Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
L. A. ROTHAND D. S. CLARK Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K I A OR6 Accepted January 17, 1975 ROTH,L. A., and D. S. CLARK.1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21: 629-632. Studies with artificially inoculated fresh beef showed that lactobacilli markedly restrict the growth ofMicrobacterium thermosphactum when the beef is vacuum-packaged. High concentrations of carbon dioxide (up to 75%) had little effect on the growth of M. thermosphactum under either aerobic or anaerobic conditions. Tests were made with samples of lean beef inoculated with pure culturesof lactobacilli and (or) M. thermosphactum and stored unpackaged in air, in air enriched with carbon dioxide, in nitrogen -carbon dioxide mixtures, or vacuum-packaged as in commercial practice. On vacuum-packaged meat, growth of M. thermosphactum was extensive in the absence of lactobacilli, but was restricted in the presence of lactobacilli. On unpackaged meat (i.e. stored in air) this inhibition did not occur, showing that anaerobic conditions are necessary for it to take place. These results show the importance of lactobacilli on the storage life of vacuum-packaged fresh beef. ROTH,L. A., et D. S. CLARK.1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21: 629-632. Des ttudes sur l'inoculation artificielle de beuf frais dtmontrent que les lactobacilles restreignent de f a ~ o nmarqute la croissance de Microbacterium thermosphactum lorsque le beuf est empaquett sous vide. Des concentrations Clevtes de bioxyde de carbone (iusqu'a 75%) ont peu d'effet sur la croissance de M. thermosphact~tmsous conditions atrobiques ou anatrobiques. Des tchantillons de beuf maigre furent inocults par des cultures pures de lactobacilles et (ou) d e M. thermosphactum et placts en entrepbt sans empaquetage, soit I'air libre, soit a I'air enrichi de bioxyde de carbone, ou dans des mtlanges d'azote et de bioxyde de carbone, ou bien ils furent empaquetts sous vide comme dans la pratique commerciale. Chez les tchantillons empaquetts sous vide, la croissance d e M. thermosphact~tmfut importante en l'absence de lactobacilles, mais restreinte en leur presence. Chez les tchantillons non empaquetts cette inhibition n'a pas eu lieu, dtmontrant que les conditions anatrobiques sont nkcessaires I'inhibition. Ces rtsultats indiquent I'importance des lactobacilles pour la prtservation en entrepBt du beuf frais empaquett sous-vide. [Traduit par le journal]
Introduction Recent studies showed that the meat-spoilage organism Microbacterium thermosphactum will not grow on the surface of fresh beef vacuumpackaged in a gas-impermeable film (9, 10). The reason for this growth failure was not apparent since the environmental conditions in the package would seem to favor proliferation of the organism; M . thermosphactum grows in the absence of oxygen (14) and in the presence of at least 10% carbon dioxide. Concentrations of CO, higher than 10% (levels up to 30% in vacuum-packaged meat have been reported (5)), or competitive inhibition exerted by the lactobacilli were thought to be contributing factors. The lactobacilli predominate in the flora of 'Received November 4, 1974. 'NRCC No. 14586.
vacuum-packaged meat and are inhibitory to some organisms (1 1). Definite information, however, on which factor is the main one.was not available and tests were therefore made to determine the specific effects of strains of lactobacilli and of high concentrations of carbon dioxide on the growth rate of M . thermosphactum on fresh beef. Since in commercial practice (12) large volumes of fresh beef are being vacuum-packaged, it is important to know the interrelation of the microflora in the package and to understand the factors that affect the growth of spoilage organisms.
