broth after two subcultures with a 24 h interval. They were resuspended in sterile neutralized saline containing 0.2% cysteine-HC1 to give approximately a doubled concentration.
21 Except where otherwise stated each tube with test medium was inoculated with 0.05 ml of inocula and incubated at 30°C. Inocula were also used for the Gram reaction and morphological examination. Gas production from glucose and gluconate and acid production from carbohydrates were determined by the method of Mitsuoka (1969) with the exception that the basal medium for fermentation with agar and indicator omitted was used for gas production. Tubes were incubated for 10 days at 30°C and observed 3, 6, and 10 days after inoculation. Catalase was tested on cultures grown on Briggs agar and nitrate reduction was examined using tryptic nitrate medium (Difco) according to the description of Cowan (1974). Hydrolysis of arginine was determined using the modified arginine broth containing 2% glucose (Hitchener et al., 1982). Hydrolysis of aesculin was determined by the method of Sharpe et al. (1966). Growth in 7.5% NaC1 was determined in MRS broth containing 7.5% NaC1, and growth at 15, 30, and 45°C examined in MRS broth. Growth on LBS agar was tested in comparison with growth on Briggs agar. The media were streak inoculated and incubated under 100% CO 2 for 3 days. Clotting of milk was determined using Litmus milk (Difco). B.C.P.-Plate count agar (Eiken Chemical Co., Japan) supplemented with 5% sucrose was streak inoculated for dextran production from sucrose and incubated at 25°C for 6 days under 100% CO2. To determine the lactic acid configuration strains were grown at 30°C for most of them but at 37°C for some in T N Y G medium. T N Y G medium had the following composition: tomato juice (Mitsuoka, 1969), 20 ml; neopeptone (Difco), 1 g; yeast extract (BBL), 0.5 g; glucose, 1 g; K 2 H P O 4, 0.3 g; Na-citrate, 0.5 g; Tween 80, 0.1 g; mineral solution B (Mitsuoka, 1969), 0.5 ml; and distilled water, 80 ml (pH 6.8). The cells were removed by centrifugation at 3000 r.p.m, for 20 min after two subcultures with a 24 h interval. L-(+)- and D-( -- )-lactate were estimated in supernatants using enzymic methods. L-(+)-Lactate was assayed using the reaction kits provided by Boehringer-Mannheim, and n - ( - ) - l a c t a t e assayed using D-( -- )-lactate dehydrogenase in place of L-(+)-lactate dehydrogenase in the kits according to the prescription.
Results and Discussion
A total of 690 isolates were examined. 13 of them were leuconostocs because they were cocci or short rods and produced gas and D - ( - )-lactate from glucose. Of the remainder, 440 strains from 45 samples were streptobacteria, 122 from 18 samples were thermobacteria, and 115 from 34 samples were betabacteria. Table I shows the properties of the groups and subgroups of streptobacteria. Groups $6 and $7 were dominant, and the strains were isolated from 32 samples each. Groups $4 and $5 were subordinate, and their isolates were from 20 and 17 samples, respectively. Groups $6 and $7 as well as groups $4 and $5 had mutually similar fermentation properties. However, groups $4 and $6 were separated from
22
TABLE
I
Characteristics of streptobacterial groups from meats and meat products
Growth
, - - , , - - ,
~
~
o
~
f
o
,~ ?.
~o ,-
Z
S1
+ +
x
(+ )
-
t,/oL
--
-
-
+
+
DL
--
--
--
zz
'~
+
+
+
DL
+
--
--
_
_
+
+
+
+
DL
+/--
d
_
_
+
+
+
DE
+
d
-
I
a
+ +
_
_
b
+
+
+
DL
+
--
--
_
_
C
+
+
+
DL
d
d
-
_
_
+
+
+
DI.
