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Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp.
International Journal o f Food Microbiology, 21 (1994) 279-292 © 1994 Elsevier Science B.V. All rights reserved 0168-1605/94/$07.00
279
FOOD 649
Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp. A . D . S u t h e r l a n d * and R. Murdoch Hannah Research Institute, Ayr KA6 5HL, Scotland, UK
(Received 22 February 1993; accepted 20 September 1993)
Mesophilic and psychrotrophic isolates of Bacillus species displayed seasonal incidences in raw and pasteurised milk. The incidence of mesophilic isolates was highest in the winter and lowest in the summer/autumn while psychrotroph incidence was conversely lowest in the winter and highest in the late summer/autumn. Spores of Bacillus sp. were isolated from raw milk taken from farm milk machines and bulk tanks, milk tankers, dairy silos and pasteurised milk. A consistent seasonal fluctuation in incidence throughout these samples suggested that spores of Bacillus sp. derived from the farm environment survived as important contaminants right through the milk chain to the pasteurised product. Up to seven mesophilic Bacillus sp. were isolated from a single sample with three species commonly occurring in most samples. The predominant mesophilic species isolated were B. purnilus, B. licheniformis and B. subtilis. The dominant psychrotrophic isolate was B. cereus. Selected mesophilic isolates were examined for possible antagonistic effects on the growth of psychrotrophic B. cereus and B. pumilus isolates. Bacillus subtilis and B. licheniformis were found to produce antagonistic factors. It was considered that these factors may influence the incidence and growth of psychrotrophic isolates in the farm environment or in milk but the factors are not yet fully characterised or identified. Key words: Bacillus; Mesophile; Psychrotroph; Milk; Seasonality; Bacterial interaction
Introduction P s y c h r o t r o p h i c B a c i l l u s sp. a r e i m p o r t a n t c o n t a m i n a n t s o f milk. S p o r e s of B a c i l l u s sp. can survive p a s t e u r i s a t i o n a n d s u b s e q u e n t o u t g r o w t h in c o l d - s t o r e d p a s t e u r i s e d p r o d u c t s c a n l e a d to spoilage. A s well as this, strains o f B a c i l l u s c e r e u s can p r o d u c e two types o f f o o d p o i s o n i n g toxins, t h e e m e t i c an d d i a r r h o e a g e n i c toxins ( K r a m e r a n d Gilbert, 1989). P s y c h r o t r o p h i c strains g r o w i n g in c o l d - s t o r e d milk p r o d u c t s co u l d t h e r e f o r e c o n c e i v a b l y pose a public h e a l t h risk ( C h r i s t i a n s s o n et al., 1989; Griffiths, 1990). P s y c h r o t r o p h i c B a c i l l i have b e e n f o u n d to o c c u r with a seasonal distribution in raw an d p a s t e u r i s e d milk. In a study by Phillips an d Griffiths (1986), psy-
* Corresponding author: Tel: 0292 76013; 0292 671052. SSDI 0168-1605(93)E0065-Y
280 chrotrophic sporeformers predominated in raw milk during the s u m m e r / a u t u m n months. This finding provided a possible reason for an earlier observation by Phillips et al. (1981) that psychrotrophic Bacilli were predominant as spoilage flora in pasteurised cream during the late s u m m e r / a u t u m n period. An understanding of the reasons for the seasonal incidence of psychrotrophic Bacillus sp. may lead to measures for decreasing the extent to which these organisms contaminate milk supplies. McKinnon and Pettipher (1983) suggested after a limited study of one winter and one summer sample from four farms that one possible reason for a seasonal distribution of psychrotrophic Bacilli in raw milk is that they are derived from summer pasture and enter milk due to increased contamination of cows' udders. This implies that the cow udder is a major source of contamination of raw milk by psychrotrophic Bacilli. Poorly sanitised milking equipment and farm bulk tanks may also, however, be sources for these organisms (Cannon, 1972; Stewart, 1975). It has also been reported that mesophilic Bacillus sp. occur with a seasonal distribution in raw milk supplies. In contrast to psychrotrophic Bacillus isolates, they are usually present in greater numbers during the winter periods (Ridgway, 1954; Waes, 1976; Johnston and Bruce, 1982; McKinnon and Pettipher, 1983) and it has been suggested that this is due to increased udder contamination with Bacillus sp. derived from soiled winter bedding. It could also be speculated that the growth of psychrotrophic strains of Bacillus sp. may be inhibited by the presence of large populations of antagonistic mesophilic Bacillus sp. The decrease in counts of mesophilic Bacillus isolates in the summer and autumn would reduce this antagonism and this could be a compounding reason for the presence of higher numbers of psychrotrophic isolates being found at this time. In this study we examined the incidence of both psychrotrophic and mesophilic Bacillus sp. across the milk supply chain to determine at what points in the chain these isolates occurred at various seasons of the year. Following the results of this survey we then examined the interaction between selected psychrotrophic and mesophilic Bacillus sp. to determine whether there were any antagonistic effects by mesophiles on psychrotroph growth.
Materials and Methods
Milk samples Triplicate raw milk samples of approximately 100 ml each were taken monthly from the milking machine and bulk tank of five farms, five tankers and five dairy silos supplied by these tankers. A pasteurised milk sample was also taken from the same dairies. The milking machinery for farms 1 and 2 were of the bucket variety while farms 3 and 4 had a parlour pipeline and farm 5 had an 'around the byre' system. Milk samples were heat-treated at 80°C for 10 rain before spore counts were made. From June 1991 until March 1992 aliquots (200-500/~1) of milk were spread
281 on duplicate nutrient agar (Difco) plates supplemented with 5 mg of MnSO4-4HeO per litre (NA + MnSO 4) (Logan and Berkeley, 1984). Plates were incubated at 30°C for up to 72 h before a mean total mesophilic spore count was made from six replicate plates of each sample. Single colonies of each colony type grown on plates were sub-cultured to purity on further NA + MnSO 4 plates and then bacteria identified to species level. Identification involved determining cell morphology, spore size and position by Gram-stain and biochemical reaction in the API 50CHB system (Logan and Berkeley, 1984). From August 1991 until March 1992 a heated 100-ml milk sample from each site was taken monthly for a total psychrotrophic Bacillus spore count. The milk samples were pre-incubated for 7 days at 6°C as described previously by others (Phillips and Griffiths, 1986). This promoted outgrowth and multiplication of the small number of psychrotrophic Bacilli expected in samples. The pre-incubated samples were then plated onto NA + MnSO 4 using a spiral plater and plates were incubated at 6°C for 7 days before counting. Colonies of psychrotrophic isolates were isolated and bacteria identified as described for mesophilic isolates.
Interaction between mesophilic and psychrotrophic Bacillus sp. The influence that selected mesophilic Bacillus sp. had on the growth of psychrotrophic Bacillus sp. was assessed in three ways. (1) Contemporaneous spot on the lawn technique: confluent lawns of psychrotrophic Bacillus isolates were sown on milk agar (Oxoid) plates by spreading 100 ~1 of a predetermined dilution of an overnight nutrient broth (NB) (Oxoid) culture of each isolate over the agar surface and allowing the inoculum to dry. To this lawn, 20-/zl drops of undiluted overnight NB culture of each mesophilic isolate were added. When dry, individual plates were incubated at 6, 21 or 30°C until colonies developed. (2) Sowing lawns or streaks of psychrotrophic &olates around already established spots or streaks of mesophilic isolates: spots of growth of mesophilic Bacillus isolates were established by dropping 20-/zl volumes of undiluted overnight NB cultures onto milk agar plate surfaces and incubating at 21 or 30°C until growth was obvious. A predetermined dilution of an overnight NB culture of each psychrotrophic isolate was then spread (100 ~1) carefully around the already established areas of mesophile growth using a sterile plastic loop. Individual plates when dry were further incubated at 6, 21 or 30°C until psychrotroph colonies developed. (3) Bactometer experiments: overnight NB cultures of mesophilic Bacillus isolates were centrifuged to pellet bacteria and the supernatant filter sterilised (spent broth). Volumes (0.5 ml) of spent broths were mixed with 0.5 ml of fresh NB inoculated with a 1 X 105 dilution of an overnight NB culture of psychrotrophic Bacillus isolate HRM 44 in wells of a Bactometer module (Biomerieux). Samples
282 were grown in a Bactometer for up to 50 h and the impedance recorded. A detection time (dt) was given for each sample which was equivalent to the time taken for cultures to reach a count of 1 × 10 6 colony forming units (cfu)/ml. Isolates of Bacillus sp. used in these interaction studies included psychrotrophic isolates: B. cereus H R M 44, G T E 257 and MRM 62; B. pumilus PM 20. Mesophilic isolates: B. licheniformis H R M 013, H R M 014; B. subtilis H R M 057, H R M 079; B. firmus N C D O 1762; B. circulans MRM 24. All isolates were from milk and all but B. firmus N C D O 1762 were isolated at Hannah Research Institute and held there in the Institute culture collection.
