Progress 10/01/98 to 09/30/04
Outputs
Salmonella sp, Listeria sp. and Escherichia coli 0157:H7 sustain injury in foods, from processing conditions such as acidification/fermentation, cooking and freezing. While these conditions can be used to inhibit these pathogens, recovery of the sublethally injured pathogens is challenging to food microbiologists and regulatory authorities. During the course of this project we demonstrated that the use of selected antimicrobial producing lactococci for cheese making can enhance food safety by accelerating the death (1-31og) of these pathogens in buttermilk and Camembert or Cheddar cheese. A combination of natural acids in apple cider and freezing for 2 weeks can cause severe damage to E. coli 0157:H7 such that when this juice if defrosted and stored at 4C could result in 5 log death of the above three pathogens. This can be an alternate to pasteurization as stipulated by the US FDA. We also developed a bacteriological medium and food homogenate preparation diluent that
would permit recovery of sublethally injured Salmonella sp. and E. coli 0157:H7 which would otherwise not be detected.
Impacts
This research remains useful to the dairy product producers in enhancing food safety and for standardizing the diluent for preparing the food homogenate for salmonella or E. coli 0157:H7 analysis to improve detection and therefore eventually pathogen control.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The previously developed plating medium (Trytic Soy agar plus yeast extract, ferric ammonium citrate and sodium thiosulfate: TSAFC) for recovery of injured Salmonella from cutured buttermilk and aged Cheddar cheese, performed well in recovery of Salmonella from frozen-defrosted orange juice and the diluent used also influenced the recovery; 20 fold higher numbers on TSAFC as compared to XLD (selective medium, Xylose Lysine Desoxycholate agar). Milk diluent recovered higher numbers on both TSAFC and XLD. Numbers recovered were intermediate with Maximum recovery diluent(MRD). TSAFC medium was more inhibitory than XLD to thermally injured Salmonella Typhimurium (60C for 20-30 seconds) limiting its applicability to use with dried foods which may contain injured salmonella and other heat resistant microflora. This can be overcome by plating on TSAFC without ferric mamonium citrate and Sodium thiosulafte(TSAYE) and after plating and incubating for 24-48 hrs, overlaying the
medium with melted-tempered XLD. Salmonella colonies will develop black color with 2 hrs for further characterization. With this approach and use of milk as the diluent, highest numbers of injured Salmonella were recovered followed by Buffered Peptone water (BPW), MRD and Butterfield's Phosphate buffer. Incorporating 0.2ml milk into 9.8 ml of MRD and BPW as diluents improved the recovery of injured Salmonella.
Impacts
This research should be useful in standardizing the diluent for preparing the food homogenate for salmonella analysis to improve detection and therefore eventually pathogen control.
Publications
- Yashodhar, B. 2003. Quantitive recovery of Salmonella from cultured dairy products. M.S. Thesis. University of Minnesota, St. Paul. 148 p.
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Progress 01/01/02 to 12/31/02
Outputs
Even though milk, buffered peptone water(BPW) and maximum recovery diluent(MRD)were of similar osmolality (270 milliosmomoles) milk diluent allowed highest recovery of salmonella stressed in buttermilk at 4C, when plated on nonselective/differential agar; one log higher with milk than BPW or MRD. Of the milk components( 2.5% casein,1% whey proteins and 5% lactose) tested,casein allowed higher recovery of stressed salmonella (0.5 log higher) from buttermilk than MRD. Commercial pasteurized skim milk allowed recovery of stressed salmonella from buttermilk (0.5log higher) than the combined MC system as the diluent. Results from a 15 of 24 month USDA funded survey of commercial foods from retail stores (luxury, economy, neghborhood, and farmers market) in the Minneapolis-St.Paul area for prevalence of antibiotic resistant Escherichia coli(ABREC)indicated the following: Regardless of the source the incidence of ABREC (resistant to at least 1 of 12 antibiotics) was highest
for poultry products(84% of 114) followed by Beef/Pork(54% of 78)and the least in other foods(fruits,vegetables,etc;3% of 871). The prevalence of E.coli strains resistant to antibiotics used for growth promotion of food animals(trimethoprim-sulfamethoxazole,nalidixic acid-a marker for quinolone,fluoroquinolone and an extended spectrum cephalosporin)was highest for poultry(20-30%vs <7% for other foods). ABREC from poultry also exhibited transmissible resistance. An appreciable minority of the food-borne ABREC exhibited virulence traits associated with pathogenicity(extra intestinal pathogens,EXPEC).
Impacts
Data from diluents used for preparing the food homogenate for enumeration of stressed salmonella from buttermilk indicate the need for standardization of diluents for optimal recovery of especially injured salmonella. Data from the retail food survey strongly suggest that retail poultry may be a significant source of virulent ABREC to food service and house-hold kitchens and to food handlers including consumers. This also threatens to undermine our ability to treat EXPEC infections in humans. Therefore,further emergence of antiobiotic resistance needs to be curtailed by halting the use of antibiotics in food animal production.
Publications
- Burgula,Y., and Tatini, S.R. 2002 A nonselective/differential medium for recovery of stressed salmonella from cultured dairy products. Program and Abstract book, p. 124 of IAFP - 2002, San Diego,CA.
- Tatini, S.R., and Kauppi, K.L. 2002 Microbiological Analyses. pp 73-79, Encyclopedia of Dairy Sciences, Editors Roginski, H., Fuquay,J.W. and Fox, P.F. Academic Press.
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Progress 01/01/01 to 12/31/01
Outputs
Selective/differential media (Tellurite MacConkey Sorbitol agar-TSMAC) did not allow recovery of E. coli 0157 from cultured buttermilk whereas a non-selective chrom agar (without selective agents) allowed recovery of these stressed cells (5-10 fold higher counts). Liquid media (buffered peptone and modified Ec broth) used as diluents caused further damage to stressed E. coli in buttermilk. Use of milk as the diluent protected these stressed E. coli and allowed higher recovery.
Impacts
This would permit more accurate determination of death kinetics of E. coli in cheese during aging, thus a safer cheese for human consumption.