+
--
--
+
a
+
+
+
DL
+
--
--
b
+
+
+
DL
+
--
--
+
+
+
DL
d
-
+
a
+
+
+
DL
d
-
+
b
+
+
+
DL
d
c
+
+
+
DL
+
--
+
+
+
+
DL
+
--/+
a
+
+
+
DL
+
b
+
+
+
DL
c
+
+
+
DL
+
+
+
+
+
+
$5
$6
$7
$8 $9
'=
o
$2
$3 $4
=
+
_
+
--
_
+
+
--
_
+
+
--
_
+
_
_
_
+
--
--
--
d-
_
_
+
_
_
_
+
+
+
--
--/+
+
--
+
+
--
_
+
+
+/--
+
+
--
_
+
+
--
+
+
d
+
+
L/DL
+
--
+
_
_
_
+
DL
+
--
+
d
-
d
+
-
+
+
All the groups fermented glucose, mannose and fructose, did not grow at 45°C, produced no a m m o n i a from arginine, and showed no clotting of milk. + , positive for 90% or more strains; - , negative for 90%
groups $5 and $7, respectively, mainly by i-arabinose fermentation. Groups $6 and $7 differed clearly from groups $4 and $5 in aesculin and salicin, and also from groups $8 and $9 in melezitose, D-mannitol, and u-sorbitol. Groups $4, $5, $6, and $7 were separated into a few subgroups each as shown in Table I. Of the predominant groups group $6 was subdivided into 3 subgroups (S6/a, S6/b, and $ 6 / c ) on the basis of D-sucrose and starch fermentation. Subgroup S 6 / a is D-sucrose and melibiose negative and Starch positive; subgroup S 6 / b is D-sucrose and starch positive and variable in melibiose and shows a little weak growth in 7.5% NaCI; subgroup $ 6 / c is D-sucrose positive and starch negative. Biochemical characteristics of group $6 resemble those of group 1 of Hitchener et al. (1982), Lactobacillus sake, or L. curvatus (Rogosa, 1974, Stetter and Stetter, 1980), and is also similar to L. alimentarius with the exception that the latter produces L-(+)-lactate (Reuter, 1983). Subgroup S 6 / a is more similar to L. curvatus except
23
Fermentation A
o
o
.
~a
-
+
-/+
-
-
-
-
+
+
d
-
d
d
-
+
+
+
-
+
+
d
d
+ d
d
+/-
d
d
d
-
-/+
+
+
d +
+
d
-
-
d +
d +
+ .
+ + +
d
+ d + --
-
d
+/-
.
+
+
+
--/+
d
-+
d
+
d
-
-
-
+
-/+
-
d
d
+
+
-
-
-
+
-
-
-
d
+
.
.
.
+/-
d
+
.
.
=
-
.
.
+
.
.
.
+
d
d
+
d
+
d
-/+
-
d
+
+
+
-
+
d
+
-
-
-
+
+
+
+
+
+
+
d
d
d
-
-
+
+
d
+
d
+
+/-
d
-
-
+
d
+
d
-
d
+
d
+
d
d
+ d
+
+
+ d
+ d
+
+
+ +
+
d
-
-
+
d
+ d
+
d
+ -
+
d
-
-
+
d
+
+
+ +
d
+
-
-
+
d
+
+
+
+
+
+
-
-
+
d
d
d
+
+
+
+
+
+
+
+
+
+
+/-
+
+
+/-
+
+
+ d
o r m o r e strains; 21-79%
+/-
o f strains; ( + ) ,
and - / +, positive and negative, respectively
!
for 80% o f strains; d, p o s i t i v e for
w e a k r e a c t i o n . $ 8 is an k casei g r o u p , a n d $ 9 m o s t p r o b a b l y
is L. plantarum.
for amygdalin fermentation. Subgroups. S6/b and $6/c resemble L. sake. Subgroup 6 / c is not distinguished from group $7, especially subgroup S7/a, except for L-arabinose fermentation. No notable difference was found in the distribution of the subgroups of group $6 in foods. Subgroups of group $7 were mutually distinguished by fermentation patterns of rhamnose and trehalose. Subgroup S7/a is rhamnose negative and trehalose positive; subgroup S7/b is negative for both sugars; subgroup $ 7 / c is positive for both. Group $7 resembles group 3 of Hitchener et al. (1982), and is similar to cluster II of Shaw and Harding (1984), or L. bavaricus and L. sake. However, L. bavaricus is an L-(+)-lactate producer and L. sake is L-arabinose negative (Stetter and Stetter, 1980). There is no different distribution of the subgroups of group $7 in foods except that subgroup $ 7 / c was present more frequently in pork than the others were.