Results
Seasonal variation in sporulating organisms in milk The mean spore counts ( c f u / m l ) of mesophilic Bacillus sp. recovered from each sample are given in Table I. The species of mesophilic Bacillus isolated and their frequency of isolation are given in Tables I and II. Fig. 1 shows the mean c f u / m l of the total mesophilic Bacillus sp. recovered from each sample over the period. Spores of mesophilic Bacillus sp. were recovered from all sample sites. Counts were generally low in all samples (range 0-965 cfu/ml), but up to seven species were isolated from single samples with the majority having at least three species (Table I). Bacillus pumilus, B. licheniformis and B. subtilis were the most frequently isolated species (Table II). The mesophile spore counts were highest in the winter periods, particularly from November until March inclusive, and lowest in the summer (Fig. 1). The mean counts of psychrotrophic Bacillus species isolated after pre-incubation and their frequency of isolation are given in Tables III and IV. Psychrotrophic Bacillus species were isolated from all samples sites but were only detectable in samples taken in the summer-autumn months (Table III, Fig. 2). Bacillus cereus was by far the most commonly isolated psychrotrophic species (Table IV). Up to two species were isolated from single samples. Interactions between mesophilic and psychrotrophic Bacillus sp. The results of interaction studies are summarised in Table V. Lawns of four psychrotrophic isolates of Bacillus sp. (three isolates of B. cereus and one of B. pumilus) were spread around established areas of growth of mesophilic Bacillus sp. and plates incubated at 21°C. Zones of inhibition were seen around growth of the two mesophilic isolates of B. subtilis and two of B. licheniformis tested. No inhibition was seen around areas of B. firmus or B. circulans growth (one isolate of each tested). This was repeated by spreading cultures of the same psychrotrophic isolates perpendicular to established streaks of mesophile growth and incubating plates at 6°C. Again areas of inhibition were seen around streaked growth of both B.
283 TABLE I S e a s o n a l s t u d y o f a e r o b i c m e s o p h i l i c s p o r e s in r a w m i l k Month
Farm/
Sample (mean cfu/ml)
dairy
Milking
Bulk tank
Tanker
Silo
Postpasteurisation
machine June
1 2 3
0.8 b:¢ 0.8 b 45.0 gbdqei
4 5
10.0 gd 14.1 bgdq
Overall mean July
16.7 gkqd 9.1 bqid 6.8
1 2 3
0.8 b 2.5 qlb 4.2 ab
35.0 c 1.7 cb U C bic
4 5
0 40.8 hie
5.0 dq 8.4 bl
Overall mean August
14.1
1.7 bgdel 3.3 b 3.3 ed
1 2 3 4
9.6
10.0
0.8 e 0 3.4 ea 20.0 bcei
8.3 becdj 13.0 13.3 29.9 5.0
eUl dbgej ebgd bde
13.9
14.8
8.4 b¢ 12.5 cqblk
3.3 15.0 6.6 5.0 8,3
10.8 bc 12.5 cb 8.3 bcq 10.5
0.8 k 0 5.0 eqa 1.7 e
6.7 eaci 5.8 ceaqt
0
0.8 q
0 3.3 eaq 3.3 ba
Overall mean
4.9
2.7
3.8
Sept.
1 2 3 4 5
3.5 0.6 6.2 1.7 1.7
Overall mean
2.7
5
Oct.
1 2 3 4 5
3.6 1.6 17.0 4.3 7.8
Overall mean
6.9
Nov,
1 2 3 4 5
Overall mean Dec. 1 2 3
90.0 1.3 14.0 3.3 15.0
hb h bh lab dqhlg
2.7 0.2 3.2 2.2 2.0
qhb h h hb gdblh
2.1 bi b qbi bq qb
6.5 0 11.0 2.5 5.0
abq ac aj ~g a
8.5 41.0 aj 1.4 abq 2.7 abe
56.0 3.5 14.0 4.0 12.0
11.2 cqb 36.8 Ibaqbc 3.5 hdbl 7.8 hbq NS
15.8 blqdd
bqi b b
blq bql bdlq bqcd
24.6
17.9 189.0 aq 1.7 cbaqa 5.0 aqc
bg lbc bec bcq qaobc
243.0 ab NS 131.0 abqc 164.0 qabc 149.0 acbapn
16.7 ba 5.0 bd
2.5 cqc 7.5 ~qbcl 7.5 agJq 11.6 uaq 12.5 abg
7.0
8.2
0.8 q 5,8 eb 5.8 bea
3.3 7.5 3.3 8.3 2.5
2.5 ab 30.8 eba
4.7 5.8 4.5 2.3
hcdbg dqhcbk dqbkq hcbqal
4.7 dacbg 15.0 Ihqk 10.8 hbdqco 3.7 hqcb NS 6.8
51.0 cqd
28.8 41.8 30.0 30.0 43.0
38.9
26.9
16.0 67.3 40.0 20.0
244.0 191.0 149.0 239.0 197.0
qec caelk becak cob eak
5.0
3.5
35.0 30.2 18.0 24.0
5.8 ebgid 12.5 ~ibd 9.2 egibc
9.8
NS
bi
aj ai ab agc acg
edjc cdjecb becdg ebdg bkejdq
9.1
11.9
5.0
10.0 6.6 29.2 13.3 15.0
cb cq qbld bcqd
abq aqb aqlb aqbc nclaqbp
137.8 235.0 afqmc 158.0 acqmp
204.0 37.0 balrnfq
676.0 q
244.0 q
280.0 abfqc
164.0 204.0 127.0 276.0 96.0
cbqd qbc qlbd cqbd qbcp
ap abc bacq abpqc cpnabq
173.4 164.0 aflqb 241.0 aflqbm 263.0 cfbaqm
284 TABLE I (continued) Month
Farm/ dairy
4 5 Overall mean Jan.
Milking machine
Bulk tank
Tanker
Silo
Postpasteurisation
3.3 qaci 160.0 caq
69.0 acqb UC cqa
360.0 q UC amcq
314.0 q UC aqcmf
182.0 mafbqc 213.0 an,
285.8
195.0
192.6
168.0 qac
193.0 acbq
41.7
52.9
143.0 ab 0.7 i 46.0 bacq 8.0 ca 956.0 ca
212.0 acqb 1.0 i 535.0 cbaq 15.0 aqb 965.0 q
182.0 acq 129.8 boa 292.0 qcab
280.0 qacb 253.0 qacb 292.0 ach 136.0 acbq 120.0 acqb
Overall mean
230.7
249.3
225.2
216.2
128.4
Feb.
1 2 3 4 5
37.0 2.0 20.0 4.0 676.0
98.0 ahabd 9.0 hqba 11.0 abcd 13.0 acqdb 637.0 daq
43.0 accb 87.0 caqb 383.0 abc 138.0 cab 103.0 qac
58.0 cbqa 105.0 acjbq 443.0 acbqj 82.0 qabcj 148.0 caq
96.0 aqbc 156.0 acbq 170.0 bacq 138.0 bacqjc 104.0 qajcb
Overall mean
147.8
153.6
150.8
167.2
132.8
11.0 aqfbj 40.0 iaq 3.0 aqb 99.0 baqfc UC
71.0 2.0 3.0 23.0 UC
139.0 acfdq 188.0 acbqfd 257.0 bcaf 215.0 qadcbf 280.0 cadq
154.0 afcdq 144.0acfdqb 140.0 cbqda 194.0 caafq 43.0 cqbadf
132.0 dabfcq 123.0acfb 148.0 qcafb 170.0 acbfqd 33.0 dcfaq
165.4
135.0
121.2
March
1 2 3 4 5
Sample (mean cfu/ml)
1 2 3 4 5
Overall mean
38.3
abah qbh adbcq a dabcq
24.8
abf ac qba qabc
355.0 aqcb
124.0 qcb
135.0 acqb 84.0 acbq 106.0 ~'q
* Letters indicate the species of Bacillus isolated and are coded as in Table II; UC, Spreading colony made sample uncountable; NS, no sample.