Publications
- Poms, R.E. and Tatini, S.R. 2001. Survival of Helicobacter pylori in ready-to-eat foods at 4 deg C. Intl. J. Food Microbiology. 63:281-286.
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Progress 01/01/00 to 12/31/00
Outputs
Selective - differential agar (xylose - lysine - desoxycholate; XLD) used for detection of Salmonella in foods is unsuitable for quantitative recovery of Salmonella especially if the organisms sustained injury as in fermented foods (cheese, buttermilk) or dry foods as toasted oats. Deleting the sodium desoxycholate from XLD enhanced the quantitative recovery of both injured and healthy cells; two to eight fold higher with healthy Salmonella cells and 20-50 fold higher with stressed cells in Cheddar cheese and buttermilk and 10 fold higher in naturally contaminated toasted oats from an outbreak. Trypticase soy agar containing yeast extract and the indicator system (ferric ammonium citrate-sodium thiosulfate) also enhanced recover of Salmonella (as black colonies) in the above foods and in a similar manner to that of XLD without desoxycholate.
Impacts
The use of nonselective medium containing differential agents would improve quantitative recovery of Salmonella from foods. This will result in a more accurate determination of infective dose and assist in quantitative microbiological risk analysis.
Publications
- Velazquez, M., Tatini, S.R. and Feirtag, J.M. 2000. Evaluation of a two-step protocol for rapid detection of Salmonella in ice-cream and cheddar cheese. Food Microbiology. 17:349-359.
- Heinitz, M.L., Ruble, R.D., Wagner, D.E. and Tatini, S.R. 2000. Incidence of Salmonella in fish and seafood. J. Food Protection. 63:579-592.
- Kersten, I.W. 2000. The use of freeze-thaw of unpasteurized apple cider as an alternative method to pasteurization. M.S. Thesis. University of Minnesota. 120 pages.
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Progress 01/01/99 to 12/31/99
Outputs
Frozen storage of unpasteurized apple cider in 1/2 gallon and 1 gallon containers for 7 days at -16.8 deg C, defrosting and holding juice at 4 deg C for 14 days did not result in the 5 log FDA mandated decrease of E. coli and Salmonella. However, the addition of the approved preservative (sodium benzoate allowable level up to 0.1%) enhanced inactivation of E. coli and Salmonella in frozen defrosted juice; 0.07% was more effective than 0.05% while both levels of sodium benzoate caused 5 log destruction of E. coli and Salmonella in 1/2 gallon containers, 0.07% was required for 1 gallon containers (7 days frozen storage, defrosting and holding at 4 deg C for 3 days).
Impacts
Freezing apple cider is a simple process that can be used by the cider producers. This will also allow marketing juice throughout the year if there is demand for unpasteurized juice.
Publications
- Elliason, D.J. and Tatini, S.R. 1999. Enhanced inactivation of inactivation of Salmonella typhimurium and verotoxigenic Escherichia coli by nisin at 6.5 deg C. Food Microbiology. 16:257-267.
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Progress 01/01/98 to 12/31/98
Outputs
Frozen storage of unpasteurized apple cider at -16.8 C for 3 weeks, defrosting and then holding at 4 C for 5 days resulted in an overall average destruction of 5.8 + or - 0.4 log for four strains of enterohemorrhagic Escherichia coli (0157 and other serotypes). When sodium benzoate was used as a preservative, 5.9 log destruction of these strains required one week of frozen storage at -16.8 C and holding thawed juice at 4 C for only 3 days. A 5.7 log destruction of three strains of Salmonella was also achieved in frozen apple cider (at -16.8 C) for 3 weeks followed by thawing and holding at 4 C for 3 days. Thus, the above process would meet the FDA guidelines (5 log destruction of E. coli) relating to alternate methods to pasteurization to achieve food safety.
Impacts
(N/A)
Publications
- KAUSHIK, B.S. 1998. Control of verotoxigenic Escherichia coli in fruit juices. M.S. Thesis. Univ. Minnesota.
- ELLIASON, D.J. 1998. Inhibition of Salmonella typhimurium and verotoxigenic Escherichia coli by low temperature and metabolites of lactic acid bacteria. M.S. Thesis. Univ. Minnesota. 108p.
- KAUPPI, K., O'SULLIVAN, D.J. and TATINI, S.R. 1998. Influence of nitrogen source on low temperature growth of verotoxigenic Escherichia coli. Food Microbiol. 15:355-364.
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Progress 01/01/97 to 12/31/97
Outputs
Verotoxigenic E. Coli (VTEC) including 0157:H7 added to commercially pasteurized juices at 1000/ml decreased to <1/ml in 1-2 days at 37 deg C. This decrease to <1/ml depended upon pH and type of juice (acid); at 22 deg C it was 4 days at pH 3.0 and 7 days at 3.4 in grape juice whereas it was 10 days in apple, grape fruit, pineapple and orange (pH 3.5-4.0). At 4 deg C, this decrease to <1/ml was 11 days and 28 days in grape juice of pH 3.0 and 3.5, respectively, in apple juice it was 42 days in apple juice of pH 3.5 and 62 days at pH 3.7. In other juices (pH 3.7-4.0) it was 62 days at 4 deg C. VTEC decreased 3 logs in 1 hour at 52 deg C in apple juice of pH 3.2. VTEC added to apple juice before freezing at -20 deg C decreased 1 log after defrosting and the survivors (2 logs) died during 7 days when the juice was held at 4 deg C. Increasing acidity of juices (blending juices), exposing to low heat (52 deg C) and freeze/thaw will cause injury and death of VTEC and a
combination of these (acidity and low heat, acidity and freeze/thaw and acidity, low heat and freeze/thaws) could be used as an alternate to pasteurization of fruit juices to enhance food safety.
Impacts
(N/A)
Publications
- HOFFMAN, C.A., WAGNER, D.E. and TATINI, S.R. 1997. Comparative study of recovery of Salmonella javiana from Mozzarella cheese by two official analytical procedures. J. Food Protection. 60:1493-1496.
