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Heat Resistant Mesophilic Microorganisms1
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEM Heat Resistant Mesophilic Microorganisms t J. H. MARTIN Dairy Science Department Clemson University Clemson, SC 29631
A number of factors are encouraging development of commercial sterilization of fluid milk. First, delivery costs are growing rapidly, and no end appears in the forseeable future. Second, costs for processing are accelerating, and these costs could be decreased by processing two or three times per week instead of every day. Finally, energy costs for cold storage of milk in processing plants, delivery trucks, and in grocery stores could be reduced or eliminated if milk could be stored unrefrigerated. Of course, when this approach for milk storage is considered, mesophilic rather than psychrophilic microorganisms become of paramount importance. And, because of the detrimental effects of high heat-long time sterilization of milk, the most likely heat treatment of choice will be ultra-high-temperature (UHT) pasteurization. When the UHT process is applied - whether it be by direct steam injection or by indirect heat exchange - the organisms of greatest concern are those with the highest degree of heat resistance, namely, the aerobic sporeforming bacteria. In 1974, a study concerned thermoduric types o f bacteria commonly in farm bulk tank milk (9). Results are summarized in Table 1. The organisms included streptococci, which included Streptococcus faecatis, S. tbermopbilus, and S. bovis; micrococci, including the species Micrococcus luteus and M. varians; Corynebacterium lacticum; some unidentified Lactobacitlus species; Alcaligenes tolerans; and most aerobic sporeformers including Bacillus cereus, B. subtilis, B. licbeniformis, and B. circulans. That so many of the common microorganisms in milk are thermoduric is of great concern, especially if they survive the common uhra-high-temperature treatments. Another study from England revealed the incidence of
Received December 7, 1979. Technical Contribution No. 1733 from the South Carolina Agricultural Experiment Station, Clemson, SC. 1981 J Dairy Sci 64:149-156
the different types of thermoduric bacteria in farm bulk tank milk supplies (10). These data (Table 2) revealed that aerobic spore forming rods comprised 27 to 42% of all cultures isolated and identified in two separate studies. Next, the micrococci comprised approximately 38% of the total cultures isolated. Streptococci and corynebacteria were the next most common, with 13 to 16% of the cultures falling in these two types. Also, a study in 1972 (2) revealed that the microbial flora of commercially pasteurized milk stored for 30 days at refrigeration temperature consisted of 84% Bacillus organisms (Table 3). Since micrococci are less heat resistant than Bacillus spores, the data confirm that organisms of greatest concern are aerobic sporeforming bacteria. Several studies to determine the incidence of sporeformers in milk have been conducted around the world. With the possible exception of some early work by US Department of Agriculture investigators in the 1920's, our work at Ohio State University in 1962 was the initial effort to determine the incidence of spores in milk in this country (5). Spore counts in 176 samples of Ohio grade A and manufacturing grade milk ranged from about 400 spores/ml to 760 spores/rot (Table 4). High count milk (manufacturing grade) did not always contain significantly greater number of spores, although it did tend to contain more spores than did low count Grade A milk. A total of 155 of the milk samples contained spores, which was 85% of all milk analyzed. Specific organisms isolated and identified in the Ohio study included: B. licheniformis, B. cereus, B. coagulans, B. purnilis, B. spbaericus, B. cereus var. mycoides, B. brevis, B. laterosporus, B. circulans, and B. stearothermopbilus (Table 5). Only 14 (4.7%) of approximately 300 isolates from milk were anaerobic, and these organisms were not identified. Aerobic sporeformers were of greatest significance. Several other studies have been conducted in different parts o f the world since the 1962 work. Workers in India in 1974 (1) showed B. subtilis, 149
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TABLE 1. Types of bacteria commonly found in farm bulk tank milk (9). Types of bacteria
Thermoduric species
Streptococci
S. faecalis S. thermophilus S. bovis M. luteus M. varians
Micrococci
Corynebacteria Other asporogenous gram positive rods Other anaerogenic gram negative rods Aerobic sporeforming rods
Streptomycetes
C. lacticum Lactobacillus A. tolerans B. cereus B. subtilis B. licbeniJbrmis B. circulans Other species Occasional
B. cereus, B. pumilis, B. megaterium, B. licheniformis, B. coagulans, and B. stearothermophilis were the m o s t c o m m o n sporeformers in Indian milk (Table 6). Of these, B. subtilis and B. licheniJbrmis exhibited both mesophilic and t h e r m o p h i l i c growth characteristics, and B. subtilis, B. cereus, and B. stearothermophilus were in 70% o f the samples in a b o u t equal proportions. Work in the Netherlands in 1974 c o m p a r e d the spore profile o f milk and cocoa (8). Results are in Table 7. Bacillus subtilis and B. licheniformis was p r e d o m i n a n t in milk with 51% of the isolates being identified as B. licheniformis. Bacillus species in milk, and a p p r o x i m a t e l y 43% of the Bacillus species in cocoa. Bacillus licheniformis were p r e d o m i n a n t in milk with 51% of the isolates being identified as B. licbeniforrnis. Cocoa also contained a large percentage of B. licheniformis, with 40% of the isolates being B. licheniformis. Bacillus pumilus, B. alvei, B. megaterium, and B. stearotherrnophilus also were in cocoa and B. pumiIus, B. alvei, B. sphaericus, and B. cereus in milk, but they appeared m u c h less frequently than B. subtilis and B. licheniformis. In 1975 a c o m b i n e d research study by the National Institute for Research in Dairying and Journal of Dairy Science Vol. 64, No. 1, 1981
TABLE 2. Incidence of different types of thermoduric bacteria in farm bulk tank milk supplies (10).