licheniformis a n d B. subtilis i s o l a t e s . Bacillus firmus a g a i n d i d n o t i n h i b i t psyc h r o t r o p h g r o w t h ( F i g s . 3a a n d b). S t u d i e s w e r e also c a r r i e d o u t with c o n t e m p e r a n e o u s lawns of the four psyc h r o t r o p h i c i s o l a t e s . M e s o p h i l i c i s o l a t e s d i d n o t g r o w r e a d i l y at 6 o r 21°C a n d o n l y s m a l l a r e a s o f i n h i b i t i o n w e r e s e e n w i t h two B. subtilis m e s o p h i l i c i s o l a t e s (Fig. 4), while
two B. licheniformis i s o l a t e s s h o w e d n o i n h i b i t i o n . A t 3 0 ° C t h e m e s o p h i l i c
i s o l a t e s g r e w r e a d i l y a n d b o t h i s o l a t e s o f B. subtilis s h o w e d s u b s t a n t i a l z o n e s o f i n h i b i t i o n w i t h all f o u r p s y c h r o t r o p h i c i s o l a t e s . T h e t w o i s o l a t e s o f B. licheniformis a n d o n e o f B. firmus a n d B. circulans h a d n o e f f e c t o n c o n t e m p o r a n e o u s p s y c h r o t r o p h l a w n s a t t h i s t e m p e r a t u r e ( a n e x a m p l e is s h o w n in Fig. 5). S p e n t b r o t h f r o m N B c u l t u r e s o f b o t h m e s o p h i l i c i s o l a t e s o f B. subtilis a n d B.
licheniforrnis w e r e m i x e d w i t h a c u l t u r e o f o n e p s y c h r o t r o p h i c B. cereus i s o l a t e ( H R M 44) i n a B a c t o m e t e r . C o n t r o l s a m p l e s o f H R M 44 in N B g r e w at 3 0 ° C w i t h a d e t e c t i o n t i m e ( d t ) o f 7.7 h in t h e B a c t o m e t e r e x p e r i m e n t ( r e s u l t s n o t s h o w n ) . Bacillus cereus H R M 44 d i d n o t g r o w at all in t h e p r e s e n c e o f s p e n t b r o t h f r o m o n e
285 T A B L E II Summary of mesophilic Bacillus species isolated from milk samples (June 1991 - March 1992) Code
Species
No. of samples from which individual species were isolated (total no. of samples = 250)
a b c d e f g h i j k 1 m n o p q
B. licheniformis B. pumilus B. subtilis B. lentus B. pantothenticus B. amyloliquifaciens B. stearothermophilus B. mycoides B. cereus B. circulans B. firmus B. megaterium B. sphaericus B. macerans B. laterosporus B. Polymyxa Unknown
131 156 127 52 33 25 19 22 16 15 10 27 6 3 2 5 120
B. subtilis mesophilic isolate (IIRM 57) while growth was substantially inhibited in the presence of spent broth from the other B. subtilis isolate and both B. licheniformis isolates with dt of 22.1, 12.2 and 11.8 h, respectively (Results not shown).
300
250
200 E = u 150 o E I O0 l
5oi 0
'
Jun •
Jul
Aug Sep
Oct
Nov Dec
Jan
Feb
Mar
milk machine ~-~ bulk tank [ ] tanker [ ] silo [ ] pasteurised milk
Fig. 1. M e a n seasonal count of mesophilic Bacillus sp. Samples were taken from five farms and five dairies.
286 T A B L E IIl Seasonal study of aerobic psychrotrophic spores in raw milk (pre-incubated count) (August 1991-March 1992) * Month
Farm/
Sample (mean c f u / m l )
dairy
Milking machine
Bulk tank
Tanker
Silo
Aug.
1 2 3 4 5
0 0 0 0 0
0 0 0 0 0
3.2x 1.0x 3.3x 6.0x 1.7x
4.6x 6.0x 6.0x 4.2x 6.0x
Sep.
1 2 3 4 5
3 . 7 x 10 sa'+ 0 0 0 1.3 × 104 ~
3 . 4 x 104~' 0 0 0 0
Oct.
1 2 3 4 5
0 4 . 6 x 105b 0 1.3X 106b 5.5 X 103a
0 3 . 5 x 104~' 0 0 1.7X 106~
104a'+ 104a 102" 105a 104a
Postpasteurisation 103a 105= 10 s~l 104a 105a
1.9x 103a 6 . 0 × 1 0 s~ 4 . 5 x 10 s~ 6 . 0 x 105a 1.3× 10 s '
0 2 . 6 x 104~' 6 . 4 x 104a 3 . 0 x 104~' 0
0 6 . 0 x 104a 3 . 7 x 104~ 6 . 8 x 104" 0
0 5 . 7 x 104~' 1.2x 104a 0 0
0 7.0× 106a 0 7.8×105a 1.0×107a
0 3 . 3 x 106a 0 2.6X 106a 1.0× 107a
2.8 x 10 ~' " 4 . 6 x 106a 1.4 x 106 b~ 2.2X 106be 1.0xl07~'
* No psychrotrophic Bacillus sp. were isolated in any sample taken during November 1991 to March 1992. + Letters indicate the species of Bacillus isolated and are coded as in Table IV.
8
6
i1 c~4
0i Aug
_ Sep
Oct
Nov
Dec
Jan
Feb
Mar
• milk machine ~ bulk tank [~ tanker ~ silo ~ paeteurised milk
Fig. 2. M e a n seasonal count of psychrotrophic Bacillus sp. recovered from milk samples after pre-incubation at 6°C for 7 days. Samples were taken from five farms and five dairies.
287 TABLE IV Summary of psychrotrophic Bacillus species isolated from milk samples June 1991-March 1992 Code
Species
No. of samples from which individual species were isolated (total no. of samples = 200)
a b c
B. cereus B. polymyxa B. circulans
39 4 1
Fig. 3. A streak plate of established mesophilic Bacillus growth (A, B. licheniformis HRM 13; B, B. licheniformis HRM 14; C, B. subtilis HRM 57; D, B. subtilis HRM 79; E, B. firmus NCDO 1752) across which has been streaked psychrotrophic Bacilli cultures (1, B. cereus HRM 44; 2, B. cereus MRM 652; 3, B. cereus GTE 257; 4, B. pumilus PM 20) which were then grown at 6°C. Mesophilic isolates of B. licheniformis showed some inhibition and B. subtilis substantial inhibition while B. firmus showed no inhibition towards growth of the four psychrotrophic isolates.
Fig. 4. Spots of mesophilic B. subtilis cultures (A, HRM 57; B, HRM 79) showing inhibitory activity when grown contemporaneously with a lawn of psychrotrophic B. cereus (HRM 44) culture at 6°C.
-
b c d e
Method of analysis
6
L
21
H R M 13 b
30
6
_+ +
-_ _
_
_
_ _
± +
30
6
i _+
+
_+
21
H R M 57 ~
21
H R M 14 b
Contemporaneous lawn culture
+ +
+
+
30
= n o inhibition; _+ = visible inhibition; + = s u b s t a n t i a l inhibition.
N o inhibition w a s s e e n w i t h B. circulans a n d B. f i r m u s isolates by a n y m e t h o d . B. licheni]ormis. B. subtilis. B. cereus. B. pumilis.
P M 20 °
T e m p e r a t u r e (°C): Psychrotrophic isolate: HRM44 d MRM62 d G T E 257 d
Mesophilic isolate:
A n t a g o n i s m of m e s o p h i l i c Bacillus isolates a for p s y c h r o t r o p h i c Bacillus isolates
TABLE V
_+ +
_+
+
6
± _+
_+
+
21
H R M 79 c
+ +
+
+
30
+ +
+
+
6
6
+ + + +
+ + + +
+ + + +
21
H R M t4
21
H R M 13
+ + + +
6
+ + + +
21
H R M 57
Psychrotrophic lawns around established mesophile growth
+ + + +
+ + + +
21
H R M 79 6
289
Fig. 5. An example of spots of mesophilic Bacilli cultures (A, B. subtilis HRM 57; B, B. licheniformis HRM 13; C, B. licheniformis HRM 14; D, B. subtilis HRM 79; E, B. firmus NCDO 1752) grown contemporaneously with a lawn of psychrotrophic B. cereus (HRM 44) culture at 30°C. Both isolates of B. subtilis were shown to inhibit the growth of the psychrotrophic isolate.