- VOUGHT, K.J. and TATINI, S.R. 1997. Salmonella enteritidis contamination of ice cream associated with a 1994 multistate outbreak. J. Food Protection. In Press.
- KAUPPI, K.L. 1997. Influence of temperature and media composition on the growth and survival of verotoxigenic Escherichia coli. M.S. Thesis. 80 pp.
- JOHNSON-M, M.B. 1997. Inhibition of gram positive and gram negative pathogens by Bifidobacteria spp. M.S. Thesis. 105 pp.
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Progress 01/01/96 to 12/30/96
Outputs
Bifidobacterium infantis strain 15702 produced antimicrobial compounds in peptone yeast extract or cheddar cheese whey supplemented with yeast extract. Methanol-ethanol (CMA) extracts from 1 liter of lyophilized growth showed low pH (4.0 ( 0.2) and caused instant destruction of Escherichia coli 0157:H7 and Helicobacter pylori. This bacteriocidal activity was in part due to acetic and lactic acids. MA extract from fermentor growth of B. infantis with constant pH (6.5). also caused instant destruction of #E. coli 0157H7 and H. pylori. Use of a combined nisin positive Lactococcus lactis and B. infantis as starter culture to make Cheddar and Camembert cheeses resulted in more rapid destruction of E. coli 0157:H7 and Salmonella typhimurium in cheese during ripening at 7(C, than that which was made with a commercial cheese culture. This indicates the potential of antimicrobial producing lactococci and bifidobacteria to enhance food safety.
Impacts
(N/A)
Publications
- RUSSANOV, B., HAWKINS, D.M. and TATINI, S.R.. 1996. Estimating bacterial densityfrom tube dilution data by a Bayesian method. Food Microbiol. 13:341-364.
- KAUPPI, K.L., TATINI, S. R., HARRELL, F. and FENG, P. 1996. Influence of substrate and low temperature on growth and survival of verotoxigenic Escherichia coli. Food Microbiol. 13: 397-405.
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Progress 01/01/95 to 12/30/95
Outputs
Methanol-acetone extracts from six Bifidobacterium species inhibited both gram negative (Salmonella and E. coli 0157:H7) and gram positive (Listeria monocytogenes and Bacillus cereus) pathogens in vitro. B. infantis 15702 showed the largest zone of inhibition with each of the four pathogens. B. bifidum 15696 showed the largest zone with L. monocytogenes but only showed a smaller zone with Salmonella or E. coli 0157:H7, perhaps suggesting a difference in the nature of inhibitor(s) produced. Thus, these data suggest the potential for use of these desirable microbes which are the flora found in healthy humans, to control gastrointestinal pathogens by consuming foods containing the Bifidobacteria. Nisin, an antimicrobial produced by lactococci and used to control gram positive spoilage or pathogens in foods (Clostridium botulinum or Listeria monocytogenes), was found to inhibit and inactivate Salmonella species and also E. coli 0157:H7, during refrigerated storage; 1-2
log decrease of E. coli over 7-14 days at 4 or 7oC in peptone broth. This effect was reduced in foods due to the effects of food components, protein, fat and especially divalent cations (calcium and magnesium). Magnesium afforded greater protection against the action of nisin than calcium.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Growth and survival behavior of verotoxigenic Escherichia coli 0157:H7 strains at refrigeration temperatures indicated that the minimum temperature was dependent upon the availability of an energy source and the medium. All 15 strains grew in milk at 6.5oC and died in Brain Heart Infusion (BHI) or Tryptic Soy Broth (TSB). Addition of 2-3% level of glucose or lactose which is present in milk, allowed survival and supported growth at a rate similar to milk in TSB; 48-50 hours generation time. Growth rate in BHI plus glucose or lactose was slower than milk or TSB + Lactose; 115 hrs vs. 48.50 hours. Verotoxigenic E. coli strains may grow in milk at abusive refrigeration temperatures such as 6.5oC that might exist in homes and within the expected shelf-life of 2 weeks. Addition of 500IU nisin or nisin producing nonlactose fermenting Lactococci to refrigerated milk caused inhibition of Salmonella strains including S. enteritidis which was involved in a recent multistate
outbreak. Salmonella strains subjected to freezing stress were susceptible to the action of nisin, however, in ice cream a single freezing and defrosting did not cause sufficient injury to enhance their susceptibility to nisin. Nisin may be useful in sea food which is subject to multiple cycles of ice, ice water and freezing, to control Salmonella.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Salmonella typhimurium cells suspended in 0.1% peptone and exposed to 100 IU of nisin exhibited injury leading to death (3 log reduction) over a 2-week period at 4(degree)C. A higher concentration of nisin (500 IU) was required to cause injury and death (1 log reduction) in whole milk at 4(degree).C. Addition of viable cells of specific non-lactose fermenting and nisin-producing lactococci (10(superscript 9) cells/ml) to raw or sterile milk caused inhibition of growth of S. typhimurium and Escherichia coli 0157:H7 at 12(degree)C. The inhibition was greater in raw milk: no increase of salmonellae or E. coli for 2 days in either raw or sterile milk with Lactococcus lactis culture (354/07) compared to a 2-log increase without lactococci; a 2-log increase of pathogens in sterile milk vs. no increase in raw milk with lactococci after 4 days at 12(degree)C. This enhanced inhibition of lactococci in raw milk varied with source of milk possibly due to differing levels of