Materials and Methods Tests were made with I-lb samples of lean meat cut from rump muscle of A-brand fresh beef (aged 4-5 days) obtained from a local packing plant. T o minimize con-
CAN. J. MICROBIOL. VOL. 21. 1975
M THERMOSPHACTUM ( W ITH
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Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
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STORAGE TIME .DAYS
F I G . 1. Growth curves of lactobacilli and M. /l1ermospl7ac/~1rn,and of mixtures of the two types on vacuum-packaged or unpackaged fresh beef at 5'C. tamination, the meat was trimmed with a sterile knife before excising samples and the knife was swabbed with ethyl alcohol and wiped dry with a sterile cloth after each cut. The samples were placed in petri dishes and the top surfaces of the samples were inoculated evenly in a spray-type inoculating chamber (1) with a uniform mixture of 10 strains of M. /l~ermospl~ac/um and (or) 15 strains of lactobacilli. The inoculum was prepared as described in an earlier publication (3), from 4-day-old cultures grown at 5'C on tryptone glucose extract agar (Difco). All strains were previously isolated from naturally contaminated beef (10). Preliminary classification studies showed that all the lactobacilli strains were homofermentative. T o determine the effect of lactobacilli on the growth rate of M. /l~ermosphac/urnon vacuum-packaged beef, samples inoculated with mixtures of both types or with each type separately were placed in separate vinyldene chloride - vinyl chloride copolymer (VC) b a g s h h i c h were then evacuated (15 in. of Hg), heat shrunk (198"F/15 s), and incubated at 5'C. T o determine growth rates on unpackaged beef for comparison with those obtained in the vacuum-packaging tests, samples inoculated as described above were incubated in air at 5'C. These samples were stored in an 8-litre desiccator which was continually flushed with sterilized and humidified air. The air was sterilized by filtering through cotton and humidified by 3Cryovac S ; 0.002 in. thick; oxygen permeability 10-30 cm3/m2 per atmosphere per 24 h ; W. R. Grace and Co., Duncan, South Carolina.
bubbling through two vertical 6-in. columns of sterile water. T o determine the effect of CO, on the growth of M. /llermospl~ac/urnunder both aerobic and anaerobic conditions, meat samples inoculated with the mixture of 10 strains were incubated up to 30 days at 5'C in air-C0, or N,-CO, mixtures in 8-litre desiccators. The C 0 , concentrations used were 0, 20, 50, and 75%. The gas mixtures were obtained by continuous mixing of metered flows o f air and CO, (99.8% pure), or nitrogen (99.99% pure), and CO,, and were sterilized and humidified as described above. The concentrations of CO, werechecked using a gas chromatograph. Eighteen samples were incubated for each test condition and duplicate samples were removed periodically (intervals of 1-6 days) for microbiological analysis. For analysis, 6 c m 2 of inoculated surface was washed with an aqueous 0.1% peptone solution using the spray gun technique (2) and the washing then plated on (a) tryptone glucose extract agar to enumerate the total number of cells, (b) STAA (streptomycin sulfate - thallous acetate actidione agar (4)) to enumerate M. /hermospltac/um. and (c) LBS agar (Lactobacillus selection agar, BBL) to enumerate the lactobacilli. The tryptone glucose extract and STAA plates were incubated at 25'C for 48 h and the LBS agar at 25'C for 4 days.
Results and Discussion Lactobacilli markedly restricted the growth of M , thermosphactun7 on the vacuum-packaged
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
ROTH AND CLARK: EFFECT OF LACTOBACILLI AND COz ON M. THERMOSPHACTUM ON BEEF
beef (anaerobic conditions) but not on beef incubated in air (Fig. 1). Actually, in the latter case it appeared that M. therrnosphacturn restricted the growth of lactobacilli somewhat, causing a log-cycle reduction in the ultimate population. High concentrations of carbon dioxide (up to 75%) did not inhibit the ultimate growth yield of M. therrnosphacturn (Fig. 2). Similar results were obtained under anaerobic conditions (N, and CO,). Under both aerobic and anaerobic conditions the lag phase was lengthened with increasing concentrations of CO, but was not so protracted as to be a factor in prolonged storage. In vacuum-packaged fresh meat the concentration of CO, builds up gradually (13), and therefore in practice the gas would have even less effect on M. tl~errnosphacturnthan in these experiments, where the concentration was constant throughout. Published studies on the com-
631
position of the gas developing around meat in a vacuum package (5, 13) show that the carbon dioxide content increases during the first few days of storage and stabilizes at between 10 and 20%; the highest reading reported was 31% (5). This study provides another example of the conditions under which members of the lactic acid group of bacteria (streptococci and lactobacilli) can suppress the growth of other types. In studies with streptococci (6, 8), the cause was shown in some cases to be antibiotic peptides of which nisin is the best known. With the lactobacilli, the cause has not yet been determined. In the present case, the fact that after inoculation M. therrnosphact~rnr began to grow in the vacuum package in the presence of lactobacilli and then stopped, and even decreased, when the lactobacilli population reached the maximum stationary phase (Fig. 1) suggests that inhibition was caused by a substance produced by the lactobacilli, rather than by competition for space or food. Lactic acid is not suspected since M. tl~errnospliacturnproduces lactic acid (7). Also, any slight depression of the pH of the meat at the surface resulting from the presence of lactic acid is not thought to be a factor because preliminary tests have shown that the organism can grow well on meat at pH 5.5, as low a pH as is normally found in stored meat.