Discussion
Mesophile spore counts in both raw milk and pasteurised milk samples were generally higher in the winter months (November to March, inclusive) than in the summer/autumn months (June to October, inclusive). This coincident increase in counts in both raw and pasteurised milk samples suggests that raw milk is likely to be a source of spores which survive pasteurisation. Generally, the increase in counts in the winter period occurred throughout the sampled areas, i.e., in the farm milk machine, bulk tank, milk tankers, dairy silos and pasteurised milk samples. Increases, however, were first detectable in tanker, silo and pasteurised milk samples. This possibly reflects growth of the mesophilic sporeformers in the latter part of the milk chain including tanker surfaces and on dairy equipment or indicates additional sources of contamination at these points as suggested by others (Canon, 1972; Stewart, 1975). Increased mesophilic spore counts from farm raw milk in winter has been detected previously by others (Waes, 1976; Johnston and Bruce, 1983; McKinnon and Pettipher, 1983) and increased mesophilic spores on udder surfaces due to contact with contaminated winter bedding has been implicated as a cause of this. The three most frequently isolated mesophilic sporeformers over the sampling period were B. pumilus, B. licheniformis and B. subtilis. These same three species were also the most frequent isolates from previous surveys of raw milk in Scotland (Phillips and Griffiths, 1986) and elsewhere (Martin, 1981). The predominant psychrotrophic species isolated was B. cereus and this again agreed with the findings of others (Coghill and Juffs, 1979; Johnstone and Bruce, 1982; Sharma et al., 1984; Phillips and Griffiths, 1986; Griffiths and Phillips, 1990). This noticeable consistency in the predominance of these organisms in milk might
290 suggest factors exist, such as increased numbers of these organisms in the environment, an increased ability to adhere to cow teat surfaces or faster growth rates compared to other Bacillus sp. which would preferentially increase counts of these organisms in milk. The seasonal distribution of samples containing psychrotrophic sporeformers was quite distinct. Psychrotrophic strains were found in samples during the late s u m m e r / a u t u m n period. This again was in agreement with the findings of others (Phillips and Griffiths, 1986). McKinnon and Pettipher (1983) found that psychrotrophic sporeformers represented about 22% of the total spore count derived from summer pasture samples. This suggested that the seasonal incidence of psychrotrophic sporeformers was due to these organisms dominating an environmental niche which milking cows were exposed to when on summer pasture. The distinct and exclusive seasonal distribution between mesophilic and psychrotrophic spore populations found in the work reported here suggested, however, that an additional reason for seasonal occurrence may also exist. It was thought conceivable that large populations of mesophilic sporeformers may have antagonistic effects upon psychrotrophc sporeformers which prevented their growth and multiplication either in environmental sources or in raw milk. To examine this possibility the incidence of individual mesophilic sporeformers (Table I) was determined and species which were absent or were present in low numbers in the s u m m e r / a u t u m n months (namely B. subtilis, B. licheniformis and B. circulans) were examined for their influence on the growth of psychrotrophic sporeformers. Bacillus firmus which was more common in the summer than in the winter was also studied as a control which would be unlikely to inhibit psychrotroph growth. Since B. cereus was by far the most common psychrotrophic sporeformer isolated in the survey, three isolates of this species and one isolate of B. pumilus were included to look for species and isolate variations. No differences were seen in the interactive effects of mesophiles between any of the psychrotroph isolates tested. Established cultures of mesophilic B. subtilis and B. licheniformis were shown in agar plate studies to produce factors which inhibited the growth of psychrotrophic B. cereus and B. pumilus isolates grown at temperatures as low as 6°C. The inhibitory factors could have been produced when mesophilic growth was established at 30°C but this method at least showed that these inhibitory factors were active at 6°C. The streak method on agar plates offered a rapid, low cost method of screening mixed cultures for antagonistic effects. Bactometer studies indicated that inhibitory factors were extracellular. Bacillus subtilis was found to be more inhibitory than B. Iicheniformis in the present examinations. When lawns of the psychrotrophic isolates were spotted with contemporaneous cultures of mesophilic isolates only B. subtilis showed inhibitory effects. This effect was more pronounced at 30°C than at 21 or 6°C which probably reflected the poor levels of growth by B. subtilis at the lower temperatures. Bacillus licheniformis showed no inhibitory effects on psychrotroph growth even at 30°C. This suggested
291 t h a t t h e i n h i b i t o r y f a c t o r d e t e c t e d in e s t a b l i s h e d c u l t u r e s of B. licheniformis was n o t p r o d u c e d quickly e n o u g h in p o t e n t q u a n t i t i e s to inhibit c o n t e m p o r a n e o u s p s y c h r o t r o p h g r o w t h w h e n s t u d i e d by this m e t h o d . It is h y p o t h e s i s e d t h a t i n h i b i t o r y factors c o u l d p l a y a role in influencing t h e s e a s o n a l i n c i d e n c e o f p s y c h r o t r o p h i c Bacilli in milk by i n f l u e n c i n g t h e relative p r o p o r t i o n s of m e s o p h i l i c a n d p s y c h r o t r o p h i c Bacilli e i t h e r in the f a r m environm e n t (e.g., soil) o r in raw milk. M o r e work, however, is r e q u i r e d to d e t e r m i n e directly h o w t h e s e a n t a g o n i s t s affect t h e g r o w t h of p s y c h r o t r o p h i c Bacilli in e n v i r o n m e n t s such as soil a n d c o l d - s t o r e d milk. It w o u l d also b e o f value to d e t e r m i n e if t h e most c o m m o n l y i s o l a t e d m e s o p h i l i c a n d p s y c h r o t r o p h i c Bacillus sp. can p r o d u c e factors which r e d u c e the i n c i d e n c e a n d growth o f o t h e r less c o m m o n Bacillus sp. in milk. T h e n a t u r e o f t h e a n t a g o n i s t i c factors d e s c r i b e d h e r e a r e u n k n o w n . Bacillus subtilis p r o d u c e s m e t a l l o a n d s e r i n e p r o t e a s e s , the a n t i b i o t i c mycobacillin a n d has s u r f a c t a n t activity which can lyse r e d b l o o d cells (Priest, 1989). Bacillus fichenif o r m & also p r o d u c e s p r o t e a s e s (Priest, 1989) a n d t h e b a c i t r a c i n antibiotics (Katz a n d D e m a i n , 1977). S o m e o f t h e s e factors m a y b e a n t a g o n i s t s a l o n e o r in c o n c e r t with e a c h o t h e r o r u n d e t e r m i n e d factors m a y b e involved. F u r t h e r r e s e a r c h into d e f i n i n g t h e a n t a g o n i s t i c factors w o u l d b e o f interest.
References Cannon, R.Y. (1972) Contamination of raw milk with psychrotrophic sporeformers. J. Dairy Sci. 55, 669. Christiansson, A., Naidu, A.S., Nilsson, I., Wadstrom, T. and Pettersson, H.-E. (1989) Toxin production by Bacillus cereus dairy isolates in milk at low temperatures. Appl. Environm. Microbiol. 55(10), 2595-2600. Coghill, D. and Juffs, H.S. (1979) Incidence of psychrotrophic sporeforming bacteria in pasteurised milk and cream products and effect of temperature on their growth. Australian J. Dairy Technol. 34, 150-153. Griffiths, M.W. (1990) Toxin production by psychrotrophic Bacillus species present in milk. J. Food Protect. 53(9), 790-792. Griffiths, M.W. and Phillips, J.D. (1990) Incidence, source and some properties of psychrotrophic Bacillus sp. found in raw and pasteurised milk. J. Soc. Dairy Technol. 43(3), 62-66. Johnstone, D.W. and Bruce, J. (1982) Incidence of thermoduric psychrotrophs in milk produced in the west of Scotland. J. Appl. Bacteriol. 52, 333-337. Katz, E. and Demain, A.L. (1977) The peptide antibiotics of Bacillus: chemistry biogenesis and possible functions. Bacteriol. Rev. 41,449-474. Kramer, J.M. and Gilbert, R.J. (1989) Bacillus cereus and other Bacillus species. In: Foodborne Bacterial Pathogens, Doyle, M.P. (Ed.) Marcel Dekker, NY. pp. 22-70. Logan, N.A. and Berkeley, R.C.W. (1984) Indentification of Bacillus strains using the API system. J. Gen. Microbiol. 130, 1871-1882. Martin, J.H. (1981) Heat resistant mesophilic microorganisms. J. Dairy Sci. 64, 149-156. McKinnon, C.H. and Pettipher, G.L. (1983) A survey of sources of heat resistant bacteria in milk with particular reference to psychrotorphic spore-forming bacteria. J. Dairy Res. 50, 163-170. Phillips, J.D. and Griffiths, M.W. (1986) Factors contributing to the seasonal variation of Bacillus sp. in pasteurised dairy products. J. Appl. Bacteriol. 61,275-285.
292
Phillips, J.D., Griffiths, M.W. and Muir, D.D. (1981) Growth and associated enzymic activity of spoilage bacteria in pasteurised double cream. J. Soc. Dairy Technol. 34, 113-118. Priest, F.G. (1989) Products and applications. In: Bacillus, Harwood, C.R. (Ed.), Plenum Press, NY and London. pp. 293-320. Sharma, J.K., Malik, R.K. and Mather, D.K. (1984) Isolation and identification of proteolytic psychrotrophic sporeforming bacteria from raw milk supplies at an experimental farm in India. J. Soc. Dairy Technol. 37, 96-98. Stewart, D.B. (1975) Factors influencing the incidence of B. cereus spores in milk. J. Soc. Dairy Technol. 28, 80-85. Waes, G. (1976) Aerobic mesophilic spores in raw milk. Milchwissenschaft 31,521-524.