lactoperoxidase. Addition of these lactococci with 200 IU of cell associated nisin to raw or sterile milk caused inactivation of psychrotrophic Bacillus cereus and Listeria monocytogenes at 7(degree)C or 12(degree)C due to cell associated nisin and lactoperoxidase. Direct addition of nisin (250 IU/ml) to milk containing B. cereus spores and heating at 72(degree) for 5 min (pasteurization) caused inhibition of outgrowth of germinated spores at 7(degree)C over a 9-day period. Nisin appears to be an effective agent in controlling pathogens at refrigeration temperatures.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Thermally stressed Listeria monocytogenes strains (55(degree)C for 60-90 min) could not be enumerated directly on selective media (oxford or palcam) primarily due to the effect of the concentration of LiCl: a 3 log difference in count on nonselective agar vs. oxford/palcam. The antibiotic supplements of these media had only a small effect (0.5 log) on stressed L. monocytogenes. Reducing LiCl to 0.5 or 0.75% and addition of defibrinated sheep blood or egg yolk enhanced recovery by 10-100 fold depending upon the strain. Repair in trypticase soy broth at 37(degree)C for 5-6 hr prior to plating on the modified media further enhanced recovery of listeria; the count was within 2 fold on modified agar and still 4 fold less on palcam or oxford compared to nonselective media. Listeria monocytogenes (Scott A and V7) attached to turkey skin (3 x 2 cm sections) and heated at 52(degree)C for 3 min in poultry scald water showed no decrease whereas addition of 100 IU visin per ml to
this scald water, resulted in a 2 log decrease of both strains of L. monocytogenes due to the combined effect of nisin and heat. Nisin alone without heat resulted in 1 log decrease of listeria. Likewise, Salmonella typhimurium and S. senftenberg attached to turkey skin also showed a 1 log decrease when heated in poultry scald water with 100 IU nisin/ml. Thus, addition of this natural antimicrobial to poultry scald tank can help reduce the subsequent cross contamination chances downstream and also reduce the overall incidence of pathogens.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Competitive inhibition of Listeria monocytogenes by lactic starters during the early stages of fermentation of milk or in Cheddar cheese makes L. monocytogenes insensitive to the lytic action of nisin (4 log destruction of pure culture vs. 1.0 log for associative culture of L. monocytogenes with non-nisin starter when these were transferred to a 10-hour culture of nisin producing lactic starter). Thus, addition of 200 IU/ml of nisin to Cheddar cheese milk and use of nisin producing lactic starter resulted in a 1.5 log decrease of L. monocytogenes in Cheddar cheese produced under the above conditions with 3 x 10 listeria/ml of cheese milk contained < 1 listeria/g in fresh and 4-week old cheese. Thermization of milk containing 100 IU of nisin/ml (65 degree C for 1 min) resulted in a 6 log destruction of L. monocytogenes (Scott A) as compared to a 3 log destruction with heat alone. Addition of 100-250 IU of nisin/ml to poultry scald water (52 degree C for 3 min)
enhanced destruction of listeria (experimentally) attached to the skin of chicken thighs (0.6 to 1 log). Exposure of chicken thighs with listeria to poultry scald water at room temperature (no heating) caused a 2 to 2.5 log decrease of listeria. Addition of nisin to raw milk or poultry could be useful in inhibiting growth at < 5 degree C and also destroy listeria in conjunction with sublethal heating (thermization or scalding temperatures).
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Associative growth of rapid acid and nisin-producing lactococci (L. lactis NCDO 1404 and L. cermoris JS102) caused a 2.5 log destruction of Listeria monocytogenes during fermentation of milk and a 2.0 log destruction during manufacture and aging (up to 1 week) of Camembert cheese when compared to non-nisin lactococci. Further, there was a 3-week lag before regrowth of listeria during aging of Camembert. Control of listeria contamination of cheese milk (<10/ml) and use of nisin lactococci should enhance the safety of aged Camembert. Nisin-producing lacotococci were ineffective for control of listeria in stirred curd Cheddar chesse at contamination levels of 10 or 10 L. monocytogenes/ml of cheese milk. Nisin enhanced thermal destruction of L. monocytogenes in poultry scald water of pH 6.8 with 25 IU/ml and 52C for 3 min and in shrimp slurry (Ph 6.5) with 250 IU/ml at 55C for 5 min. In poultry scald water there was a 3 log decrease of listeria by the combined effect
of nisin and heat vs. a maximum of 1.2 log decrease with nisin or heat. In shrimp there was a 2.5 log decrease with nisin plus heat vs. 1.3 log with nisin or heat alone. Inhibition of lactics with LiCl (0.3-0.5) in the FDA and IDF listeria enrichment broth was essential for recovery of 1-2 listeria present in 25 ml of fermented milk which otherwise yields a false negative result. Initial adjustment of the pH of Cheddar cheese/enrichment blends to 7.0-7.5 was adequate to recover 1-2 L. monocytogenes/25 g.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Nisin was bactericidal and/or bacteriostatic to Listeria monocytogenes (7 strains) depending upon the pH and temperature. Minimum inhibitory concentration in tryptic soy broth plus yeast extract was 512-1024 and 128-256 i.u./ml, respectively, at pH 6-7 and 5.0 at 35 degrees C. At 4 degrees C and pH 7.0, listeria survived 6 months without growth. At 15 degrees C and pH 4.5 and 5.0 (lactic acid), listeria decreased from 1 x 10 to less than 1/ml within 10 days in Ellikers broth and 21 days in fermented whole milk. Associative growth of one of two nisin producing Lactococcus lactis subsp. lactis (11454) in reconstituted nonfat dry milk or whole milk at 35 degrees C, inhibited growth of L. monocytogenes as compared to commercial cheese cultures which allowed a 1-2 log increase. L. lactis (11454), when used as starter culture to make Camembert cheese, inhibited listeria growth during manufacture (24 h) and also enhanced decrease of listeria during aging and/or storage at
4-13 degrees C (0.5 log decrease with commercial cheese culture vs. 1.2 log with 11454). Addition of 0.1% diacetyl, to brine used for soaking nisin containing cheese made with 11454, enhanced decrease of Salmonella typhimurium during aging at 13 degrees C and storage at 4 degrees C over a 3-week period (0.5 log lower in the crust and 1.5 log lower in the interior of cheese).