Acknowledgment The authors thank N. U. Cholette for technical assistance.
tl
0
FIG.2.
I
5
I
I
I
I0 I5 20 STORAGE TIME, DAYS
I
25'
Effect of carbon dioxide o n the growth of M. fresh beef.
tl~ermosphactumo n
I. C L A R KD. , S. 1963. Uniform inoculation of nutrient surfaces. Biotechnol. Bioeng. 5: 123-129. 2. C L A R KD. , S. 1965. Improvement of spray gun method of estimating bacterial populations on surfaces. Can. J . Microbiol. 11: 1021-1022. 3. C L A R KD. , S . 1968. Growthofpsychrotolerantpseudomonads and achromobacter on chicken skin. Poult. Sci. 47: 1575-1578. 4. GARDNER,G. A. 1966. A selective medium for the enumeration of Mioobncteriirnz tl~ertnosplzactlrmin meat and meat products. J. Appl. Bacteriol. 29: 455460. 5. GARDNER, G . A , , A. W. CARSON,and J. PATTON. 1967. Bacteriology of prepackaged pork with reference to the gas composition within the pack. J. Appl. Bacteriol. 30: 321-333. 6. HURST,A. 1966. Biosynthesis of the antibiotic nicin and other basic peptides by Streptococciis lactis grown in batch culture. J. Gen. Microbiol. 45: 503513. 1953. 7. MCLEAN, R. A,, and W. L. SULZBACHER. Microbacteri~rm thermospl~actirrn. spec. nov.; a nonheat-resistant bacterium from fresh pork sausage. J . Bacteriol. 65: 428.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
632
CAN. J. MICROBIOL. VOL. 21, 1975
8. MATTICK, A. T. R., and A. HIRSCH.1944. A powerful inhibitory substance produced by group N streptococci. Nature (Lond.), 154: 55 1. 9. PIERSON, M. D., D. L. COLLINS-THOMPSON, and 2. J. ORDAL.1970. Microbiological, sensory and pigment changes in aerobically and anaerobically packaged beef. Food Technol. 24: 129-133. 10. ROTH,L. A., and D. S. CLARK.1972. Studies on the bacterial flora of vacuum-packaged fresh beef. Can. J. Microbiol. 18: 1761-1766. 11. SANDINE, W. E., K. S. MURALIDHARA, P. R. ELLIKER, and D. C. ENGLAND. 1972. Lactic acid bacteria in food and health: A review with special reference to
enteropathogenic Escherichia coli as well as certain enteric diseases and their treatment with antibiotics and lactobacilli. J. Milk Food Technol. 35: 691-702. 12. SHAW,S . T. 1973. Past half-way point, move to boxed beef is accelerating. The National Provisioner, November 3. 13. TAYLOR,A. A. 1971. The gaseous environment of packaged fresh meat. R o c . 17-Eur. Meet. Meat Res. Work. p. 662. 14. WEIDMANN, J. F. 1965. A note on the microflora of beef muscle stored in nitrogen atOO.J. Appl. Bacterial. 28: 365-367.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
L. A. ROTHAND D. S. CLARK Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K I A OR6 Accepted January 17, 1975 ROTH,L. A., and D. S. CLARK.1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21: 629-632. Studies with artificially inoculated fresh beef showed that lactobacilli markedly restrict the growth ofMicrobacterium thermosphactum when the beef is vacuum-packaged. High concentrations of carbon dioxide (up to 75%) had little effect on the growth of M. thermosphactum under either aerobic or anaerobic conditions. Tests were made with samples of lean beef inoculated with pure culturesof lactobacilli and (or) M. thermosphactum and stored unpackaged in