279
FOOD 649
Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp. A . D . S u t h e r l a n d * and R. Murdoch Hannah Research Institute, Ayr KA6 5HL, Scotland, UK
(Received 22 February 1993; accepted 20 September 1993)
Mesophilic and psychrotrophic isolates of Bacillus species displayed seasonal incidences in raw and pasteurised milk. The incidence of mesophilic isolates was highest in the winter and lowest in the summer/autumn while psychrotroph incidence was conversely lowest in the winter and highest in the late summer/autumn. Spores of Bacillus sp. were isolated from raw milk taken from farm milk machines and bulk tanks, milk tankers, dairy silos and pasteurised milk. A consistent seasonal fluctuation in incidence throughout these samples suggested that spores of Bacillus sp. derived from the farm environment survived as important contaminants right through the milk chain to the pasteurised product. Up to seven mesophilic Bacillus sp. were isolated from a single sample with three species commonly occurring in most samples. The predominant mesophilic species isolated were B. purnilus, B. licheniformis and B. subtilis. The dominant psychrotrophic isolate was B. cereus. Selected mesophilic isolates were examined for possible antagonistic effects on the growth of psychrotrophic B. cereus and B. pumilus isolates. Bacillus subtilis and B. licheniformis were found to produce antagonistic factors. It was considered that these factors may influence the incidence and growth of psychrotrophic isolates in the farm environment or in milk but the factors are not yet fully characterised or identified. Key words: Bacillus; Mesophile; Psychrotroph; Milk; Seasonality; Bacterial interaction
Introduction P s y c h r o t r o p h i c B a c i l l u s sp. a r e i m p o r t a n t c o n t a m i n a n t s o f milk. S p o r e s of B a c i l l u s sp. can survive p a s t e u r i s a t i o n a n d s u b s e q u e n t o u t g r o w t h in c o l d - s t o r e d p a s t e u r i s e d p r o d u c t s c a n l e a d to spoilage. A s well as this, strains o f B a c i l l u s c e r e u s can p r o d u c e two types o f f o o d p o i s o n i n g toxins, t h e e m e t i c an d d i a r r h o e a g e n i c toxins ( K r a m e r a n d Gilbert, 1989). P s y c h r o t r o p h i c strains g r o w i n g in c o l d - s t o r e d milk p r o d u c t s co u l d t h e r e f o r e c o n c e i v a b l y pose a public h e a l t h risk ( C h r i s t i a n s s o n et al., 1989; Griffiths, 1990). P s y c h r o t r o p h i c B a c i l l i have b e e n f o u n d to o c c u r with a seasonal distribution in raw an d p a s t e u r i s e d milk. In a study by Phillips an d Griffiths (1986), psy-
* Corresponding author: Tel: 0292 76013; 0292 671052. SSDI 0168-1605(93)E0065-Y
280 chrotrophic sporeformers predominated in raw milk during the s u m m e r / a u t u m n months. This finding provided a possible reason for an earlier observation by Phillips et al. (1981) that psychrotrophic Bacilli were predominant as spoilage flora in pasteurised cream during the late s u m m e r / a u t u m n period. An understanding of the reasons for the seasonal incidence of psychrotrophic Bacillus sp. may lead to measures for decreasing the extent to which these organisms contaminate milk supplies. McKinnon and Pettipher (1983) suggested after a limited study of one winter and one summer sample from four farms that one possible reason for a seasonal distribution of psychrotrophic Bacilli in raw milk is that they are derived from summer pasture and enter milk due to increased contamination of cows' udders. This implies that the cow udder is a major source of contamination of raw milk by psychrotrophic Bacilli. Poorly sanitised milking equipment and farm bulk tanks may also, however, be sources for these organisms (Cannon, 1972; Stewart, 1975). It has also been reported that mesophilic Bacillus sp. occur with a seasonal distribution in raw milk supplies. In contrast to psychrotrophic Bacillus isolates, they are usually present in greater numbers during the winter periods (Ridgway, 1954; Waes, 1976; Johnston and Bruce, 1982; McKinnon and Pettipher, 1983) and it has been suggested that this is due to increased udder contamination with Bacillus sp. derived from soiled winter bedding. It could also be speculated that the growth of psychrotrophic strains of Bacillus sp. may be inhibited by the presence of large populations of antagonistic mesophilic Bacillus sp. The decrease in counts of mesophilic Bacillus isolates in the summer and autumn would reduce this antagonism and this could be a compounding reason for the presence of higher numbers of psychrotrophic isolates being found at this time. In this study we examined the incidence of both psychrotrophic and mesophilic Bacillus sp. across the milk supply chain to determine at what points in the chain these isolates occurred at various seasons of the year. Following the results of this survey we then examined the interaction between selected psychrotrophic and mesophilic Bacillus sp. to determine whether there were any antagonistic effects by mesophiles on psychrotroph growth.
Materials and Methods
Milk samples Triplicate raw milk samples of approximately 100 ml each were taken monthly from the milking machine and bulk tank of five farms, five tankers and five dairy silos supplied by these tankers. A pasteurised milk sample was also taken from the same dairies. The milking machinery for farms 1 and 2 were of the bucket variety while farms 3 and 4 had a parlour pipeline and farm 5 had an 'around the byre' system. Milk samples were heat-treated at 80°C for 10 rain before spore counts were made. From June 1991 until March 1992 aliquots (200-500/~1) of milk were spread
281 on duplicate nutrient agar (Difco) plates supplemented with 5 mg of MnSO4-4HeO per litre (NA + MnSO 4) (Logan and Berkeley, 1984). Plates were incubated at 30°C for up to 72 h before a mean total mesophilic spore count was made from six replicate plates of each sample. Single colonies of each colony type grown on plates were sub-cultured to purity on further NA + MnSO 4 plates and then bacteria identified to species level. Identification involved determining cell morphology, spore size and position by Gram-stain and biochemical reaction in the API 50CHB system (Logan and Berkeley, 1984). From August 1991 until March 1992 a heated 100-ml milk sample from each site was taken monthly for a total psychrotrophic Bacillus spore count. The milk samples were pre-incubated for 7 days at 6°C as described previously by others (Phillips and Griffiths, 1986). This promoted outgrowth and multiplication of the small number of psychrotrophic Bacilli expected in samples. The pre-incubated samples were then plated onto NA + MnSO 4 using a spiral plater and plates were incubated at 6°C for 7 days before counting. Colonies of psychrotrophic isolates were isolated and bacteria identified as described for mesophilic isolates.
Interaction between mesophilic and psychrotrophic Bacillus sp. The influence that selected mesophilic Bacillus sp. had on the growth of psychrotrophic Bacillus sp. was assessed in three ways. (1) Contemporaneous spot on the lawn technique: confluent lawns of psychrotrophic Bacillus isolates were sown on milk agar (Oxoid) plates by spreading 100 ~1 of a predetermined dilution of an overnight nutrient broth (NB) (Oxoid) culture of each isolate over the agar surface and allowing the inoculum to dry. To this lawn, 20-/zl drops of undiluted overnight NB culture of each mesophilic isolate were added. When dry, individual plates were incubated at 6, 21 or 30°C until colonies developed. (2) Sowing lawns or streaks of psychrotrophic &olates around already established spots or streaks of mesophilic isolates: spots of growth of mesophilic Bacillus isolates were established by dropping 20-/zl volumes of undiluted overnight NB cultures onto milk agar plate surfaces and incubating at 21 or 30°C until growth was obvious. A predetermined dilution of an overnight NB culture of each psychrotrophic isolate was then spread (100 ~1) carefully around the already established areas of mesophile growth using a sterile plastic loop. Individual plates when dry were further incubated at 6, 21 or 30°C until psychrotroph colonies developed. (3) Bactometer experiments: overnight NB cultures of mesophilic Bacillus isolates were centrifuged to pellet bacteria and the supernatant filter sterilised (spent broth). Volumes (0.5 ml) of spent broths were mixed with 0.5 ml of fresh NB inoculated with a 1 X 105 dilution of an overnight NB culture of psychrotrophic Bacillus isolate HRM 44 in wells of a Bactometer module (Biomerieux). Samples
282 were grown in a Bactometer for up to 50 h and the impedance recorded. A detection time (dt) was given for each sample which was equivalent to the time taken for cultures to reach a count of 1 × 10 6 colony forming units (cfu)/ml. Isolates of Bacillus sp. used in these interaction studies included psychrotrophic isolates: B. cereus H R M 44, G T E 257 and MRM 62; B. pumilus PM 20. Mesophilic isolates: B. licheniformis H R M 013, H R M 014; B. subtilis H R M 057, H R M 079; B. firmus N C D O 1762; B. circulans MRM 24. All isolates were from milk and all but B. firmus N C D O 1762 were isolated at Hannah Research Institute and held there in the Institute culture collection.