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Purified nisin and that produced by Streptococcus lactis (strains DL16 and 11454) in Elliker's broth were bacteriostatic and/or bacteriocidal to Listeria monocytogenes depending upon the temperature (4, 15 and 34C), pH (7, 6 and 5) and the type of acid (HC1 vs. lactic acid). The minimum inhibitory concentration of nisi in tryptic soy broth was 512-1024 ppm at pH 7, 256-512 at pH 6, and 128-256 at pH 5.0 and 35C; 256 at pH 6.0 and 64-128 at pH 5.0 and 15C. Nisin delayed growth of listeria at pH 7.0 and 4C; 11-16 days for visible growth in control vs. 45-70 days with nisin. At low pH (5.0-5.5) lactic acid enhanced the bacteriocidal effect of nisin; listeria (3 strains) died out within 3 days with 256 ppm nisin at pH 5.0 (lactic acid), whereas they were able to grow after 4 days at pH 5.0 (HC1) and 35C. Two strains of listeria (Scott A and V(7)) were killed by heating at 55-57C for 6 minutes in the presence of nisin naturally produced in the supernatant of S. lactis
(11454) cultured in Elliker's broth (pH adjusted to 5.5), whereas both strains survived this heat treatment in Elliker's broth of pH 6.5 and no nisin.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Four of eight Staphylococcus aureus cultures claimed to be enterotoxigenic for six different enterotoxins (SE) by enzyme linked immunosorbent assay (ELISA) were found to be positive for only one SE (SEC) with microouchterlony and reverse passive latex assays. The other four were negative with these assays. Diacetyl was found to enhance thermal destruction of Listeria monocytogenes even in the presence of 60% sucrose. L. monocytogenes survived 24 hours in commercial chocolate at 60C. Enterotoxigenic strains of Bacillus cereus grew in Brain Heart Infusion broth within a range of 10-45C, within 6-9 hours at 21C, and after 5-7 days at 10-13C. Abusive refrigeration may result in growth and toxin production.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Purified tannins from persimmon and plantain did not cause complete inactivationof biological activity of staphylococcal enterotoxin B in monkeys. Additional components present in crude persimmon extract perhaps are required. Diacetyl and commercial starter distillate containing diacetyl caused more rapid inactivation of Listeria monocytogenes at 50C, 55C, 60C, or 70C even in the presence of 30% sucrose in Trypticase Soy broth containing yeast extract. These data indicate the potential use of starter distillate to control foodborne pathogens in high-sugar-containing products such as chocolate and candies. Conditions of growth of S. aureus (temperature and pH) resulted in production of other antigens, along with staphylococcal enterotoxin A (SEA), which showed binding to polyclonal or monoclonal antibodies in immunoblots. Pretreatment with trypsin and/or normal rabbit serum reduced this nonspecific binding of monoclonal antibodies by eliminating other antigens and not
SEA. This approach should make the monoclonal antibody assays more specific for rapid detection of SEA in foods.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
Purified tannins from persimmon and plantain showed differences in both the ratio of prodelphinidin-procyanidin (PD:PC) and binding capacity to staphylococcal enterotoxins A and B (SEA, SEB). Plantain tannin showed PD:PC of 72:28 vs. 65.35 for persimmon. Both tannins bound more effectively to SEB than SEA (1 mg of SEA required ca. 1 mg of tannin vs. only 0.6 mg for SEB to completely precipitate toxins). Diacetyl was found to be bacteriostatic to Salmonella typhimurium and Staphylococcus aureus at levels at 0.01-0.10% and 15 and 37C and was bactericidal at higher temperatures (55C for 15 min.) and levels of 0.25 to 0.50%, a 4 to 6 log enhancement in death of both organisms. Commercial starter distillate (SD) containing diacetyl showed similar effect on inhibition or destruction of spores of putrefactive anaerobe (PA3679) in cream style corn and sirloin burger. In cream style corn, survival at 110C for 28 minutes with 10 spores/10 g, growth in 2 weeks in the absence
of SD, and no growth even after 16 weeks under similar conditions with SD, were noted when incubated at 37C.
Impacts
(N/A)
Publications
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Progress 01/01/84 to 12/30/84
Outputs
Production of staphylococcal enterotoxin A (SEA) was proportional to growth at 14, 30, 37 or 45C under shaking or static cultural conditions in NZ amine broth. Release of SEA was greater at higher temperature (45C), at higher pH (8.5) and shaking conditions. Plate count agar containing 7.5% NaC1, 5% defribinated sheep's blood and Tryptone (PBS) was found to be a less expensive substitute for Baird-Parker agar (BPA) in recovery of Staphylococcus aureus from naturally contaminated commercial products such as cheddar cheese, genoa salami, ground pork and noodles (P<0.05). Recovery of heat stressed S. aureus though somewhat less efficient in PBS than PBA, from a practical standpoint is adequate. Monoclonal antibodies (McAb) produced against staphylococcal enterotoxin A showed very low reactivity with SEB; the titer with 1 mug SEA was 10E1-10E7 and only 10Ey with 10 mug SEB. Nonspecific binding of antimouse conjugates with crude extracts of S. aureus would not permit
the use of McAb to detect SEB in food extracts.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
Soluble tannins present in quebracho, black or green tea and black or red currant leaves did not inactivate the biological activity of staphylococcal enterotoxins A or B in Rhesus monkeys. Staphylococcus hyicus strains which share coagulase, thermonuclease and protein A characteristics with S. aureus were correctly identified through use of "Serostat" test. "Serostat" test would not only save time in enumerating S. aureus (2 min vs 6 hrs) from foods but also avoid false positive speciation ont he basis of tube coagulase test (current official test). Monoclonal antibodies developed against staphylococcal enterotoxin A (SEA) showed reactivity with SEE in the enzyme linked immunosorbent assay (ELISA). Studies with crude enterotoxins (A and E) suggest the potential existence of multiple molecular forms of each enterotoxin. This is useful in further refining enterotoxin assays.
Impacts
(N/A)
Publications
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Progress 01/01/82 to 12/30/82
Outputs
Soluble tannins naturally present in unripe American persimmon inactivated emetic activity of crude staphylococcal enterotoxins (SEA, B, C, and D) when these toxins were mixed with tannins and fed intragastrically to rhesus monkeys; 4 of 4, 6 of 6, 6/6 and 4 of 4 responding monkeys, respectively. This work might have potential application in processing enterotoxin contaminated foods to prevent economic losses. Plantain pulp inactivated SEA and not SEB; this would help in identifying specific tannins involved in inactivation and permit characterization of active sites. "Serostat" test showed 100% correlation with tube coagulase test, therefore, this test could save 1-1 1/2 days in enumeration of S. aureus in foods. Monoclonal antibodies specific for SEA were produced and these can be used for rapid assay of SEA in foods (4 hours vs. 3-5 days with traditional microouchterlony). These antibodies would also help in characterizing angigenic sites on SEA. This work is
useful in control of food poisoning from staphylococcal toxins.