24
TABLE
II
Characteristics
of thermobacterial
and
betabacterial
groups
from
meats
and
meat
products
Growth
^
..
~
~
~
8
~
2 ¢-,
,--
"*
t--
.~
,z
~
©
¢~
~
<
<
Therrnobacterium T1
-
+
-
+
DL
.
T2
-
+
-
+
DL
--
. --
.
d
-
T3
-
+
-
+
DL
--
--
d
-
+
T4
-
+
-
+
L
--
-
+ / -
-
d
T5
-
+
-
+
L
.
-
+
DL
+
-
+ +
t)L I)L
+ +
. --
DL
4-
if-//--
and
melezitose
.
.
.
d -
+
.
Betabacterium
B1
-
+
B3
+ +
-
B4
+
-
B2
All the groups as for Table
+ (+ ) -
fermented I. T 3
and
(q-)
glucose and T4
are
were rhamnose
identified
with
the
L. acidophilus
-
.
. ---
negative. group,
+
-
+
--
q.-
q-
.
Reaction
probably
marks
shown
L. crispatus
and
arc L.
Groups $6 and $7 were difficult to distinguish morphologically, their strains grow singly or in short chains in MRS broth, being straight, or curved, occasionally horseshoe-like. Their surface colonies on Briggs agar are about 1 to 2 m m in diameter, circular, convex, and greyish-white. Groups $6 and $7 were separated only by L-arabinose fermentation in this report, so further studies may be needed to justify this separation. In chicken, Group $6 was found more frequently than Group $7, while there was no difference in occurrence in the others (Table III). At any rate, the two groups have the common and important characteristics that D-ribose and salicin fermentation and aesculin hydrolysis are positive and melezitose and D-mannitol fermentation are negative, and that they were present at remarkably high frequency in meat and meat products (Table III). Such properties are shared by described species and groups such as L. curvatus (Rogosa, 1974), L. sake, L. bavaricus, and L. alimentarius (Rogosa, 1974; Stetter and Stetter, 1980; Reuter, 1983), and atypical streptobacteria of Cavett (1963), biotype A 1 / 2 of Reuter (1970), groups 1 and 3 of Hitchener et al. (1982), and cluster II of Shaw and Harding (1984). In addition, these streptobacteria are most likely to be important components of the lactobacilli in meat and meat products (Cavett, 1963, Reuter, 1970; Bickstad et al., 1981; Hitchener et al., 1982; Vanderzant et al., 1982; Shaw and Harding, 1984; Morishita et al., 1985b). Of the subgroups of streptobacteria, group $4 was separated into 3 subgroups on the basis of the fermentation of D-sucrose, trehalose, melibiose, and starch. Subgroup S 4 / a is a main subgroup which is positive for D-sucrose, trehalose, and
25
Fermentation A.,
•-
~
.g
~
~
~
~
2
~
~
o
o '-
.~
~
z
~
~.
~
~
,~
~
d +
d +
-/+ -
-
d
+
d
d
+
d
-
d
d
+ +
+ +
+ +
+ +
+ +
d +
+ +
-
d +/-
+
+
-
+
+
+
+
+
-
+
+/-
+
+
-
+
+
+
+
+
+
+
+
+
d
+
+
+
--
+
-
+
+
+ -
+
+
d
d
d
d +
+
d +
+ -
+ +
+
+
+
d
+
+
+
+
+
-
+
+
+
+
salivarius, r e s p e c t i v e l y ; confusus, r e s p e c t i v e l y .
BI,
<
d
-
-
+
d
d
d
-
d
+
+
-
+
+
+
B2, B3, and
B4 are
L. fermentum, L. viridescens, L. brevis,
and
probably
L.