Types of thermoduric bacteria
Streptococci Micrococci Corynebacteria Other gram-positive nonsporing rods Gram-negative rods Aerobic sporeforming rods Streptomycetes Unclassified
Incidence of cultures Thomas Buchman et al. (1961) a (1967) b ~ (%) 15.9 38.4 8.6
1.1 39.5 13.4
7.0 0 27.4 2.5 .2
2.8 1.0 41.9 0 .3
No. of cultures
986
709
No. of milk samples
100
30
acultures isolated at 32°C & 35°C. bCultures isolated at 30°C.
the Food and Agriculture Organization was conducted to d e t e r m i n e the sporeforming species in milk f r o m countries where high average temperatures prevail (3). Bacillus subtilis (48.5%), B. licheniformis (16.3%), and B. cereus (11.85%) were the m o s t c o m m o n species with B. lentus, B. pumilus, B. coagulans, B. stearotbermophilus, B. circulans, B. firmus, B. brevis, B. megaterium, B. laterosporus, and B. macerans comprising the remainder of the spore flora of milk f r o m these countries (Table 8). It appears that essentially the same species of sporeforming microorganisms are involved
TABLE 3. Microbial flora of commercially pasteurized milk following 30 days of storage at 4.5°C (2).
Species
Percent of total isolations
Bacillus Microbacterium Micrococcus Acbromobacter Alcaligenes Streptococcus
84 9 2 2 2 I
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS
151
TABLE 4. Standard plant counts and aerobic spore counts of raw milk (5). Mesophilic spore counts a
Class ! 11 I11 IV
SPC range (per ml) < 50,000 >50,000 <200,000 >200,000 < 1,000,000 >1,000,000 < 5,000,000
No. of samples analyzed
Avg SPC (per ml)
Thermophilic spore counts a
Average (per ml)
No. of positive samples
Average (per ml)
19
32,000
400
16
46
36
98,000
400
35
45
48
580,000
710
40
55
73
2,300,000
760
60
41
aSpore counts were determined after heating milk at 80°C for 10 rain. Mesophilic counts were determined by a pour plate procedure; thermophilic counts by a most probable number dilution tube technique.
worldwide with the m o s t c o m m o n being B. subtilis, B. licbeniformis, and B. cereus. The only major difference appears to be different p r o p o r t i o n s in some countries f r o m others. What a b o u t the h e a t resistance of the different species? Our w o r k in 1966 evaluated effects of three U H T t r e a t m e n t s on spores of the various species isolated f r o m Ohio milk (6). Results are summarized in Tables 9, 10, and 11. The temperatures were 104.5°C, 121°C, and 137.8°C. Holding time was approxim a t e l y 1 s in all cases. A p p r o x i m a t e l y o n e million spores per milliliter were added to raw milk, which then was pasteurized by indirect U H T heating for 1 s at the different U H T temperatures. Percent destruction at 104.5°C ranged from n o n e with B. laterosporus to 50% with B. p u m i l u s . The t e m p e r a t u r e t r e a t m e n t of 121°C was only 97.7% effective with B. licbeniformis, 98.4% with B. laterosporus, and 98.7% with B. megaterium. At 13 7.8°C the percentage destruction was at least 99.99% with all organisms tested, but surviving spores ranged f r o m 2/ml for B. sphaericus to 15/ml for B. megaterium. The data indicate that any UHT t r e a t m e n t selected must be greater than a c o m b i n a t i o n of 137.8°C for 1 s to approach commercial sterilization. Otherwise, if certain sporeforming Bacillus organisms are in the milk, there is a very strong possibility some will survive. Our idea at the t i m e was that possibly UHT-treated milk could be stored w i t h o u t
refrigeration, so we determined the recovery and o u t g r o w t h o f the surviving spores at 35°C, a t e m p e r a t u r e approaching the o p t i m u m for m o s t of the Bacillus species. Results are presented also in Tables 9, 10, and 11. The spores that survived germinated and grew rapidly in the UHT-treated milks. The vegetative cell c o u n t for all species was in the millions within 6 h in the milk heated at 104.5°C. Counts for spores also were very high in this milk, ranging f r o m 56,000/ml for B. p u m i l u s to 1,050,000/ml
TABLE 5. Organisms isolated from raw milk supplies (4). Organism B licheniformis a B. cereus B. coagulans B. pumilus B. sphaericus B. cereus var. mycoides B. brevis B. laterosporus B. circulans B. stearotbermopbilus Clostridium (species unidentified)
Total
Number isolated 129 111 11 10 6 5 4 3 2 2
% Total 43.3 37.4 3.7 3.4 2.0 1.7 1.3 1.0 .7 .7
14
4.7
297
100.0
aDifferentiated from Bacillus subtilis by anaerobic growth in glucose broth and by colony characteristics on nutrient agar. Journal of Dairy Science Vol. 64, No. 1, 1981
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MARTIN
TABLE 6. Distribution of spore forming bacterial isolates according to milk samples (1). Source of samples Bacillus species
Incubation Temp (°C)
Organized dairy farm Cattle Bulk milk yard from dairy
Pasteurized milk
Market milk
37 37
50 45
20 34
22 38
37
7
2
•, .
46 26 6
37 37 55 55 55 63
3 10 ... 15 5 20
1 7 9 10 3 24
... 11 7 I5 9 45
Commercial dairy
Total number .
B. B. B. B. B. B. B. B. B.
subtilis cereus pumilus megaterium licbeniformis subtilis coagulans licheniformis stearotbermopbilus
7 5 1
145 148 16
5
...
9
4
6 ...
38 23
2
67
7
25 I
...