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Progress 01/01/81 to 12/30/81
Outputs
Proteolyisis of milk or Cotto salami containing S. aureus cells with intracellular enterotoxins, with pepsin for 16 hrs. at 37 degrees C followed by lysostaphin treatment (50-500 units) for 6 hrs. did not result in release and detection of enterotoxins. However, this treatment resulted in detection of enterotoxins in cheddar cheese. Crude enterotoxin A was found to be negative in 5 Cynomologus and 2 Rhesus monkeys when fed with bananas. Feeding toxin A with bananna slurry and Ketamine sedation also did not result in positive assay (no vomiting) whereas all the monkeys vomited when fed saline diluted toxin with Ketamin sedation. These preliminary data may suggest the potential inactivation of enterotoxin A by bananas. More data using a larger number of fresh monkeys is needed to confirm this. Application of numerical taxonomy (NIH-FDA computer systems) to several Staphylococcal and few other strains resulted in 17 groups at 80% similarity level. Four groups
contained the majority of the strains. Of the 61 features used, the following practical tests were most useful in separating or speciating these four groups: Tube coagulase or protein A in cell wall, formation of acid from maltose or trehalose and protein A in cell wall, formation of acid maltose or trehalose and resistance to novobiocin.
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Progress 01/01/80 to 12/30/80
Outputs
Two strains of Staphylococcus hyicus subsp. chromogenes were found enterotoxigenic by monkey feeding tests. Intracellular enterotoxin A released from S. aureus cells by lysostaphin was found to be biologically active in monkeys when fed at levels comparable to extracellular enterotoxin. Feeding viable S. aureus cells containing intracellular enterotoxin A, caused vomition in 3 of 5 monkeys within 3 hours. S. aureus cells grown at low a(w) (0.90) released intracellular enterotoxins A and D only when resuspended at a higher a(w) (0.99). This release was temperature and pH dependent; release was greatest at 45 degrees C followed by 37 degrees C, 21 degrees C, and 4 degrees C, and was fastest at pH 8.5 as compared to 5 at each temperature. Though toxin was released faster at higher temperatures of 60 and 100 degrees C it was inactivated. S. aureus cells grown in pork at lower a(w) (adjusted with sucrose, 0.91) showed higher level of intracellular enterotoxin A than
that accumulated at 0.95 or 0.98. S. aureus grown on pork, ham or beef at 21 degrees C showed intracellular enterotoxins A and D. Cotto salami and Cheddar cheese agar systems supported growth and production of extra and intracellular enterotoxins A and D at 37 and 21 degrees C.
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Progress 01/01/79 to 12/30/79
Outputs
DNA-DNA hybridization studies with 20 staphylococcal strains against the 3 knownspecies (Bergey's Manual; 8th ed., 1974) indicate and confirm the existence of additional species as S. hyicus which shares thermonuclease, cell wall protein A and coagulase with S. aureus. S. hyicus strains showed insignificant DNA-homology (less than 20%) with S. aureus, S. epidermidis or S. saprophyticus. Three S. hyicus strains were confirmed as enterotoxin producers by monkey feeding tests. S. aureus cells grown on food agar plates at 37 C (beef, pork, or ham) showed presence of intracellular enterotoxins A, B, and E. Cells grown at pH 4.5 in pork agar showed 2-3-fold higher intracellular enterotoxin A than cells grown at pH 5.5 or 6.0. Cynomologus monkeys responded to oral feeding of enterotoxin A by vomiting within 4 hours and varied in sensitivity; the most sensitive ones (among the 18 fed) responded to crude enterotoxin A levels as low as 7.5 Mug regardless of the body
weight. S. aureus cells released enterotoxin A without death or lysis by pancreatic enzyme(s) activity within 30 min.
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Progress 01/01/78 to 12/30/78
Outputs
Several staphylococcal strains (150) acquired from various sources were examinedfor cultural, physiological, and biochemical characteristics. Speciating on the basis of the commonly used diagnostic tests as coagulase and thermonuclease (TNase) in conjunction with other tests did not result in a clear cut differentiation of Staphylococcus aureus from S. epidermidis. Tritium-labeled DNA was prepared from neotype strains of S. aureus and S. epidermidis for further work to establish species designation with DNA-DNA hybridization. Five of 16 strains of S. aureus produced enterotoxin D and 3 of the 5 were weak TNase producers. Biological activity (in cats) of enterotoxin E in Similac was inactivated between 5 and 10 min at 121.1 degrees C. Crude enterotoxin E, in Brain Heart Infusion (BHI) saline of pH 7.5 and 6.5, heated for 15 min at 121.1 degrees C while showing the same serological residual titer in both was biologically active only with pH 7.5. S. aureus (z88) grown
in BHI with added food preservatives as propyl, methyl, or butyl esters of para droxybenzoic acid and propylene or butylene glycols produced smaller amounts of extracellular enterotoxins (2-5 fold) and higher levels of intracellular enterotoxins (5-10 fold) over BHI control per unit growth. S. aureus cells grown at 15 and 21 degrees C showed presence of intracellular enterotoxin A at higher levels (per unit growth) than at 37 degrees C.