melibiose and negative for starch; subgroup S 4 / b is trehalose positive and negative in the other three sugars mentioned; subgroup $ 4 / c is positive for D-sucrose and starch, and negative for the others. Subgroup S 4 / a is physiologically similar to group 2 of Hitchener et al. (1982). Subgroup S 4 / a was distributed widely in meat and meat products, whereas the other subgroups were found mainly in meat. Group $5 was separated into 2 subgroups by fermentation of melibiose. Both subgroups show o-sucrose negative and trehalose positive reactions, and appear physiologically similar except for melibiose. They are unlikely to be identical to known species. There was a difference in distribution in meat and meat products: subgroup S 5 / a was widely distributed in meat and meat products, and subgroup S 5 / b was present only in meat, especially chicken and pork. Separation of $4 from $5 remains to be studied further because the main key to this separation was only L-arabinose fermentation. Of the minor groups of streptobacteria, group S1 showed o-galactose and o-ribose negative reactions, and no growth on LBS agar. The colony forms of the strains of this group were different from those of the other groups of streptobacteria and were rather similar to that of L. viridescens. This group is a combined group of S1 and $2 from our previous report (Morishita et al., 1985b,). Six strains, which belonged to a previous group $2, produced o - ( - ) - l a c t a t e from glucose, and slightly resemble L. homohiochi (Rogosa, 1974). Group S1 was present almost exclusively in meat products (Table III). Group $2 is negative for D-ribose, cellobiose, lactose, trehalose, and positive for
26 D-mannitol, resembling L. coryniformis subsp, torquens (Rogosa, 1974), but this group is composed of DL-lactate producers, whereas the corresponding species is D - ( - ) - l a c t a t e producer. This group was present in meats. Group $3 is positive for rhamnose and D-ribose and produces gas from gluconate. These points differentiate this group from group $2. Group $3 was present in meats. Groups $8 is not distinguished from the L. casei group. Two of the three isolates were identical to L. casei subsp, casei, and one is subsp, pseudoplantarum. The three strains were isolated from meat. Group $9 shows morphological and biochemical properties identical to that of L. plantarum. It was found mainly in meat. L. casei and L. plantarum were reported to be present in meat and meat products by many workers (Cavett, 1963; Reuter, 1970; Mol et al., 1971; Enfors et al., 1979; Blickstad et al., 1981; Vanderzant et al., 1982; Blickstad and Molin, 1983). Thermobacterium isolates, which were all isolated from meat, were separated into 5 groups as shown in Table II. Groups T2, T3, and T4 were dominant of thermobacteria from meats. All of the thermobacterial strains were isolated from chicken and pork, but not from beef and meat products (Table III). Chicken was the main source of these isolates. Group T1 is negative for lactose fermentation and is heterogeneous. Group T2 is close in physiology to L. helveticus. This group, however, is positive for D-sucrose, raffinose, and starch. Group T2 was found more frequently in pork than the others were, Group T3 belongs to the L. acidophilus group which consists of L. acidophilus, L. crispatus, L. gasseri, and some unnamed groups (Johnson et al., 1980; Lauer et al., 1980; Lauer and Kandler, 1980; Cato et al., 1983). Since it fermented raffinose and starch but not trehalose, group T3 is most likely to be L. crispatus, although the L. acidophilus group was reported not to be distinguished by sugar fermentation (Johnson et al., 1980; Lauer et al., 1980; Kuroshima and Kodaira, 1983). G r o u p T3 was found most frequently in chicken. Group T4 is identical with L. saliuarius. This group was also found mainly in chicken. Group T4 as well as group T3 are intestinal bacteria, and were isolated with remarkable frequency from chicken in this study. This suggests that the sanitory quality of chicken in the Japanese market should be improved. Group T5 produces L-(+)-lactate and is positive for D-ribose, cellobiose, starch and amygdalin but not for D-mannitol. This group, therefore, apparently differs from L. saliuarius. Of the 122 thermobacteria strains, 104 were isolated at 37°C incubation temperature. This indicates that 37°C incubation is necessary to recover thermobacteria from meat. Betabacterium isolates were separated into 4 groups (Table III). Group B1 is most likely to be L. fermentum since this group grew at 45°C but not at 15°C and showed biochemical characteristics identical to those of L. fermentum. All strains of group B1 were isolated from meat at an incubation temperature of 37°C. The occurrence of this group in meat, as mentioned above for groups T3 and T4, indicates that the meat was under poor sanitary conditions. L. fermentum was reported to occur in meat and meat products (Reuter, 1970; Blickstad et al., 1981).