18
14
22
125
for B. circulans. A f t e r i n c u b a t i o n at 35°C for 6 h, s p o r e c o u n t s were in the 10's of t h o u s a n d s in milk h e a t e d at 104.5°C for 1 s. F o r t h e milk t r e a t e d at 121°C, the n u m b e r s surviving were m u c h lower, b u t t h o s e s p o r e s t h a t survived g e r m i n a t e d a n d grew rapidly in t h e milk i n c u b a t e d at 35°C. N u m b e r s of vegetative cells o f B. l i c b e n i f o r m i s , B. p u m i l u s , B. cereus var m y c o i d e s , B. laterosporus, a n d B. m e g a t e r i u m were all in the millions at the e n d of t h e 6-h i n c u b a t i o n . A t t h e highest t e m p e r a t u r e used, 137.8°C, t h e p e r c e n t d e s t r u c t i o n was 99.9% or g r e a t e r for all species tested. However, t h e few t h a t did survive, with the e x c e p t i o n o f B. l i c h e n i f o r m i s , g e r m i n a t e d a n d grew to sigonificant n u m b e r s in t h e U T H milk s t o r e d at 35 C for 6 h. T h e d a t a i n d i c a t e t h a t p r o x i m a t e d e s t r u c t i o n of Bacillus spores will n o t suffice if milk is to be s t o r e d
unrefrigerated. The h e a t i n g process m u s t prod u c e an a b s o l u t e l y sterile p r o d u c t for storage at r o o m t e m p e r a t u r e t o b e successful. We also calculated t h e n u m b e r of g e n e r a t i o n s d u r i n g t h e 6 h at 35°C to d e t e r m i n e if t h e h e a t t r e a t m e n t was s t i m u l a t o r y to s p o r e g e r m i n a t i o n a n d s u b s e q u e n t g r o w t h . The d a t a are in T a b l e 12. T h e t e m p e r a t u r e o f 1 2 1 ° C was s t i m u l a t o r y to all species, with t h e n u m b e r o f g e n e r a t i o n s d u r i n g 6 h o f i n c u b a t i o n a f t e r h e a t i n g at this t e m p e r a t u r e always m o r e t h a n t h e n u m b e r at 104.5°C. F o r a few species, B. spbaericus, B. m e g a t e r i u m , B. laterosporus, a n d B. circulans, t h e rate o f g r o w t h was s t i m u l a t e d even m o r e at
TABLE 7. Species of Bacillus isolated in the survey (8).
Species
Number isolated
Percentage of total isolates
B. B. B. B. B. B. B. B. B. B. B. B. B.
131 44 32 14 13 13 7 4 4 3 2 2 1
48.52 16.30 11.85 5.15 4.81 4.81 2.56 1.49 1.49 1.13 .76 .76 .37
% Total isolates
B. B. B. B. B. B. B. B.
subtilis licbeniformis pumilus alvei nlegaterium stearotbermopbilus sphaericus cereus
Cocoa
Milk
42.8 40.0 8.5 2.8 2.8
35.8 50.9 5.7 4.7
2.8 .... ....
Journal of Dairy Science Vol. 64, No. 1, 1981
.... ....
1.9 3.8
TABLE 8. Total number and relative proportions of species isolated from milks of all countries in NIRD/ FAO survey (3).
subtilis licbeniformis cereus lentus pumilus coagulans stearothermopbilus circulans litmus brevis megaterium macerans laterosporus
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS
153
TABLE 9. Effect of UHT treatment at 104.5°C on bacterial spores (6).
Species
Spore count/ml a Before After U HT U HT
Approximate percent destruction
UHT milk after 6 h at 35°C Vegetative cell Spore count/ml count/ml
B. B. B. B. B. B. B. B. B.
767,000 547,000 813,000 803,000 870,000 1,530,000 1,240,000 1,190,000 957,000
50 45 45 27 25 19 16 4 0
28,200,000 28,000,000 43,500,000 30,300,000 34,600,000 18,700,000 22,300,000 71,000,000 38,000,000
pumilus coagulans spbaericus cereus var. mycoides cereus circutans licbeniformis megateriurn laterosporus
381,000 301,000 450,000 583,000 657,000 1,240,000 1,050,000 1,140,000 1,060,000
55,700 87,400 68,300 483,000 497,000 1,050,000
363,000 827,000 687,000
aAverage of three trials. Before addition of spores, the spore count of the raw milk was less than 60/ml.
O
1 3 7 . 8 C. T h e s p o r e s t h a t survived g e r m i n a t e d a n d grew f a s t e r at 35°C t h a n did t h e survivors f r o m e i t h e r 104.5°C or 1 2 1 ° C heat treatment. T h e s e d a t a i n d i c a t e t h a t certain U H T t e m p e r a t u r e t r e a t m e n t s a c t i v a t e t h e bacterial spore so t h a t it g e r m i n a t e s a n d t h e vegetative cells r e p r o d u c e faster t h a n cells w h i c h have n o t b e e n s u b j e c t e d t o high h e a t t r e a t m e n t s . A n y U H T t r e a t m e n t utilized c o m m e r c i a l l y m u s t be in excess o f a n y s t i m u l a t o r y t e m p e r a t u r e t h a t
m i g h t r e d u c e n u m b e r s drastically, b u t t h a t m i g h t cause t h e few survivors to grow even m o r e rapidly t h a n n o r m a l . W o r k p u b l i s h e d in 1 9 7 8 revealed t h a t raw m i l k o f average m i c r o b i a l q u a l i t y h e a t e d a t 8 0 ° C f o r 12 rain still c o n t a i n e d viable organisms a f t e r this h e a t t r e a t m e n t (7). S u m m a r y d a t a are in T a b l e 13. T h e r e were 170 m e s o p h i l i c a n d 7 p s y c h r o t r o p h i c o r g a n i s m s surviving per milliliter o f milk, a n d 7 6 / m l surviving t h a t were interm e d i a t e in g r o w t h t e m p e r a t u r e b e t w e e n m e s o -
TABLE 10. Effect of UHT treatment at 121°C on bacterial spores (6).
Species
Spore count/ml a Before After UHT UHT
Approximate percent destruction
B. B. B. B. B. B. B. B. B.
1,240,000 870,000 547,000 767,000 813,000 803,000 957,000 1,190,000 1,530,000
87.70 99.98 99.97 99.99
licbeniforrnis cereus coagulans pumilus spbaericus cereus vat. mycoides laterosporus megaterium circulans
28,000 139 171 62 38 76 14,900 15,600 147
99.99
99.99 98.40 98.70 99.99
UHT milk after 6 h at 35°C Vegetative cell Spore count/ml count/ml 1,370,000 119,000 111,000 1,490,000 651,000 3,730,000 7,970,000 29,700,000 285,000
6,630 102 158 49 33 33 4,690 10,100 194
aAverage of three trials. Before addition of spores, the spore count of the raw milk was tess than 60/ml. Journal of Dairy Science Vol. 64, No. 1, 1981
154
MARTIN
TABLE 11. Effect of UHT treatment at 137.8°C on bacterial spores (6). UHT milk after 6 h at 35°C Spore count/mla Species
Before UHT
After UHT
Approximate percent destruction
Vegetative cell eount/ml
Spore count/ml
B. licbeniformis B. cereus B. coagulans B. pumilus B. spbaericus B. cereus var. mycoides B. laterosporus B. megaterium B. circulans
1,240,000 870,000 547,000 767,000 813,000 803,000 957,000 1,190,000 1,530,000
6 8 3 8 2 9 6 15 3
99.99
99.99 99.99 99.99 99.99 99.99 99.99 99.99 99.99
29 5,800 1,200 85,000 73,300 44,300 57,600 518,000 35,100
13 8 9 11 6 5 11 3 17
aAverage of three trials. Before addition of spores, the spore count of the raw milk was less than 60/ml.