air, in air enriched with carbon dioxide, in nitrogen -carbon dioxide mixtures, or vacuum-packaged as in commercial practice. On vacuum-packaged meat, growth of M. thermosphactum was extensive in the absence of lactobacilli, but was restricted in the presence of lactobacilli. On unpackaged meat (i.e. stored in air) this inhibition did not occur, showing that anaerobic conditions are necessary for it to take place. These results show the importance of lactobacilli on the storage life of vacuum-packaged fresh beef. ROTH,L. A., et D. S. CLARK.1975. Effect of lactobacilli and carbon dioxide on the growth of Microbacterium thermosphactum on fresh beef. Can. J. Microbiol. 21: 629-632. Des ttudes sur l'inoculation artificielle de beuf frais dtmontrent que les lactobacilles restreignent de f a ~ o nmarqute la croissance de Microbacterium thermosphactum lorsque le beuf est empaquett sous vide. Des concentrations Clevtes de bioxyde de carbone (iusqu'a 75%) ont peu d'effet sur la croissance de M. thermosphact~tmsous conditions atrobiques ou anatrobiques. Des tchantillons de beuf maigre furent inocults par des cultures pures de lactobacilles et (ou) d e M. thermosphactum et placts en entrepbt sans empaquetage, soit I'air libre, soit a I'air enrichi de bioxyde de carbone, ou dans des mtlanges d'azote et de bioxyde de carbone, ou bien ils furent empaquetts sous vide comme dans la pratique commerciale. Chez les tchantillons empaquetts sous vide, la croissance d e M. thermosphact~tmfut importante en l'absence de lactobacilles, mais restreinte en leur presence. Chez les tchantillons non empaquetts cette inhibition n'a pas eu lieu, dtmontrant que les conditions anatrobiques sont nkcessaires I'inhibition. Ces rtsultats indiquent I'importance des lactobacilles pour la prtservation en entrepBt du beuf frais empaquett sous-vide. [Traduit par le journal]
Introduction Recent studies showed that the meat-spoilage organism Microbacterium thermosphactum will not grow on the surface of fresh beef vacuumpackaged in a gas-impermeable film (9, 10). The reason for this growth failure was not apparent since the environmental conditions in the package would seem to favor proliferation of the organism; M . thermosphactum grows in the absence of oxygen (14) and in the presence of at least 10% carbon dioxide. Concentrations of CO, higher than 10% (levels up to 30% in vacuum-packaged meat have been reported (5)), or competitive inhibition exerted by the lactobacilli were thought to be contributing factors. The lactobacilli predominate in the flora of 'Received November 4, 1974. 'NRCC No. 14586.
vacuum-packaged meat and are inhibitory to some organisms (1 1). Definite information, however, on which factor is the main one.was not available and tests were therefore made to determine the specific effects of strains of lactobacilli and of high concentrations of carbon dioxide on the growth rate of M . thermosphactum on fresh beef. Since in commercial practice (12) large volumes of fresh beef are being vacuum-packaged, it is important to know the interrelation of the microflora in the package and to understand the factors that affect the growth of spoilage organisms.