27 TABLE III Distribution of groups of lactobacilli on meats and meat products Bacterial
Food
group
Chicken
Pork
Beef
Ham
Sausage
Bacon
Streptobacterium
104 (11) a 0 (0) 5 (4) 0 (0) 9 (6) 25 (6) 36 (9) 23 (5) 1 (1) 2 (2)
171 (17) 1 (1) I (1) 3 (1) 8 (7) 21 (7) 70(15) 54 (15) 2 (2) 11 (5)
74 (3) 0 (0) 0 (0) 3 (1) 2 (1) 0 (0) 30(3) 37 (3) 0(0) 2 (2)
25 (4) 7 (3) 0 (0) 0 (0) 3 (1) 2 (2) 4(1) 4 (2) 0(0) 5 (1)
20 (4) 2(1) 0 (0) 0 (0) 2 (2) 2 (1) 3(1) 5 (2) 0(0) 0 (0)
46 (7) 3 (1) 0 (0) 0 (0) 17 (2) 3 (1) 10(3) 13 (5) 0(0) 0 (0)
440 (45) 13 (6) 6 (5) 6 (2) 47 (20) 53 (17) 156(32) 136 (32) 3 (3) 20 (10)
(7) (2) (5) (2) (3) (1)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0(0) 0(0) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0(0) 0 (0) 0 (0) 0 (0)
122 (18) 6 (6) 24(11) 60 (12) 25 (11) 7 (4)
(8) (8) (0) (1) (1)
23 (10) 12 (2) 2 (2) 7 (3) 2 (2)
10 (3)
23 (3)
9 (3)
22 (7)
115 (34)
1 (1)
0 (0)
0 (0)
0 (0)
39 (11)
1 (1) 8 (2) 0 (0)
22 (3) 1 (1) 0(0)
2 (2) 0(0) 7 (2)
22 (7) 0(0) 0 (0)
49 (15) 17 (7) 10 (5)
235 (12)
213 (17)
84 (3)
48 (4)
29 (5)
68 (8)
677 (49)
SI $2 $3 $4 $5 $6 $7 $8 $9
Thermobacteriurn T1 T2 T3 T4 T5
Betabacterium B1 B2 B3 B4 Total
Total
103 (11) 4 (4) 15 (6) 57 (10) 22 (8) 5 (3) 28 26 0 1 1
19 2 9 3 3 2
a No. of strains (No. of positive samples).
G r o u p B2 is i d e n t i c a l to L. viridescens. T h i s g r o u p grew in 7.5% NaC1 a n d p r o d u c e d n o a m m o n i a f r o m a r g i n i n e . S t r a i n s of it were i s o l a t e d f r o m 12 s a m p l e s of m e a t p r o d u c t s a n d f r o m o n l y 3 s a m p l e s of meat. Since N i v e n a n d E v a n s (1957) d e s c r i b e d L. viridescens, the p r e s e n c e of this species in m e a t a n d m e a t p r o d u c t s was r e p o r t e d b y m a n y w o r k e r s ( A l l e n a n d Foster, 1960; C a v e t t , 1963; R e u t e r , 1970; M o l et al., 1971; E n f o r s et al., 1979; V a n d e r z a n t et al., 1982; Blickstad a n d M o l i n , 1983). G r o u p B3 is likely to b e L. brevis. T h i s g r o u p was f o u n d m a i n l y in p o r k a n d beef, a n d grew w e a k l y i n 7.5% NaC1. T h e o c c u r r e n c e of L. brevis i n m e a t a n d m e a t p r o d u c t s was r e p o r t e d b e f o r e (Cavett, 1963; R e u t e r , 1970; E n f o r s et al., 1979). G r o u p B4 shows b i o c h e m i c a l a n d m o r p h o l o g i c a l s i m i l a r i t y to L. confusus ( S h a r p e et al., 1972; H o l z a p f e l a n d v a n W y k , 1982), b u t a n a l y s i s of the cell wall is r e q u i r e d for full j u s t i f i c a t i o n of the d e s i g n a t i o n of this g r o u p ( H o l z a p f e l a n d v a n W y k , 1982). T h e m a j o r i t y of the s t r a i n s of this g r o u p p r o d u c e d d e x t r a n f r o m sucrose. W e d i d n o t isolate L-( + ) - l a c t a t e - p r o d u c i n g b e t a b a c t e r i a , w h i c h were r e p o r t e d b y H i t c h e n e r et al. (1982) a n d H o l z a p f e l a n d G e r b e r (1983), f r o m either m e a t or m e a t products.