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Progress 01/01/77 to 12/30/77
Outputs
Adjustment of a(w) of the growth medium (PAP plus amine) to 0.98-95 with sucroseor glycerol repressed enterotoxin A synthesis by Staphylococcus aureus (Z88). A further decrease of a(w) from 0.95 to 0.90 increased the ratio of intracellular to extracellular enterotoxin A indicating the restrictive influence of a(w) on release of enterotoxin. Restriction to release occurred at 0.92-0.88 in NaCl medium. Cells grown in glycerol medium of 0.90 a(w) released amost 40% of intracellular enterotoxin upon resuspending in a medium of 0.99 a(w) and shaking for 15 hours at 4 degrees C and none at 0.90 a(w). High concentration of glycerol (A(w) 0.90) stimulated production of total extracellular proteins (TEP) other than enterotoxin A and thermonuclease. This stimulative effect of glycerol on TEP was observed with several staphylococcus aureus strains and it varied 2-26 fold over the control Propylene glycol and butylene glycol inhibited growth and extracellular enterotoxin A
production by S. aureus (Z88). However, the amount of intracellular enterotoxin A per cell was higher in cells grown in the presence of these glycols than in the control. Seventy percent of thermonuclease positive strains of S. epidermidis biotype 2 produced trypsin resistant biologically active toxins to cats (I/P injection) and only 2% produced serological type E toxin.
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Progress 01/01/76 to 12/30/76
Outputs
Humectant used to adjust a(w) of PHP+NZ amine medium influenced growth rate of Staphylococcus aureus (Z88). Generation time at 37C was shorter with sucrose or NaCl as compared to glycerol or restricted water system at each a(w); 25 vs.40 min at 0.95 a(w) and 50 vs 120 min at 0.90 a(w), respectively. Amount of extracellular enterotoxin A was substantially reduced with sucrose and increasesd with NaC1 as compared to either glycerol or water restricted system at each a(w). Only trace amount of intracellular enterotoxin was detected at 0.99 a(w) and in NaCl system of 0.95 or 0.90 a(w), whereas cells from glycerol system showed higher amount of intracellular enterotoxin. A diffusible component from brewer's yeast stimulated enterotoxin A and not B production. Brewer's yeast enhanced enterotoxin A production in ground beef and bread dough. A rapid method for detection of enterotoxin A in food which yields confirmed results within 30 hrs. was developed. The time saving
step was concentrating enterotoxi by TCA precipitation and resolubilizing in a small volume for assay. 99% of 95 coagulase positive and only 5% of 84 coagulase negative staphylococci from vari sources produced thermonuclease (results from this year and last year). Enterotoxin E is extremely heat stable; its biological activity in cats withstood 20 min. heating, whereas the serological activity withstood 22 min. heating at 121.1C with 1 mu g toxin/ml initially.
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Progress 01/01/75 to 12/30/75
Outputs
Both pH and a of the growth medium (protein hydrolysate powder plus NZ amine NAK) influenced the production of enterotoxins A and D by Staphylococcus aureus (Z88). Enterotoxins were produced in detectable amounts when the a was 0.99 atall pH values tested (7, 6 or 5). Though S. aureus grew well when a was 0.94 at all pH values, enterotoxins were detected at pH 6 and not at 5. when a was 0.89, pH 6 supported enterotoxins production. When a was 0.86, despite good growth (4x10(8)/ml), at pH 7 enterotoxins were not produced in detectable amount. The limiting a for production of enterotoxins varied with S. aureus strain. Only 1 of 6 strains (F625) tested produced enterotoxin A when was 0.86 and pH was 7 at 37C. Though isolated soy proteins did not support enterotoxin Aproduction by S. aureus (Z88, Fyae1 or 418), soy flours (from three different commercial sources) supported enterotoxin A production. Following conditions were found optimal for micrococcal nuclease (E.C.
No. 3.1.4.7; Sigma Chemical Co.) as well as crude staphylococcal nuclease from growth of S. aureau (Z88): pHof 10, calcium level of 0.0005M and incubation temperature of 45 or 50C. S. aureus strains (600) from various ecological sources produced heat-stable nuclease. Porcine plasma yields a more complete and rapid clot formation than rabbit plasma in the tube coagulase test for identification of S. aureus.
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Progress 01/01/74 to 12/30/74
Outputs
Neither growth nor production of heat-stable deoxyribonuclease (DNase) and enterotoxins by Staphylococcus aureus occurred during curing, smoking or drying (4 weeks at 10C) of pepperoni inoculated initially with 10,-10ae cells/g. Duringfermentation of Thuringer (37C for 14-18 hur), S. aureus (101-10>/g) grew and produced detectable amounts of DNase in the inner and outer parts. However, enterotoxin A was produced in detectable amounts only in the outer part with 2x10> initial and 1x1014 final S. aureus/g. Enterotoxin A and DNase survived cooking of Thuringer to an internal temperature of 65C. S. aureus grew faster and reached a higher final population with brewer's yeast as the source of protein as compared to fish protein concentrate or soy; generation time of 30, 56 and 50 min respectively. Addition of yeast to whole milk enhanced growth of S. aureus and production of DNase and enterotoxins. Reduction of Aw of PHP+NZ amine from 0.99 to 0.94 resulted in a 3-fold
increase in generation time of S. aureus; however, detectable amounts of enterotoxin A were produced in both caseswith a final population of 2-5x10ae/ml. Enterotoxins A and D (added at 0.005 ug/ml) survived heating of whole milk at 143.3C for 9 sec. All the 400 strains of staphylococci produced heat-stable DNase.
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Progress 01/01/73 to 12/30/73
Outputs
Detectable amounts of enterotoxins (A and D) and heat-stable nuclease (DNase) were produced in smoked sausage between 2 and 3 hours of processing (45C internal temperature of product) with 1 x 10> Staphylococcus aureus/g. Cooked bologna supported growth and production of DNase and enterotoxins by S. aureus at 10 and 45C, whereas production of neither occurred during curing, smoking andcooking of bologna (9 hours processing at 10-45C). Production of DNase and enterotoxin A was better in cooked than in raw pork or beef. Sodium nitrate (1,000 or 2,000 ppm) and sodium nitrite (200 or 400 ppm) showed lack of inhibitory effect on production of enterotoxins. Three of fourteen spices (garlic, ginger and cloves) which showed inhibition of S. aureus growth on Trypticase Soy Agar exerted no inhibitory effect when tested at levels of normalusage (0.2-0.4%) in sausage and bologna. S. aureus exhibited a long lag (9-12 hours) in production of enterotoxins and DNase under shaken
conditions at 45C. Commercially processed foods (butter, dried malted milk, nonfat dry milk and fermented sausage) involved in food poisoning showed > 5 ug DNase 20g. 96% of 112 strains of staphylococci produced heat-stable deoxyribonuclease (DNase) and 4% produced neither heat-stable nor heat-labile DNase.