philic and psychrotrophic. A f t e r 7 days storage at 7°C, numbers were 1800 mesophilic, 700 intermediate, and 340 psychrotrophic organisms per milliliter of milk. Surely these organisms were sporeformers and the entity surviving the heating was the spore. We did m u c h w o r k with spores for several years and f o u n d nothing but spores surviving 80°C for 12 rain. This w o r k also points up the i m p o r t a n c e of c o m p l e t e destruction if U H T is to be successful. M e n t i o n e d earlier was the j o i n t study of
Bacillus species by the National Institute for
Research in Dairying and F A O f r o m countries with high average temperatures (3). The isolates f r o m these studies were subjected to f o u r temperatures, 100°C for 30 min, 110°C for 10 min, 120°C for 20 min, and 130°C for 10 min. Results are in Table 14. At least 20% of all the species isolated survived 100°C for 30 rain, with 21.9% of B. cereus surviving and ranging upwards to 100% for B. coagulans, B. brevis, B. megaterium, B.
TABLE 12. Effect of UHT treatment on outgrowth of Bacillus spores in milk (6). No. of generations during 6 h of incubation at 35°C after UHT treatment a at
B. licheniformis B. cereus B. coagulans B. pumilus B. spbaericus R cereus var. mycoides B. laterosporus B. megaterium B. circulans
Average no. of generations a
. final no. No. generations = 3.3 log 10 initial no.
Journal of Dairy Science Vol. 64, No. 1, 1981
104.5°C
121°C
137.8°C
4.4 5.7 6.5 2.9 6.6 5.7 5.1 5.9 3.9 5.2
5.6 9.7 9.3 14.5 14.0 15.5 9.0 10.8 10.8 11.0
2.3 9.4 8.6 13.3 15.1 12.2 13.1 15.0 13.4 11.4
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS TABLE 13. Microbial quality of raw milk (109 samples) of the same milk upon heat treatment at 80°C for 12 min and subsequent storage at 7°C (7). Sample and plating procedure Raw milk Standard plate count Gram-negative count
155
%E 0
0
0
0
0
0
0
0
0
0
0
0
0
Microbial numbers (average CFU/ml) v
110,000 72~000 e~
Heated milk (80°C-12 min)
eq
0
oooo~o~oooooo,~
Initial
Mesophilic Intermediate Psychrotrophic 7-Day storage (7°C) Mesophilic Intermediate Psychrotrophic
170 76 7
ca
e"
1,800 700 340 e" -t 0
laterosporus, and B. stearotbermopbilus. The 110°C for 10 min heat treatment reduced Bacillus cereus numbers by approximately 94% and completely eliminated B. laterosporus and B. macerans. However, all spores of B. coagulans and B. stearotbermopbilus survived the l l 0 ° C for 10 min heat treatment. Approximately 80% of the B. subtilis, 67% of the B. brevis, and 50% of the B. megaterium organisms survived the 110°C for 10 rain treatment. The 120°C for 20 min treatment inactivated all species except 15.4% of B. coagulans and 71.4% of the B. stearotbermopbilus organisms. Heat treatment of 130°C for 10 min resulted in negative counts for all species isolated from the various countries with high average temperatures. Therefore, certain species of sporeforming microorganisms are capable of surviving tremendous heat treatments. One wonders if any ultra-high-temperature treatment utilized to date would destroy consistently all spores that might possibly be in milk. Research should continue with the high heat resistant species as the major test organisms, and storage of milk on grocery shelves without refrigeration should be approached with caution until the timetemperature relationship for complete destruction has been successfully demonstrated many times over.
,"~0
} O 0
Z e~
! e~
[.,.
e~
Journal of Dairy Science Vol. 64, No. 1, 1981
156
MARTIN REFERENCES
1 Atwal, J. S., D. N. Slaroff, R a t t a n Chand, and R. A. Srinivasan. 1974. Studies on aerobic spore forming bacteria in milk and milk products. I. Occurrence in raw and pasteurized milk. Indian J. Dairy Sci. 27:22. 2 Credit, C., R. Hedeman, P. Heywood, and D. Westhoff. 1972. Identification of bacteria isolated from pasteurized milk following refrigerated storage. J. Milk Food Technol. 35:708. 3 Davies, F. L. 1975. Heat resistance of Baci2lus species. J. Soc. Dairy Technol. 28:69. 4 Martin, J. H. 1974. Significance of bacterial spores in milk. J. Milk Food Technol. 37:94. 5 Martin, J. H., D. P. Stahly, W. J. Harper, and
Journal of Dairy Science Vol. 64, No. 1, 1981
6
7
8
9 10
I. A. Gould. 1962. Spore forming microorganisms in selected milk supplies. Proc. 16th Int. Dairy Congr., Copenhagen, 295:304. Martin, J. H., W. J. Harper, and I. A. Gould. 1966. Ultra-high temperature effects on selected Bacillus species. J. Dairy Sc L 49:1367. Mikolajcik, E. M., and N. T. Simon. 1978. Heat resistant psychrotrophic bacteria in raw milk and their growth at 7C. J. Food Prot. 41(2):93. Mossel, D.A.A., E. H. Meursing, and H. Slot. 1974. An investigation on the n u m b e r s and types of aerobic spores in cocoa powder and whole milk. Neth. Milk and Dairy J. 28:149. T h o m a s , S. B. 1974. The microflora of bulk collected milk - Part 1. Dairy Ind. 39(7):237. T h o m a s , S. B. 1974. The microflora of bulk collected milk - Part 2. Dairy Ind. 39(8):279.