28
As shown in Table III thermobacteria were found in chicken in approximately the same frequency as streptobacteria, while in pork and beef streptobacteria were isolated at a much higher frequency than thermobacteria. Such a peculiar composition of the lactobacillus populations in chicken suggests that chicken is exposed to heavy contamination by bacteria originating from the chicken itself, especially the intestines. In meat products streptobacteria and betabacteria were found at a similar frequency but no thermobacteria. These bacteria probably proliferated during the processing or preservation at low temperature after they had survived the curing or heating (Kempton and Bobier, 1970; Mol et al., 1971; Kitchell and Shaw, 1975).
References Allen, J.R. and E.M. Foster, 1960. Spoilage of vacuum-packed slices processed meats during refrigerated storage. Food Res. 25, 19-25. Blickstad, E. S., S.-O. Enfors and G. Molin, 1981. Effect of hyperbaric dioxide pressure on the microbial flora of pork stored at 4°C or 14°C. J. Appl, Bacteriol. 50, 493-504. Blickstad, E. and G. Molin, 1983. The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4°C. J. Appl. Bacteriol. 54, 45-56. Cato, E., W.E.C. Moore and J.L. Johnson, 1983. Synonymy of strains of "Lactobacillus acidophilus" group A2 (Johnson et al. 1980) with the type strain of Lactobacillus crispatus (Brygoo and Aladame, 1953) Moore and Holdeman, 1970. Int. J. Syst. Bacteriol. 33, 426-428. Cavett, J.J., 1963. A diagnostic key for identifying the lactic acid bacteria of vacuum-packed bacon. J. Appl. Bacteriol. 26, 453-470. Cowan, S.T., 1974. Cowan and Steel's Mant,al for the identification of medical bacteria. Cambridge University Press, Cambridge, 238 pp. De Man, J.C., M. Rogosa and M.E. Sharpe, 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135. Enfors, S.-O., G. Molin and A. Ternstrom, 1979. Effect of packaging under carbon dioxide, nitrogen or air on the microbial flora of pork stored at 4°C. J. Appl. Bacteriol. 47, 197-208. Gardner, G.A., 1968. Effects of pasteurization or added sulphite on the microbiology of stored vacuum package bacon burgers. J. Appl. Bacteriol. 31,462-478, Genigeorgis, C.A., 1976. Quality control for fermented meats. J. Am. Vet. Assoc. 169, 1220 1228. Hitchener, B.J., A.F. Egan and P.J. Rogers, 1982. Characteristics of lactic acid bacteria isolated from vacuum-packaged beef. J. Appl. Bacteriol. 52, 31-37. Holzapfel, W.H. and E.S. Gerber, 1983. Lactobacillus divergens sp. nov., a new heterofermentative Lactobacillus species producing L-( + )-lactate. Syst. Appl. Microbiol. 4, 522-534. Holzapfel, W.H. and E. van Wyk, 1982. Lactobacillus kandeleri sp. nov., a new species of the subgenus Betabacterium with glycine in the peptidoglycan. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig, C3, 495-502. Johnson, J.L., C.F. Phelps, C.S. Cummins, J. London and F. Gasser, 1980 Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30, 53-68. Kempton, A.G. and S.R. Bobier, 1970. Bacterial growth in refrigerated, vacuum-packed luncheon meats. Can. J. Microbiol. 16, 287 297. Kitchell, A.G. and B.G. Shaw, 1975. Lactic acid bacteria in fresh and cured meat. In: Lactic acid bacteria in beverages and food, edited by J.G. Carr, C,V. Cutting and G.C. Whiting, Academic Press, London, pp. 209-220. Kuroshima, T. and S. Kodaira, 1983. Differentiation between Lactobacillus acidophilus and L. gasseri. Japan. J. Bacteriol. 38, 809-816. Lauer, E., C. Helming and O. Kandler, 1980. Heterogeneity of the species Lactobacillus acidophilus (Moro) Hansen and Moquot as revealed by biochemical characteristics and D N A - D N A hybridisation. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. C1, 150-168.