Results
Seasonal variation in sporulating organisms in milk The mean spore counts ( c f u / m l ) of mesophilic Bacillus sp. recovered from each sample are given in Table I. The species of mesophilic Bacillus isolated and their frequency of isolation are given in Tables I and II. Fig. 1 shows the mean c f u / m l of the total mesophilic Bacillus sp. recovered from each sample over the period. Spores of mesophilic Bacillus sp. were recovered from all sample sites. Counts were generally low in all samples (range 0-965 cfu/ml), but up to seven species were isolated from single samples with the majority having at least three species (Table I). Bacillus pumilus, B. licheniformis and B. subtilis were the most frequently isolated species (Table II). The mesophile spore counts were highest in the winter periods, particularly from November until March inclusive, and lowest in the summer (Fig. 1). The mean counts of psychrotrophic Bacillus species isolated after pre-incubation and their frequency of isolation are given in Tables III and IV. Psychrotrophic Bacillus species were isolated from all samples sites but were only detectable in samples taken in the summer-autumn months (Table III, Fig. 2). Bacillus cereus was by far the most commonly isolated psychrotrophic species (Table IV). Up to two species were isolated from single samples. Interactions between mesophilic and psychrotrophic Bacillus sp. The results of interaction studies are summarised in Table V. Lawns of four psychrotrophic isolates of Bacillus sp. (three isolates of B. cereus and one of B. pumilus) were spread around established areas of growth of mesophilic Bacillus sp. and plates incubated at 21°C. Zones of inhibition were seen around growth of the two mesophilic isolates of B. subtilis and two of B. licheniformis tested. No inhibition was seen around areas of B. firmus or B. circulans growth (one isolate of each tested). This was repeated by spreading cultures of the same psychrotrophic isolates perpendicular to established streaks of mesophile growth and incubating plates at 6°C. Again areas of inhibition were seen around streaked growth of both B.
283 TABLE I S e a s o n a l s t u d y o f a e r o b i c m e s o p h i l i c s p o r e s in r a w m i l k Month
Farm/
Sample (mean cfu/ml)
dairy
Milking
Bulk tank
Tanker
Silo
Postpasteurisation
machine June
1 2 3
0.8 b:¢ 0.8 b 45.0 gbdqei
4 5
10.0 gd 14.1 bgdq
Overall mean July
16.7 gkqd 9.1 bqid 6.8
1 2 3
0.8 b 2.5 qlb 4.2 ab
35.0 c 1.7 cb U C bic
4 5
0 40.8 hie
5.0 dq 8.4 bl
Overall mean August
14.1
1.7 bgdel 3.3 b 3.3 ed
1 2 3 4
9.6
10.0
0.8 e 0 3.4 ea 20.0 bcei
8.3 becdj 13.0 13.3 29.9 5.0
eUl dbgej ebgd bde
13.9
14.8
8.4 b¢ 12.5 cqblk
3.3 15.0 6.6 5.0 8,3
10.8 bc 12.5 cb 8.3 bcq 10.5
0.8 k 0 5.0 eqa 1.7 e
6.7 eaci 5.8 ceaqt
0
0.8 q
0 3.3 eaq 3.3 ba
Overall mean
4.9
2.7
3.8
Sept.
1 2 3 4 5
3.5 0.6 6.2 1.7 1.7
Overall mean
2.7
5
Oct.
1 2 3 4 5
3.6 1.6 17.0 4.3 7.8
Overall mean
6.9
Nov,
1 2 3 4 5
Overall mean Dec. 1 2 3
90.0 1.3 14.0 3.3 15.0
hb h bh lab dqhlg
2.7 0.2 3.2 2.2 2.0
qhb h h hb gdblh
2.1 bi b qbi bq qb
6.5 0 11.0 2.5 5.0
abq ac aj ~g a
8.5 41.0 aj 1.4 abq 2.7 abe
56.0 3.5 14.0 4.0 12.0
11.2 cqb 36.8 Ibaqbc 3.5 hdbl 7.8 hbq NS
15.8 blqdd
bqi b b
blq bql bdlq bqcd
24.6
17.9 189.0 aq 1.7 cbaqa 5.0 aqc
bg lbc bec bcq qaobc
243.0 ab NS 131.0 abqc 164.0 qabc 149.0 acbapn
16.7 ba 5.0 bd
2.5 cqc 7.5 ~qbcl 7.5 agJq 11.6 uaq 12.5 abg
7.0
8.2
0.8 q 5,8 eb 5.8 bea
3.3 7.5 3.3 8.3 2.5
2.5 ab 30.8 eba
4.7 5.8 4.5 2.3
hcdbg dqhcbk dqbkq hcbqal
4.7 dacbg 15.0 Ihqk 10.8 hbdqco 3.7 hqcb NS 6.8
51.0 cqd
28.8 41.8 30.0 30.0 43.0
38.9
26.9
16.0 67.3 40.0 20.0
244.0 191.0 149.0 239.0 197.0
qec caelk becak cob eak
5.0
3.5
35.0 30.2 18.0 24.0
5.8 ebgid 12.5 ~ibd 9.2 egibc
9.8
NS
bi
aj ai ab agc acg
edjc cdjecb becdg ebdg bkejdq
9.1
11.9
5.0
10.0 6.6 29.2 13.3 15.0
cb cq qbld bcqd
abq aqb aqlb aqbc nclaqbp
137.8 235.0 afqmc 158.0 acqmp
204.0 37.0 balrnfq
676.0 q
244.0 q
280.0 abfqc
164.0 204.0 127.0 276.0 96.0
cbqd qbc qlbd cqbd qbcp
ap abc bacq abpqc cpnabq
173.4 164.0 aflqb 241.0 aflqbm 263.0 cfbaqm
284 TABLE I (continued) Month
Farm/ dairy
4 5 Overall mean Jan.
Milking machine
Bulk tank
Tanker
Silo
Postpasteurisation
3.3 qaci 160.0 caq
69.0 acqb UC cqa
360.0 q UC amcq
314.0 q UC aqcmf
182.0 mafbqc 213.0 an,
285.8
195.0
192.6
168.0 qac
193.0 acbq
41.7
52.9
143.0 ab 0.7 i 46.0 bacq 8.0 ca 956.0 ca
212.0 acqb 1.0 i 535.0 cbaq 15.0 aqb 965.0 q
182.0 acq 129.8 boa 292.0 qcab
280.0 qacb 253.0 qacb 292.0 ach 136.0 acbq 120.0 acqb
Overall mean
230.7
249.3
225.2
216.2
128.4
Feb.
1 2 3 4 5
37.0 2.0 20.0 4.0 676.0
98.0 ahabd 9.0 hqba 11.0 abcd 13.0 acqdb 637.0 daq
43.0 accb 87.0 caqb 383.0 abc 138.0 cab 103.0 qac
58.0 cbqa 105.0 acjbq 443.0 acbqj 82.0 qabcj 148.0 caq
96.0 aqbc 156.0 acbq 170.0 bacq 138.0 bacqjc 104.0 qajcb
Overall mean
147.8
153.6
150.8
167.2
132.8
11.0 aqfbj 40.0 iaq 3.0 aqb 99.0 baqfc UC
71.0 2.0 3.0 23.0 UC
139.0 acfdq 188.0 acbqfd 257.0 bcaf 215.0 qadcbf 280.0 cadq
154.0 afcdq 144.0acfdqb 140.0 cbqda 194.0 caafq 43.0 cqbadf
132.0 dabfcq 123.0acfb 148.0 qcafb 170.0 acbfqd 33.0 dcfaq
165.4
135.0
121.2
March
1 2 3 4 5
Sample (mean cfu/ml)
1 2 3 4 5
Overall mean
38.3
abah qbh adbcq a dabcq
24.8
abf ac qba qabc
355.0 aqcb
124.0 qcb
135.0 acqb 84.0 acbq 106.0 ~'q
* Letters indicate the species of Bacillus isolated and are coded as in Table II; UC, Spreading colony made sample uncountable; NS, no sample.