A number of factors are encouraging development of commercial sterilization of fluid milk. First, delivery costs are growing rapidly, and no end appears in the forseeable future. Second, costs for processing are accelerating, and these costs could be decreased by processing two or three times per week instead of every day. Finally, energy costs for cold storage of milk in processing plants, delivery trucks, and in grocery stores could be reduced or eliminated if milk could be stored unrefrigerated. Of course, when this approach for milk storage is considered, mesophilic rather than psychrophilic microorganisms become of paramount importance. And, because of the detrimental effects of high heat-long time sterilization of milk, the most likely heat treatment of choice will be ultra-high-temperature (UHT) pasteurization. When the UHT process is applied - whether it be by direct steam injection or by indirect heat exchange - the organisms of greatest concern are those with the highest degree of heat resistance, namely, the aerobic sporeforming bacteria. In 1974, a study concerned thermoduric types o f bacteria commonly in farm bulk tank milk (9). Results are summarized in Table 1. The organisms included streptococci, which included Streptococcus faecatis, S. tbermopbilus, and S. bovis; micrococci, including the species Micrococcus luteus and M. varians; Corynebacterium lacticum; some unidentified Lactobacitlus species; Alcaligenes tolerans; and most aerobic sporeformers including Bacillus cereus, B. subtilis, B. licbeniformis, and B. circulans. That so many of the common microorganisms in milk are thermoduric is of great concern, especially if they survive the common uhra-high-temperature treatments. Another study from England revealed the incidence of
Received December 7, 1979. Technical Contribution No. 1733 from the South Carolina Agricultural Experiment Station, Clemson, SC. 1981 J Dairy Sci 64:149-156
the different types of thermoduric bacteria in farm bulk tank milk supplies (10). These data (Table 2) revealed that aerobic spore forming rods comprised 27 to 42% of all cultures isolated and identified in two separate studies. Next, the micrococci comprised approximately 38% of the total cultures isolated. Streptococci and corynebacteria were the next most common, with 13 to 16% of the cultures falling in these two types. Also, a study in 1972 (2) revealed that the microbial flora of commercially pasteurized milk stored for 30 days at refrigeration temperature consisted of 84% Bacillus organisms (Table 3). Since micrococci are less heat resistant than Bacillus spores, the data confirm that organisms of greatest concern are aerobic sporeforming bacteria. Several studies to determine the incidence of sporeformers in milk have been conducted around the world. With the possible exception of some early work by US Department of Agriculture investigators in the 1920's, our work at Ohio State University in 1962 was the initial effort to determine the incidence of spores in milk in this country (5). Spore counts in 176 samples of Ohio grade A and manufacturing grade milk ranged from about 400 spores/ml to 760 spores/rot (Table 4). High count milk (manufacturing grade) did not always contain significantly greater number of spores, although it did tend to contain more spores than did low count Grade A milk. A total of 155 of the milk samples contained spores, which was 85% of all milk analyzed. Specific organisms isolated and identified in the Ohio study included: B. licheniformis, B. cereus, B. coagulans, B. purnilis, B. spbaericus, B. cereus var. mycoides, B. brevis, B. laterosporus, B. circulans, and B. stearothermopbilus (Table 5). Only 14 (4.7%) of approximately 300 isolates from milk were anaerobic, and these organisms were not identified. Aerobic sporeformers were of greatest significance. Several other studies have been conducted in different parts o f the world since the 1962 work. Workers in India in 1974 (1) showed B. subtilis, 149
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MARTIN
TABLE 1. Types of bacteria commonly found in farm bulk tank milk (9). Types of bacteria
Thermoduric species
Streptococci
S. faecalis S. thermophilus S. bovis M. luteus M. varians
Micrococci
Corynebacteria Other asporogenous gram positive rods Other anaerogenic gram negative rods Aerobic sporeforming rods
Streptomycetes
C. lacticum Lactobacillus A. tolerans B. cereus B. subtilis B. licbeniJbrmis B. circulans Other species Occasional
B. cereus, B. pumilis, B. megaterium, B. licheniformis, B. coagulans, and B. stearothermophilis were the m o s t c o m m o n sporeformers in Indian milk (Table 6). Of these, B. subtilis and B. licheniJbrmis exhibited both mesophilic and t h e r m o p h i l i c growth characteristics, and B. subtilis, B. cereus, and B. stearothermophilus were in 70% o f the samples in a b o u t equal proportions. Work in the Netherlands in 1974 c o m p a r e d the spore profile o f milk and cocoa (8). Results are in Table 7. Bacillus subtilis and B. licheniformis was p r e d o m i n a n t in milk with 51% of the isolates being identified as B. licheniformis. Bacillus species in milk, and a p p r o x i m a t e l y 43% of the Bacillus species in cocoa. Bacillus licheniformis were p r e d o m i n a n t in milk with 51% of the isolates being identified as B. licbeniforrnis. Cocoa also contained a large percentage of B. licheniformis, with 40% of the isolates being B. licheniformis. Bacillus pumilus, B. alvei, B. megaterium, and B. stearotherrnophilus also were in cocoa and B. pumiIus, B. alvei, B. sphaericus, and B. cereus in milk, but they appeared m u c h less frequently than B. subtilis and B. licheniformis. In 1975 a c o m b i n e d research study by the National Institute for Research in Dairying and Journal of Dairy Science Vol. 64, No. 1, 1981
TABLE 2. Incidence of different types of thermoduric bacteria in farm bulk tank milk supplies (10).
Types of thermoduric bacteria
Streptococci Micrococci Corynebacteria Other gram-positive nonsporing rods Gram-negative rods Aerobic sporeforming rods Streptomycetes Unclassified
Incidence of cultures Thomas Buchman et al. (1961) a (1967) b ~ (%) 15.9 38.4 8.6
1.1 39.5 13.4
7.0 0 27.4 2.5 .2
2.8 1.0 41.9 0 .3
No. of cultures
986
709
No. of milk samples
100
30
acultures isolated at 32°C & 35°C. bCultures isolated at 30°C.
the Food and Agriculture Organization was conducted to d e t e r m i n e the sporeforming species in milk f r o m countries where high average temperatures prevail (3). Bacillus subtilis (48.5%), B. licheniformis (16.3%), and B. cereus (11.85%) were the m o s t c o m m o n species with B. lentus, B. pumilus, B. coagulans, B. stearotbermophilus, B. circulans, B. firmus, B. brevis, B. megaterium, B. laterosporus, and B. macerans comprising the remainder of the spore flora of milk f r o m these countries (Table 8). It appears that essentially the same species of sporeforming microorganisms are involved
TABLE 3. Microbial flora of commercially pasteurized milk following 30 days of storage at 4.5°C (2).