Materials and Methods Tests were made with I-lb samples of lean meat cut from rump muscle of A-brand fresh beef (aged 4-5 days) obtained from a local packing plant. T o minimize con-
CAN. J. MICROBIOL. VOL. 21. 1975
M THERMOSPHACTUM ( W ITH
lxloa
F
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
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STORAGE TIME .DAYS
F I G . 1. Growth curves of lactobacilli and M. /l1ermospl7ac/~1rn,and of mixtures of the two types on vacuum-packaged or unpackaged fresh beef at 5'C. tamination, the meat was trimmed with a sterile knife before excising samples and the knife was swabbed with ethyl alcohol and wiped dry with a sterile cloth after each cut. The samples were placed in petri dishes and the top surfaces of the samples were inoculated evenly in a spray-type inoculating chamber (1) with a uniform mixture of 10 strains of M. /l~ermospl~ac/um and (or) 15 strains of lactobacilli. The inoculum was prepared as described in an earlier publication (3), from 4-day-old cultures grown at 5'C on tryptone glucose extract agar (Difco). All strains were previously isolated from naturally contaminated beef (10). Preliminary classification studies showed that all the lactobacilli strains were homofermentative. T o determine the effect of lactobacilli on the growth rate of M. /l~ermosphac/urnon vacuum-packaged beef, samples inoculated with mixtures of both types or with each type separately were placed in separate vinyldene chloride - vinyl chloride copolymer (VC) b a g s h h i c h were then evacuated (15 in. of Hg), heat shrunk (198"F/15 s), and incubated at 5'C. T o determine growth rates on unpackaged beef for comparison with those obtained in the vacuum-packaging tests, samples inoculated as described above were incubated in air at 5'C. These samples were stored in an 8-litre desiccator which was continually flushed with sterilized and humidified air. The air was sterilized by filtering through cotton and humidified by 3Cryovac S ; 0.002 in. thick; oxygen permeability 10-30 cm3/m2 per atmosphere per 24 h ; W. R. Grace and Co., Duncan, South Carolina.
bubbling through two vertical 6-in. columns of sterile water. T o determine the effect of CO, on the growth of M. /llermospl~ac/urnunder both aerobic and anaerobic conditions, meat samples inoculated with the mixture of 10 strains were incubated up to 30 days at 5'C in air-C0, or N,-CO, mixtures in 8-litre desiccators. The C 0 , concentrations used were 0, 20, 50, and 75%. The gas mixtures were obtained by continuous mixing of metered flows o f air and CO, (99.8% pure), or nitrogen (99.99% pure), and CO,, and were sterilized and humidified as described above. The concentrations of CO, werechecked using a gas chromatograph. Eighteen samples were incubated for each test condition and duplicate samples were removed periodically (intervals of 1-6 days) for microbiological analysis. For analysis, 6 c m 2 of inoculated surface was washed with an aqueous 0.1% peptone solution using the spray gun technique (2) and the washing then plated on (a) tryptone glucose extract agar to enumerate the total number of cells, (b) STAA (streptomycin sulfate - thallous acetate actidione agar (4)) to enumerate M. /hermospltac/um. and (c) LBS agar (Lactobacillus selection agar, BBL) to enumerate the lactobacilli. The tryptone glucose extract and STAA plates were incubated at 25'C for 48 h and the LBS agar at 25'C for 4 days.
Results and Discussion Lactobacilli markedly restricted the growth of M , thermosphactun7 on the vacuum-packaged
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
ROTH AND CLARK: EFFECT OF LACTOBACILLI AND COz ON M. THERMOSPHACTUM ON BEEF
beef (anaerobic conditions) but not on beef incubated in air (Fig. 1). Actually, in the latter case it appeared that M. therrnosphacturn restricted the growth of lactobacilli somewhat, causing a log-cycle reduction in the ultimate population. High concentrations of carbon dioxide (up to 75%) did not inhibit the ultimate growth yield of M. therrnosphacturn (Fig. 2). Similar results were obtained under anaerobic conditions (N, and CO,). Under both aerobic and anaerobic conditions the lag phase was lengthened with increasing concentrations of CO, but was not so protracted as to be a factor in prolonged storage. In vacuum-packaged fresh meat the concentration of CO, builds up gradually (13), and therefore in practice the gas would have even less effect on M. tl~errnosphacturnthan in these experiments, where the concentration was constant throughout. Published studies on the com-
631
position of the gas developing around meat in a vacuum package (5, 13) show that the carbon dioxide content increases during the first few days of storage and stabilizes at between 10 and 20%; the highest reading reported was 31% (5). This study provides another example of the conditions under which members of the lactic acid group of bacteria (streptococci and lactobacilli) can suppress the growth of other types. In studies with streptococci (6, 8), the cause was shown in some cases to be antibiotic peptides of which nisin is the best known. With the lactobacilli, the cause has not yet been determined. In the present case, the fact that after inoculation M. therrnosphact~rnr began to grow in the vacuum package in the presence of lactobacilli and then stopped, and even decreased, when the lactobacilli population reached the maximum stationary phase (Fig. 1) suggests that inhibition was caused by a substance produced by the lactobacilli, rather than by competition for space or food. Lactic acid is not suspected since M. tl~errnospliacturnproduces lactic acid (7). Also, any slight depression of the pH of the meat at the surface resulting from the presence of lactic acid is not thought to be a factor because preliminary tests have shown that the organism can grow well on meat at pH 5.5, as low a pH as is normally found in stored meat.