licheniformis a n d B. subtilis i s o l a t e s . Bacillus firmus a g a i n d i d n o t i n h i b i t psyc h r o t r o p h g r o w t h ( F i g s . 3a a n d b). S t u d i e s w e r e also c a r r i e d o u t with c o n t e m p e r a n e o u s lawns of the four psyc h r o t r o p h i c i s o l a t e s . M e s o p h i l i c i s o l a t e s d i d n o t g r o w r e a d i l y at 6 o r 21°C a n d o n l y s m a l l a r e a s o f i n h i b i t i o n w e r e s e e n w i t h two B. subtilis m e s o p h i l i c i s o l a t e s (Fig. 4), while
two B. licheniformis i s o l a t e s s h o w e d n o i n h i b i t i o n . A t 3 0 ° C t h e m e s o p h i l i c
i s o l a t e s g r e w r e a d i l y a n d b o t h i s o l a t e s o f B. subtilis s h o w e d s u b s t a n t i a l z o n e s o f i n h i b i t i o n w i t h all f o u r p s y c h r o t r o p h i c i s o l a t e s . T h e t w o i s o l a t e s o f B. licheniformis a n d o n e o f B. firmus a n d B. circulans h a d n o e f f e c t o n c o n t e m p o r a n e o u s p s y c h r o t r o p h l a w n s a t t h i s t e m p e r a t u r e ( a n e x a m p l e is s h o w n in Fig. 5). S p e n t b r o t h f r o m N B c u l t u r e s o f b o t h m e s o p h i l i c i s o l a t e s o f B. subtilis a n d B.
licheniforrnis w e r e m i x e d w i t h a c u l t u r e o f o n e p s y c h r o t r o p h i c B. cereus i s o l a t e ( H R M 44) i n a B a c t o m e t e r . C o n t r o l s a m p l e s o f H R M 44 in N B g r e w at 3 0 ° C w i t h a d e t e c t i o n t i m e ( d t ) o f 7.7 h in t h e B a c t o m e t e r e x p e r i m e n t ( r e s u l t s n o t s h o w n ) . Bacillus cereus H R M 44 d i d n o t g r o w at all in t h e p r e s e n c e o f s p e n t b r o t h f r o m o n e
285 T A B L E II Summary of mesophilic Bacillus species isolated from milk samples (June 1991 - March 1992) Code
Species
No. of samples from which individual species were isolated (total no. of samples = 250)
a b c d e f g h i j k 1 m n o p q
B. licheniformis B. pumilus B. subtilis B. lentus B. pantothenticus B. amyloliquifaciens B. stearothermophilus B. mycoides B. cereus B. circulans B. firmus B. megaterium B. sphaericus B. macerans B. laterosporus B. Polymyxa Unknown
131 156 127 52 33 25 19 22 16 15 10 27 6 3 2 5 120
B. subtilis mesophilic isolate (IIRM 57) while growth was substantially inhibited in the presence of spent broth from the other B. subtilis isolate and both B. licheniformis isolates with dt of 22.1, 12.2 and 11.8 h, respectively (Results not shown).
300
250
200 E = u 150 o E I O0 l
5oi 0
'
Jun •
Jul
Aug Sep
Oct
Nov Dec
Jan
Feb
Mar
milk machine ~-~ bulk tank [ ] tanker [ ] silo [ ] pasteurised milk
Fig. 1. M e a n seasonal count of mesophilic Bacillus sp. Samples were taken from five farms and five dairies.
286 T A B L E IIl Seasonal study of aerobic psychrotrophic spores in raw milk (pre-incubated count) (August 1991-March 1992) * Month
Farm/
Sample (mean c f u / m l )
dairy
Milking machine
Bulk tank
Tanker
Silo
Aug.
1 2 3 4 5
0 0 0 0 0
0 0 0 0 0
3.2x 1.0x 3.3x 6.0x 1.7x
4.6x 6.0x 6.0x 4.2x 6.0x
Sep.
1 2 3 4 5
3 . 7 x 10 sa'+ 0 0 0 1.3 × 104 ~
3 . 4 x 104~' 0 0 0 0
Oct.
1 2 3 4 5
0 4 . 6 x 105b 0 1.3X 106b 5.5 X 103a
0 3 . 5 x 104~' 0 0 1.7X 106~
104a'+ 104a 102" 105a 104a
Postpasteurisation 103a 105= 10 s~l 104a 105a
1.9x 103a 6 . 0 × 1 0 s~ 4 . 5 x 10 s~ 6 . 0 x 105a 1.3× 10 s '
0 2 . 6 x 104~' 6 . 4 x 104a 3 . 0 x 104~' 0
0 6 . 0 x 104a 3 . 7 x 104~ 6 . 8 x 104" 0
0 5 . 7 x 104~' 1.2x 104a 0 0
0 7.0× 106a 0 7.8×105a 1.0×107a
0 3 . 3 x 106a 0 2.6X 106a 1.0× 107a
2.8 x 10 ~' " 4 . 6 x 106a 1.4 x 106 b~ 2.2X 106be 1.0xl07~'
* No psychrotrophic Bacillus sp. were isolated in any sample taken during November 1991 to March 1992. + Letters indicate the species of Bacillus isolated and are coded as in Table IV.
8
6
i1 c~4
0i Aug
_ Sep
Oct
Nov
Dec
Jan
Feb
Mar
• milk machine ~ bulk tank [~ tanker ~ silo ~ paeteurised milk
Fig. 2. M e a n seasonal count of psychrotrophic Bacillus sp. recovered from milk samples after pre-incubation at 6°C for 7 days. Samples were taken from five farms and five dairies.
287 TABLE IV Summary of psychrotrophic Bacillus species isolated from milk samples June 1991-March 1992 Code
Species
No. of samples from which individual species were isolated (total no. of samples = 200)
a b c
B. cereus B. polymyxa B. circulans
39 4 1
Fig. 3. A streak plate of established mesophilic Bacillus growth (A, B. licheniformis HRM 13; B, B. licheniformis HRM 14; C, B. subtilis HRM 57; D, B. subtilis HRM 79; E, B. firmus NCDO 1752) across which has been streaked psychrotrophic Bacilli cultures (1, B. cereus HRM 44; 2, B. cereus MRM 652; 3, B. cereus GTE 257; 4, B. pumilus PM 20) which were then grown at 6°C. Mesophilic isolates of B. licheniformis showed some inhibition and B. subtilis substantial inhibition while B. firmus showed no inhibition towards growth of the four psychrotrophic isolates.
Fig. 4. Spots of mesophilic B. subtilis cultures (A, HRM 57; B, HRM 79) showing inhibitory activity when grown contemporaneously with a lawn of psychrotrophic B. cereus (HRM 44) culture at 6°C.
-
b c d e
Method of analysis
6
L
21
H R M 13 b
30
6
_+ +
-_ _
_
_
_ _
± +
30
6
i _+
+
_+
21
H R M 57 ~
21
H R M 14 b
Contemporaneous lawn culture
+ +
+
+
30
= n o inhibition; _+ = visible inhibition; + = s u b s t a n t i a l inhibition.
N o inhibition w a s s e e n w i t h B. circulans a n d B. f i r m u s isolates by a n y m e t h o d . B. licheni]ormis. B. subtilis. B. cereus. B. pumilis.
P M 20 °
T e m p e r a t u r e (°C): Psychrotrophic isolate: HRM44 d MRM62 d G T E 257 d
Mesophilic isolate:
A n t a g o n i s m of m e s o p h i l i c Bacillus isolates a for p s y c h r o t r o p h i c Bacillus isolates
TABLE V
_+ +
_+
+
6
± _+
_+
+
21
H R M 79 c
+ +
+
+
30
+ +
+
+
6
6
+ + + +
+ + + +
+ + + +
21
H R M t4
21
H R M 13
+ + + +
6
+ + + +
21
H R M 57
Psychrotrophic lawns around established mesophile growth
+ + + +
+ + + +
21
H R M 79 6
289
Fig. 5. An example of spots of mesophilic Bacilli cultures (A, B. subtilis HRM 57; B, B. licheniformis HRM 13; C, B. licheniformis HRM 14; D, B. subtilis HRM 79; E, B. firmus NCDO 1752) grown contemporaneously with a lawn of psychrotrophic B. cereus (HRM 44) culture at 30°C. Both isolates of B. subtilis were shown to inhibit the growth of the psychrotrophic isolate.