29 Lauer, E. and O. Kandler, 1980. Lactobacillus gasseri sp. nov., a new species of the subgenus Thermobacterium. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. C1, 77-78. Lerche, M. and G. Reuter, 1960. Beitrag zur Methodik der Isolierung und Differenzierung von aerob wachsenden "Laktobazillen" (Genus "'Lactobacillus Beijerinck"). Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. I. Abt. Orig. 179, 354-370. Mitsuoka, T., 1969. Vergleichende Untersuchungen i~ber die Laktobazillen aus den Faeces yon Menschen, Schweinen und Hi)hnern. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 1. Abt. Orig. 210, 32-51. Mol, J.H.H., J.E.A. Hietbrink, H.W.M. Molin and J. van Tienteren, 1971. Observations on the microflora of vacuum packed sliced cooked meat products. J. Appl. Bacteriol. 34, 377 397. Morishita, Y., K. Shiromizu and T. Hori, 1985a. Lactobacillus flora of meats and meat products from retail stores. J. Food Hyg. Soc. Japan 26, 331-336 (in Japanese). Morishita, Y., K. Shiromizu and T. Hori, 1985b. Characteristics and distribution of lactobacilli in meat and meat products. J. Food Hyg. Soc. Japan 26, 365-370 (in Japanese). Newton, K.G., J.C.L. Harrison and K.M. Smith, 1977. The effect of storage in various gaseous atmospheres on the microflora of lamb chops held at - I°C. J. Appl. Bacteriol. 43, 53-59. Niven, C.F. and J.B. Evans, 1957. Lactobacillus viridescens nov. spec., a heterofermentative species that produces a green discoloration of cure meat pigments, J. Bacteriol. 73, 758-759. Patterson, J.T. and P.A. Gibbs, 1977. Incidence and spoilage potential of isolates from vacuum-packaged meat of high pH value. J. Appl. Bacteriol. 43, 25-38. Reuter, G., 1970. Laktobazillen und eng verwandte Mikroorganismen in Fleisch and Fleischwaren 2. Mitteilung: Die Charakterisierung der isolierten Laktobazillenstamme. Fleischwirtschaft 50, 954-962. Reuter, G., 1983. Lactobacillus alimentarius sp. nov., nom. rev. and Lactobacillusfarciminis sp. nov., nora. rev. Syst. Appl. Microbiol. 4, 277-279. Rogosa, M., 1974. Genus Lactobacillus. In: Bergey's Manual of Determinative Bacteriology, 8th edition, edited by R.E. Buchanan and N.E. Gibbons, Williams & Wilkins, Baltimore, pp. 576 593. Roth, L.A. and D.S. Clark, 1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21,629-632. Sharpe, M.E., T.F. Fryer and D.G. Smith, 1966. Identification of the lactic acid bacteria. In: Identification methods for microbiologists, edited by B.M. Gibbs and F.A. Skinner, Academic Press, London, pp. 65-79. Sharpe, M.E., E.I. Garvie and R.H. Tilbury, 1972. Some slime-forming heterofermentative species of the genus Lactobacillus. Appl. Microbiol. 23, 389-397. Shaw, B.G. and C.D. Harding, 1984. A numerical taxonomic study of lactic acid bacteria from vacuum-packed beef, pork, lamb and bacon. J. Appl. Bacteriol. 56, 25 40. Stetter, H. and K.O. Stetter, 1980. Lactobacillus bavaricus sp. nov. a new species of the subgenus Streptobacterium. Zentralbl. Bakteriol. Parasitenkd. Infektionstr. Hyg. 1. Abt. Orig. C1.70 74. Sutherland, J.P., J.T. Patterson and J.G. Murray, 1975. Changes in the microbiology of vacuum-packaged beef. J. Appl. Bacteriol. 39, 227-237. Vanderzant, C., M.O. Hanna, J.G. Ehlers, J.W. Savell, G.C. Smith, D.B. Griffin, R.N. Terrell, K.D. Lind and D.E. Galloway, 1982. Centralized packaging of beef loin steaks with different oxygen-barrier films: microbiological characteristics. J. Food Sci. 47, 1070-1079.