Acknowledgment The authors thank N. U. Cholette for technical assistance.
tl
0
FIG.2.
I
5
I
I
I
I0 I5 20 STORAGE TIME, DAYS
I
25'
Effect of carbon dioxide o n the growth of M. fresh beef.
tl~ermosphactumo n
I. C L A R KD. , S. 1963. Uniform inoculation of nutrient surfaces. Biotechnol. Bioeng. 5: 123-129. 2. C L A R KD. , S. 1965. Improvement of spray gun method of estimating bacterial populations on surfaces. Can. J . Microbiol. 11: 1021-1022. 3. C L A R KD. , S . 1968. Growthofpsychrotolerantpseudomonads and achromobacter on chicken skin. Poult. Sci. 47: 1575-1578. 4. GARDNER,G. A. 1966. A selective medium for the enumeration of Mioobncteriirnz tl~ertnosplzactlrmin meat and meat products. J. Appl. Bacteriol. 29: 455460. 5. GARDNER, G . A , , A. W. CARSON,and J. PATTON. 1967. Bacteriology of prepackaged pork with reference to the gas composition within the pack. J. Appl. Bacteriol. 30: 321-333. 6. HURST,A. 1966. Biosynthesis of the antibiotic nicin and other basic peptides by Streptococciis lactis grown in batch culture. J. Gen. Microbiol. 45: 503513. 1953. 7. MCLEAN, R. A,, and W. L. SULZBACHER. Microbacteri~rm thermospl~actirrn. spec. nov.; a nonheat-resistant bacterium from fresh pork sausage. J . Bacteriol. 65: 428.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/26/14 For personal use only.
632
CAN. J. MICROBIOL. VOL. 21, 1975
8. MATTICK, A. T. R., and A. HIRSCH.1944. A powerful inhibitory substance produced by group N streptococci. Nature (Lond.), 154: 55 1. 9. PIERSON, M. D., D. L. COLLINS-THOMPSON, and 2. J. ORDAL.1970. Microbiological, sensory and pigment changes in aerobically and anaerobically packaged beef. Food Technol. 24: 129-133. 10. ROTH,L. A., and D. S. CLARK.1972. Studies on the bacterial flora of vacuum-packaged fresh beef. Can. J. Microbiol. 18: 1761-1766. 11. SANDINE, W. E., K. S. MURALIDHARA, P. R. ELLIKER, and D. C. ENGLAND. 1972. Lactic acid bacteria in food and health: A review with special reference to
enteropathogenic Escherichia coli as well as certain enteric diseases and their treatment with antibiotics and lactobacilli. J. Milk Food Technol. 35: 691-702. 12. SHAW,S . T. 1973. Past half-way point, move to boxed beef is accelerating. The National Provisioner, November 3. 13. TAYLOR,A. A. 1971. The gaseous environment of packaged fresh meat. R o c . 17-Eur. Meet. Meat Res. Work. p. 662. 14. WEIDMANN, J. F. 1965. A note on the microflora of beef muscle stored in nitrogen atOO.J. Appl. Bacterial. 28: 365-367.