Species
Percent of total isolations
Bacillus Microbacterium Micrococcus Acbromobacter Alcaligenes Streptococcus
84 9 2 2 2 I
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS
151
TABLE 4. Standard plant counts and aerobic spore counts of raw milk (5). Mesophilic spore counts a
Class ! 11 I11 IV
SPC range (per ml) < 50,000 >50,000 <200,000 >200,000 < 1,000,000 >1,000,000 < 5,000,000
No. of samples analyzed
Avg SPC (per ml)
Thermophilic spore counts a
Average (per ml)
No. of positive samples
Average (per ml)
19
32,000
400
16
46
36
98,000
400
35
45
48
580,000
710
40
55
73
2,300,000
760
60
41
aSpore counts were determined after heating milk at 80°C for 10 rain. Mesophilic counts were determined by a pour plate procedure; thermophilic counts by a most probable number dilution tube technique.
worldwide with the m o s t c o m m o n being B. subtilis, B. licbeniformis, and B. cereus. The only major difference appears to be different p r o p o r t i o n s in some countries f r o m others. What a b o u t the h e a t resistance of the different species? Our w o r k in 1966 evaluated effects of three U H T t r e a t m e n t s on spores of the various species isolated f r o m Ohio milk (6). Results are summarized in Tables 9, 10, and 11. The temperatures were 104.5°C, 121°C, and 137.8°C. Holding time was approxim a t e l y 1 s in all cases. A p p r o x i m a t e l y o n e million spores per milliliter were added to raw milk, which then was pasteurized by indirect U H T heating for 1 s at the different U H T temperatures. Percent destruction at 104.5°C ranged from n o n e with B. laterosporus to 50% with B. p u m i l u s . The t e m p e r a t u r e t r e a t m e n t of 121°C was only 97.7% effective with B. licbeniformis, 98.4% with B. laterosporus, and 98.7% with B. megaterium. At 13 7.8°C the percentage destruction was at least 99.99% with all organisms tested, but surviving spores ranged f r o m 2/ml for B. sphaericus to 15/ml for B. megaterium. The data indicate that any UHT t r e a t m e n t selected must be greater than a c o m b i n a t i o n of 137.8°C for 1 s to approach commercial sterilization. Otherwise, if certain sporeforming Bacillus organisms are in the milk, there is a very strong possibility some will survive. Our idea at the t i m e was that possibly UHT-treated milk could be stored w i t h o u t
refrigeration, so we determined the recovery and o u t g r o w t h o f the surviving spores at 35°C, a t e m p e r a t u r e approaching the o p t i m u m for m o s t of the Bacillus species. Results are presented also in Tables 9, 10, and 11. The spores that survived germinated and grew rapidly in the UHT-treated milks. The vegetative cell c o u n t for all species was in the millions within 6 h in the milk heated at 104.5°C. Counts for spores also were very high in this milk, ranging f r o m 56,000/ml for B. p u m i l u s to 1,050,000/ml
TABLE 5. Organisms isolated from raw milk supplies (4). Organism B licheniformis a B. cereus B. coagulans B. pumilus B. sphaericus B. cereus var. mycoides B. brevis B. laterosporus B. circulans B. stearotbermopbilus Clostridium (species unidentified)
Total
Number isolated 129 111 11 10 6 5 4 3 2 2
% Total 43.3 37.4 3.7 3.4 2.0 1.7 1.3 1.0 .7 .7
14
4.7
297
100.0
aDifferentiated from Bacillus subtilis by anaerobic growth in glucose broth and by colony characteristics on nutrient agar. Journal of Dairy Science Vol. 64, No. 1, 1981
15 2
MARTIN
TABLE 6. Distribution of spore forming bacterial isolates according to milk samples (1). Source of samples Bacillus species
Incubation Temp (°C)
Organized dairy farm Cattle Bulk milk yard from dairy
Pasteurized milk
Market milk
37 37
50 45
20 34
22 38
37
7
2
•, .
46 26 6
37 37 55 55 55 63
3 10 ... 15 5 20
1 7 9 10 3 24
... 11 7 I5 9 45
Commercial dairy
Total number .
B. B. B. B. B. B. B. B. B.
subtilis cereus pumilus megaterium licbeniformis subtilis coagulans licheniformis stearotbermopbilus
7 5 1
145 148 16
5
...
9
4
6 ...
38 23
2
67
7
25 I
...