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Progress 01/01/72 to 12/30/72
Outputs
Studies on biosynthesis of enterotoxins and deoxyribonuclease (DNase) by Staphylococcus aureus showed production of toxins A and D and DNase per cell notaffected by stage of growth (lag, early or late log phase). Non-replicating cells produced trace amounts of toxins and DNase and this was prevented by metabolic inhibitors (NaN(3), 2,4-dinitrophenol and chloremphenicol). With 8 strains of S. aureus, production of toxins (A, B, C, D) and DNase decreased at 25 C or lower as compared to 35 or 45 C in cooked meat medium (BBL). Limited growth (2-4 fold increase in population) and DNase production occurred at 1-6 hrs (but not at 6-15 hrs) during smoking of commercial sausage. Inactivation oftoxins A and D was affected by pH and temperature. Toxin A inactivated faster at lower pH (4.5, 5.0, 5.5) and toxin D at higher pH (6.0, 6.5, 7.5) regardless of concentration. Toxin A was inactivated more rapidly at 70 C than at 80 or 90C and toxin D at 80 C than at 70 or 90 C.
This rapid loss of serological activity was apparently due to aggregation of toxin protein, because in the presence of 6 M urea inactivation at 70 C was much slower.
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Progress 01/01/71 to 12/30/71
Outputs
The usefulness of heat stable deoxyribonuclease (DNAase) assay for assessment ofstaphylococcal growth and the likely presence of enterotoxin in cheese was evaluated. DNAase was assayed by a modified Lachica method which detected as little as 0.1 ug/ml purified DNAase and yielded a pink zone of 4.0 mm. The zonesize varied linearly with log. concentration of DNAase. The amount of DNAase varied with the strain of S. aureus, the type and amount of starter used, the presence of normal or inhibited starter, and the type of cheese. However, cheeses (Cheddar, Colby and Brick) positive for enterotoxin always showed a zoneof 4.5 mm or greater. This size zone was equivalent to 3-30 million S. aureus per gram in cheeses of partial or completely inhibited starter and to 15-50 million in normal starter cheeses. In Blue cheese, a zone of 5.0 mm was equivalent to 50 million S. aureus per gram with no detectable enterotoxin. In Swiss cheese, toxin was detectable at 7 million
per gram and 4.0 mm zone with a heat resistant strain and no toxin was detectable at 35 million and 4.5 mm zone with a heat sensitive strain.
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Progress 01/01/70 to 12/30/70
Outputs
To determine if detection and quantification of staphylococcal nuclease could beused for assessment of staphylococcal growth and thus, the likely presence of enterotoxin in cheese, Cheddar and Colby cheeses and reconstituted nonfat dry milk in which S. aureus grew to varying populations were examined by a microslide procedure developed for assay of nuclease in broth. Preliminary results indicate that this procedure can be adapted for testing cheese and milk.Cheddar and Colby cheeses made with normal and inhibited starter in which S. aureus grew to over 70 million per gram and produced enterotoxin A were nucleasepositive. Cheeses in which S. aureus reached less than 12 million per gram showed no nuclease by this procedure.
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Progress 01/01/69 to 12/30/69
Outputs
Work was continued to gather additional information on factors influencing staphylococcal enterotoxin A production in milk and in various types of cheese; particularly to develop the relationship between population and enterotoxin production, and to establish the minimum enterotoxigenic staphylococcus populations to result in detectable enterotoxin. Growth of S. aureus to a levelof 2-3 million per ml in low count raw or heated milk resulted in toxin. S. aureus populations of 3-8 million per ml in commercial raw milks did not result in toxin. The minimum population of S. aureus associated with the presence of enterotoxin in Cheddar and Colby cheese was 3-5 million per gram where starter failure occurred early in the manufacturing process. With normal starter activity, toxin appeared at 15 million per gram in Colby and at 30 million per gram in Cheddar. Toxin production under similar conditions was demonstrated in Brick and Swiss cheeses. Toxin production
occurred at substantially lower S. aureus populations (7-13 million per gram) in Swiss cheese than in Cheddar, Colby or Brick cheeses of normal starter activity. Enterotoxin could not be demonstrated in Blue or Mozzarella cheeses in which the S. aureus reached a maximum count of 25-50 million and 15-20 million, respectively, during cheese manufacture.
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Progress 01/01/68 to 12/30/68
Outputs
Studies were conducted relative to the effects of the initial bacterial count and sub-pasteurization heat treatment of raw whole milk and the initial pH of sterile nonfat milk solids (10%) on growth and enterotoxin A production by S. aureus, strains 196E and F265. Enterotoxin A production occurred in low count (< 1,000/ml) raw whole milk and sterile reconstituted NFMS at approximately the same lowest population of S. aureus (3-5x10ae per ml) there was some growth inhibition of S. aureus but enterotoxin A was still produced. Growth and enterotoxin A production were inhibited in raw whole milk (15x10, per ml) even when S. aureus was inoculated at 22x10, per ml and 9.7x10, per ml. Good growth and enterotoxin A production occurred in the same milk after heat treatment (150oF or 161oF for 16 sec). Growth and enterotoxin A production were observed at pH values of 4.3.-4.5, 5.0, 5.5, 6.0 and 6.5 in sterile NFMS. There was destruction of S. aureus and no enterotoxin A
production when pH was adjusted to4.3-4.5 with lactic acid but not with HC1. Colby cheese supports enterotoxin A production better than Cheddar. Enterotoxin A was observed at about 15x10ae /g S. aureus in Colby and at about 35x10ae /g in Cheddar made under normal starter activity. Even a partial starter failure resulted in presence of enterotoxin A at population levels of 3.3 million-11 million per gram.
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