Discussion
Mesophile spore counts in both raw milk and pasteurised milk samples were generally higher in the winter months (November to March, inclusive) than in the summer/autumn months (June to October, inclusive). This coincident increase in counts in both raw and pasteurised milk samples suggests that raw milk is likely to be a source of spores which survive pasteurisation. Generally, the increase in counts in the winter period occurred throughout the sampled areas, i.e., in the farm milk machine, bulk tank, milk tankers, dairy silos and pasteurised milk samples. Increases, however, were first detectable in tanker, silo and pasteurised milk samples. This possibly reflects growth of the mesophilic sporeformers in the latter part of the milk chain including tanker surfaces and on dairy equipment or indicates additional sources of contamination at these points as suggested by others (Canon, 1972; Stewart, 1975). Increased mesophilic spore counts from farm raw milk in winter has been detected previously by others (Waes, 1976; Johnston and Bruce, 1983; McKinnon and Pettipher, 1983) and increased mesophilic spores on udder surfaces due to contact with contaminated winter bedding has been implicated as a cause of this. The three most frequently isolated mesophilic sporeformers over the sampling period were B. pumilus, B. licheniformis and B. subtilis. These same three species were also the most frequent isolates from previous surveys of raw milk in Scotland (Phillips and Griffiths, 1986) and elsewhere (Martin, 1981). The predominant psychrotrophic species isolated was B. cereus and this again agreed with the findings of others (Coghill and Juffs, 1979; Johnstone and Bruce, 1982; Sharma et al., 1984; Phillips and Griffiths, 1986; Griffiths and Phillips, 1990). This noticeable consistency in the predominance of these organisms in milk might
290 suggest factors exist, such as increased numbers of these organisms in the environment, an increased ability to adhere to cow teat surfaces or faster growth rates compared to other Bacillus sp. which would preferentially increase counts of these organisms in milk. The seasonal distribution of samples containing psychrotrophic sporeformers was quite distinct. Psychrotrophic strains were found in samples during the late s u m m e r / a u t u m n period. This again was in agreement with the findings of others (Phillips and Griffiths, 1986). McKinnon and Pettipher (1983) found that psychrotrophic sporeformers represented about 22% of the total spore count derived from summer pasture samples. This suggested that the seasonal incidence of psychrotrophic sporeformers was due to these organisms dominating an environmental niche which milking cows were exposed to when on summer pasture. The distinct and exclusive seasonal distribution between mesophilic and psychrotrophic spore populations found in the work reported here suggested, however, that an additional reason for seasonal occurrence may also exist. It was thought conceivable that large populations of mesophilic sporeformers may have antagonistic effects upon psychrotrophc sporeformers which prevented their growth and multiplication either in environmental sources or in raw milk. To examine this possibility the incidence of individual mesophilic sporeformers (Table I) was determined and species which were absent or were present in low numbers in the s u m m e r / a u t u m n months (namely B. subtilis, B. licheniformis and B. circulans) were examined for their influence on the growth of psychrotrophic sporeformers. Bacillus firmus which was more common in the summer than in the winter was also studied as a control which would be unlikely to inhibit psychrotroph growth. Since B. cereus was by far the most common psychrotrophic sporeformer isolated in the survey, three isolates of this species and one isolate of B. pumilus were included to look for species and isolate variations. No differences were seen in the interactive effects of mesophiles between any of the psychrotroph isolates tested. Established cultures of mesophilic B. subtilis and B. licheniformis were shown in agar plate studies to produce factors which inhibited the growth of psychrotrophic B. cereus and B. pumilus isolates grown at temperatures as low as 6°C. The inhibitory factors could have been produced when mesophilic growth was established at 30°C but this method at least showed that these inhibitory factors were active at 6°C. The streak method on agar plates offered a rapid, low cost method of screening mixed cultures for antagonistic effects. Bactometer studies indicated that inhibitory factors were extracellular. Bacillus subtilis was found to be more inhibitory than B. Iicheniformis in the present examinations. When lawns of the psychrotrophic isolates were spotted with contemporaneous cultures of mesophilic isolates only B. subtilis showed inhibitory effects. This effect was more pronounced at 30°C than at 21 or 6°C which probably reflected the poor levels of growth by B. subtilis at the lower temperatures. Bacillus licheniformis showed no inhibitory effects on psychrotroph growth even at 30°C. This suggested
291 t h a t t h e i n h i b i t o r y f a c t o r d e t e c t e d in e s t a b l i s h e d c u l t u r e s of B. licheniformis was n o t p r o d u c e d quickly e n o u g h in p o t e n t q u a n t i t i e s to inhibit c o n t e m p o r a n e o u s p s y c h r o t r o p h g r o w t h w h e n s t u d i e d by this m e t h o d . It is h y p o t h e s i s e d t h a t i n h i b i t o r y factors c o u l d p l a y a role in influencing t h e s e a s o n a l i n c i d e n c e o f p s y c h r o t r o p h i c Bacilli in milk by i n f l u e n c i n g t h e relative p r o p o r t i o n s of m e s o p h i l i c a n d p s y c h r o t r o p h i c Bacilli e i t h e r in the f a r m environm e n t (e.g., soil) o r in raw milk. M o r e work, however, is r e q u i r e d to d e t e r m i n e directly h o w t h e s e a n t a g o n i s t s affect t h e g r o w t h of p s y c h r o t r o p h i c Bacilli in e n v i r o n m e n t s such as soil a n d c o l d - s t o r e d milk. It w o u l d also b e o f value to d e t e r m i n e if t h e most c o m m o n l y i s o l a t e d m e s o p h i l i c a n d p s y c h r o t r o p h i c Bacillus sp. can p r o d u c e factors which r e d u c e the i n c i d e n c e a n d growth o f o t h e r less c o m m o n Bacillus sp. in milk. T h e n a t u r e o f t h e a n t a g o n i s t i c factors d e s c r i b e d h e r e a r e u n k n o w n . Bacillus subtilis p r o d u c e s m e t a l l o a n d s e r i n e p r o t e a s e s , the a n t i b i o t i c mycobacillin a n d has s u r f a c t a n t activity which can lyse r e d b l o o d cells (Priest, 1989). Bacillus fichenif o r m & also p r o d u c e s p r o t e a s e s (Priest, 1989) a n d t h e b a c i t r a c i n antibiotics (Katz a n d D e m a i n , 1977). S o m e o f t h e s e factors m a y b e a n t a g o n i s t s a l o n e o r in c o n c e r t with e a c h o t h e r o r u n d e t e r m i n e d factors m a y b e involved. F u r t h e r r e s e a r c h into d e f i n i n g t h e a n t a g o n i s t i c factors w o u l d b e o f interest.
References Cannon, R.Y. (1972) Contamination of raw milk with psychrotrophic sporeformers. J. Dairy Sci. 55, 669. Christiansson, A., Naidu, A.S., Nilsson, I., Wadstrom, T. and Pettersson, H.-E. (1989) Toxin production by Bacillus cereus dairy isolates in milk at low temperatures. Appl. Environm. Microbiol. 55(10), 2595-2600. Coghill, D. and Juffs, H.S. (1979) Incidence of psychrotrophic sporeforming bacteria in pasteurised milk and cream products and effect of temperature on their growth. Australian J. Dairy Technol. 34, 150-153. Griffiths, M.W. (1990) Toxin production by psychrotrophic Bacillus species present in milk. J. Food Protect. 53(9), 790-792. Griffiths, M.W. and Phillips, J.D. (1990) Incidence, source and some properties of psychrotrophic Bacillus sp. found in raw and pasteurised milk. J. Soc. Dairy Technol. 43(3), 62-66. Johnstone, D.W. and Bruce, J. (1982) Incidence of thermoduric psychrotrophs in milk produced in the west of Scotland. J. Appl. Bacteriol. 52, 333-337. Katz, E. and Demain, A.L. (1977) The peptide antibiotics of Bacillus: chemistry biogenesis and possible functions. Bacteriol. Rev. 41,449-474. Kramer, J.M. and Gilbert, R.J. (1989) Bacillus cereus and other Bacillus species. In: Foodborne Bacterial Pathogens, Doyle, M.P. (Ed.) Marcel Dekker, NY. pp. 22-70. Logan, N.A. and Berkeley, R.C.W. (1984) Indentification of Bacillus strains using the API system. J. Gen. Microbiol. 130, 1871-1882. Martin, J.H. (1981) Heat resistant mesophilic microorganisms. J. Dairy Sci. 64, 149-156. McKinnon, C.H. and Pettipher, G.L. (1983) A survey of sources of heat resistant bacteria in milk with particular reference to psychrotorphic spore-forming bacteria. J. Dairy Res. 50, 163-170. Phillips, J.D. and Griffiths, M.W. (1986) Factors contributing to the seasonal variation of Bacillus sp. in pasteurised dairy products. J. Appl. Bacteriol. 61,275-285.
292
Phillips, J.D., Griffiths, M.W. and Muir, D.D. (1981) Growth and associated enzymic activity of spoilage bacteria in pasteurised double cream. J. Soc. Dairy Technol. 34, 113-118. Priest, F.G. (1989) Products and applications. In: Bacillus, Harwood, C.R. (Ed.), Plenum Press, NY and London. pp. 293-320. Sharma, J.K., Malik, R.K. and Mather, D.K. (1984) Isolation and identification of proteolytic psychrotrophic sporeforming bacteria from raw milk supplies at an experimental farm in India. J. Soc. Dairy Technol. 37, 96-98. Stewart, D.B. (1975) Factors influencing the incidence of B. cereus spores in milk. J. Soc. Dairy Technol. 28, 80-85. Waes, G. (1976) Aerobic mesophilic spores in raw milk. Milchwissenschaft 31,521-524.