18
14
22
125
for B. circulans. A f t e r i n c u b a t i o n at 35°C for 6 h, s p o r e c o u n t s were in the 10's of t h o u s a n d s in milk h e a t e d at 104.5°C for 1 s. F o r t h e milk t r e a t e d at 121°C, the n u m b e r s surviving were m u c h lower, b u t t h o s e s p o r e s t h a t survived g e r m i n a t e d a n d grew rapidly in t h e milk i n c u b a t e d at 35°C. N u m b e r s of vegetative cells o f B. l i c b e n i f o r m i s , B. p u m i l u s , B. cereus var m y c o i d e s , B. laterosporus, a n d B. m e g a t e r i u m were all in the millions at the e n d of t h e 6-h i n c u b a t i o n . A t t h e highest t e m p e r a t u r e used, 137.8°C, t h e p e r c e n t d e s t r u c t i o n was 99.9% or g r e a t e r for all species tested. However, t h e few t h a t did survive, with the e x c e p t i o n o f B. l i c h e n i f o r m i s , g e r m i n a t e d a n d grew to sigonificant n u m b e r s in t h e U T H milk s t o r e d at 35 C for 6 h. T h e d a t a i n d i c a t e t h a t p r o x i m a t e d e s t r u c t i o n of Bacillus spores will n o t suffice if milk is to be s t o r e d
unrefrigerated. The h e a t i n g process m u s t prod u c e an a b s o l u t e l y sterile p r o d u c t for storage at r o o m t e m p e r a t u r e t o b e successful. We also calculated t h e n u m b e r of g e n e r a t i o n s d u r i n g t h e 6 h at 35°C to d e t e r m i n e if t h e h e a t t r e a t m e n t was s t i m u l a t o r y to s p o r e g e r m i n a t i o n a n d s u b s e q u e n t g r o w t h . The d a t a are in T a b l e 12. T h e t e m p e r a t u r e o f 1 2 1 ° C was s t i m u l a t o r y to all species, with t h e n u m b e r o f g e n e r a t i o n s d u r i n g 6 h o f i n c u b a t i o n a f t e r h e a t i n g at this t e m p e r a t u r e always m o r e t h a n t h e n u m b e r at 104.5°C. F o r a few species, B. spbaericus, B. m e g a t e r i u m , B. laterosporus, a n d B. circulans, t h e rate o f g r o w t h was s t i m u l a t e d even m o r e at
TABLE 7. Species of Bacillus isolated in the survey (8).
Species
Number isolated
Percentage of total isolates
B. B. B. B. B. B. B. B. B. B. B. B. B.
131 44 32 14 13 13 7 4 4 3 2 2 1
48.52 16.30 11.85 5.15 4.81 4.81 2.56 1.49 1.49 1.13 .76 .76 .37
% Total isolates
B. B. B. B. B. B. B. B.
subtilis licbeniformis pumilus alvei nlegaterium stearotbermopbilus sphaericus cereus
Cocoa
Milk
42.8 40.0 8.5 2.8 2.8
35.8 50.9 5.7 4.7
2.8 .... ....
Journal of Dairy Science Vol. 64, No. 1, 1981
.... ....
1.9 3.8
TABLE 8. Total number and relative proportions of species isolated from milks of all countries in NIRD/ FAO survey (3).
subtilis licbeniformis cereus lentus pumilus coagulans stearothermopbilus circulans litmus brevis megaterium macerans laterosporus
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS
153
TABLE 9. Effect of UHT treatment at 104.5°C on bacterial spores (6).
Species
Spore count/ml a Before After U HT U HT
Approximate percent destruction
UHT milk after 6 h at 35°C Vegetative cell Spore count/ml count/ml
B. B. B. B. B. B. B. B. B.
767,000 547,000 813,000 803,000 870,000 1,530,000 1,240,000 1,190,000 957,000
50 45 45 27 25 19 16 4 0
28,200,000 28,000,000 43,500,000 30,300,000 34,600,000 18,700,000 22,300,000 71,000,000 38,000,000
pumilus coagulans spbaericus cereus var. mycoides cereus circutans licbeniformis megateriurn laterosporus
381,000 301,000 450,000 583,000 657,000 1,240,000 1,050,000 1,140,000 1,060,000
55,700 87,400 68,300 483,000 497,000 1,050,000
363,000 827,000 687,000
aAverage of three trials. Before addition of spores, the spore count of the raw milk was less than 60/ml.
O
1 3 7 . 8 C. T h e s p o r e s t h a t survived g e r m i n a t e d a n d grew f a s t e r at 35°C t h a n did t h e survivors f r o m e i t h e r 104.5°C or 1 2 1 ° C heat treatment. T h e s e d a t a i n d i c a t e t h a t certain U H T t e m p e r a t u r e t r e a t m e n t s a c t i v a t e t h e bacterial spore so t h a t it g e r m i n a t e s a n d t h e vegetative cells r e p r o d u c e faster t h a n cells w h i c h have n o t b e e n s u b j e c t e d t o high h e a t t r e a t m e n t s . A n y U H T t r e a t m e n t utilized c o m m e r c i a l l y m u s t be in excess o f a n y s t i m u l a t o r y t e m p e r a t u r e t h a t
m i g h t r e d u c e n u m b e r s drastically, b u t t h a t m i g h t cause t h e few survivors to grow even m o r e rapidly t h a n n o r m a l . W o r k p u b l i s h e d in 1 9 7 8 revealed t h a t raw m i l k o f average m i c r o b i a l q u a l i t y h e a t e d a t 8 0 ° C f o r 12 rain still c o n t a i n e d viable organisms a f t e r this h e a t t r e a t m e n t (7). S u m m a r y d a t a are in T a b l e 13. T h e r e were 170 m e s o p h i l i c a n d 7 p s y c h r o t r o p h i c o r g a n i s m s surviving per milliliter o f milk, a n d 7 6 / m l surviving t h a t were interm e d i a t e in g r o w t h t e m p e r a t u r e b e t w e e n m e s o -
TABLE 10. Effect of UHT treatment at 121°C on bacterial spores (6).
Species
Spore count/ml a Before After UHT UHT
Approximate percent destruction
B. B. B. B. B. B. B. B. B.
1,240,000 870,000 547,000 767,000 813,000 803,000 957,000 1,190,000 1,530,000
87.70 99.98 99.97 99.99
licbeniforrnis cereus coagulans pumilus spbaericus cereus vat. mycoides laterosporus megaterium circulans
28,000 139 171 62 38 76 14,900 15,600 147
99.99
99.99 98.40 98.70 99.99
UHT milk after 6 h at 35°C Vegetative cell Spore count/ml count/ml 1,370,000 119,000 111,000 1,490,000 651,000 3,730,000 7,970,000 29,700,000 285,000
6,630 102 158 49 33 33 4,690 10,100 194
aAverage of three trials. Before addition of spores, the spore count of the raw milk was tess than 60/ml. Journal of Dairy Science Vol. 64, No. 1, 1981
154
MARTIN
TABLE 11. Effect of UHT treatment at 137.8°C on bacterial spores (6). UHT milk after 6 h at 35°C Spore count/mla Species
Before UHT
After UHT
Approximate percent destruction
Vegetative cell eount/ml
Spore count/ml
B. licbeniformis B. cereus B. coagulans B. pumilus B. spbaericus B. cereus var. mycoides B. laterosporus B. megaterium B. circulans
1,240,000 870,000 547,000 767,000 813,000 803,000 957,000 1,190,000 1,530,000
6 8 3 8 2 9 6 15 3
99.99
99.99 99.99 99.99 99.99 99.99 99.99 99.99 99.99
29 5,800 1,200 85,000 73,300 44,300 57,600 518,000 35,100
13 8 9 11 6 5 11 3 17
aAverage of three trials. Before addition of spores, the spore count of the raw milk was less than 60/ml.
philic and psychrotrophic. A f t e r 7 days storage at 7°C, numbers were 1800 mesophilic, 700 intermediate, and 340 psychrotrophic organisms per milliliter of milk. Surely these organisms were sporeformers and the entity surviving the heating was the spore. We did m u c h w o r k with spores for several years and f o u n d nothing but spores surviving 80°C for 12 rain. This w o r k also points up the i m p o r t a n c e of c o m p l e t e destruction if U H T is to be successful. M e n t i o n e d earlier was the j o i n t study of
Bacillus species by the National Institute for
Research in Dairying and F A O f r o m countries with high average temperatures (3). The isolates f r o m these studies were subjected to f o u r temperatures, 100°C for 30 min, 110°C for 10 min, 120°C for 20 min, and 130°C for 10 min. Results are in Table 14. At least 20% of all the species isolated survived 100°C for 30 rain, with 21.9% of B. cereus surviving and ranging upwards to 100% for B. coagulans, B. brevis, B. megaterium, B.
TABLE 12. Effect of UHT treatment on outgrowth of Bacillus spores in milk (6). No. of generations during 6 h of incubation at 35°C after UHT treatment a at
B. licheniformis B. cereus B. coagulans B. pumilus B. spbaericus R cereus var. mycoides B. laterosporus B. megaterium B. circulans
Average no. of generations a
. final no. No. generations = 3.3 log 10 initial no.
Journal of Dairy Science Vol. 64, No. 1, 1981
104.5°C
121°C
137.8°C
4.4 5.7 6.5 2.9 6.6 5.7 5.1 5.9 3.9 5.2
5.6 9.7 9.3 14.5 14.0 15.5 9.0 10.8 10.8 11.0
2.3 9.4 8.6 13.3 15.1 12.2 13.1 15.0 13.4 11.4
SYMPOSIUM: HEAT RESISTANT MICROORGANISMS IN DAIRY FOOD SYSTEMS TABLE 13. Microbial quality of raw milk (109 samples) of the same milk upon heat treatment at 80°C for 12 min and subsequent storage at 7°C (7). Sample and plating procedure Raw milk Standard plate count Gram-negative count
155
%E 0
0
0
0
0
0
0
0
0
0
0
0
0
Microbial numbers (average CFU/ml) v
110,000 72~000 e~
Heated milk (80°C-12 min)
eq
0
oooo~o~oooooo,~
Initial
Mesophilic Intermediate Psychrotrophic 7-Day storage (7°C) Mesophilic Intermediate Psychrotrophic
170 76 7
ca
e"
1,800 700 340 e" -t 0
laterosporus, and B. stearotbermopbilus. The 110°C for 10 min heat treatment reduced Bacillus cereus numbers by approximately 94% and completely eliminated B. laterosporus and B. macerans. However, all spores of B. coagulans and B. stearotbermopbilus survived the l l 0 ° C for 10 min heat treatment. Approximately 80% of the B. subtilis, 67% of the B. brevis, and 50% of the B. megaterium organisms survived the 110°C for 10 rain treatment. The 120°C for 20 min treatment inactivated all species except 15.4% of B. coagulans and 71.4% of the B. stearotbermopbilus organisms. Heat treatment of 130°C for 10 min resulted in negative counts for all species isolated from the various countries with high average temperatures. Therefore, certain species of sporeforming microorganisms are capable of surviving tremendous heat treatments. One wonders if any ultra-high-temperature treatment utilized to date would destroy consistently all spores that might possibly be in milk. Research should continue with the high heat resistant species as the major test organisms, and storage of milk on grocery shelves without refrigeration should be approached with caution until the timetemperature relationship for complete destruction has been successfully demonstrated many times over.
,"~0
} O 0
Z e~
! e~
[.,.
e~
Journal of Dairy Science Vol. 64, No. 1, 1981
156
MARTIN REFERENCES
1 Atwal, J. S., D. N. Slaroff, R a t t a n Chand, and R. A. Srinivasan. 1974. Studies on aerobic spore forming bacteria in milk and milk products. I. Occurrence in raw and pasteurized milk. Indian J. Dairy Sci. 27:22. 2 Credit, C., R. Hedeman, P. Heywood, and D. Westhoff. 1972. Identification of bacteria isolated from pasteurized milk following refrigerated storage. J. Milk Food Technol. 35:708. 3 Davies, F. L. 1975. Heat resistance of Baci2lus species. J. Soc. Dairy Technol. 28:69. 4 Martin, J. H. 1974. Significance of bacterial spores in milk. J. Milk Food Technol. 37:94. 5 Martin, J. H., D. P. Stahly, W. J. Harper, and
Journal of Dairy Science Vol. 64, No. 1, 1981
6
7
8
9 10
I. A. Gould. 1962. Spore forming microorganisms in selected milk supplies. Proc. 16th Int. Dairy Congr., Copenhagen, 295:304. Martin, J. H., W. J. Harper, and I. A. Gould. 1966. Ultra-high temperature effects on selected Bacillus species. J. Dairy Sc L 49:1367. Mikolajcik, E. M., and N. T. Simon. 1978. Heat resistant psychrotrophic bacteria in raw milk and their growth at 7C. J. Food Prot. 41(2):93. Mossel, D.A.A., E. H. Meursing, and H. Slot. 1974. An investigation on the n u m b e r s and types of aerobic spores in cocoa powder and whole milk. Neth. Milk and Dairy J. 28:149. T h o m a s , S. B. 1974. The microflora of bulk collected milk - Part 1. Dairy Ind. 39(7):237. T h o m a s , S. B. 1974. The microflora of bulk collected milk - Part 2. Dairy Ind. 39(8):279.