BEHAVIOR OF STAPHYLOCOCCUS AUREUS IN REFRIGERATED AND FROZEN GROUND BEEF AND IN TURKISH STYLE SAUSAGE AND BROTH WITH AND WITHOUT ADDITIVES

Staphylococcus aureus 42E was inoculated into raw ground beef which was stored at 4C and -27C. Turkish style sausage and Brain Heart Infusion broth (BHIB), with and without additives were also inoculated with S. aureus and held at 4C. Samples of ground beef and sausage were examined for S. aureus, aerobic plate count and pH values. The numbers of S. aureus cells were decreased by 1.64 and 2.02 log units in ground beef held at 4C after 7 days and -27C after 7 weeks, respectively. A decrease in the number of S. aureus cells was observed in sausage and BHIB with addition of 0.5% trisodium phosphate (TSP), 78 mg/kg sodium nitrate, 78 mg/kg sodium nitrate plus 156 mg/kg sodium nitrate and 78 mg/kg sodium nitrite plus 0.5% TSP. Overall, sodium nitrite either alone or in combination with sodium nitrate was more effective on S. aureus and aerobic bacteria than TSP either alone or in combination with sodium nitrite.     

INCIDENCE OF STAPHYLOCOCCUS AUREUS IN MEALS FROM CAFETERIAS

A study to determine the incidence of Staphylococccus aureus in 705 meals from several cafeterias in Valencia, Spain was undertaken. Out of the 705 studied meals, 81 samples (11.1%) had coagulase positive staphylococci with a range of S. aureus from 100 to 53000 cfu/g. The four highest values (> 10,000 S. aureus cfu/g) were obtained with Russian type salad, meatballs, chicken croquettes and salad. These products involved more handling stages. These data show that food handlers may contribute to food poisoning and that there are some handling practices that require more attention.     

INFLUENCE OF pH ON FREEZE-THAW LETHALITY IN STAPHYLOCOCCUS AUREUS

The influence of pH on the susceptibility of Staphylococcus aureus 196E to repeated freeze-thaw stress was studied in 0.2 Macetate buffer (pH 3.0–7.8) and ground beef (adjusted to pH 4.2–6.3). In acetate buffer in the pH range of 4.4–7.0, repeated freeze-thaw stress did not decrease the viability of the cells; at pH values below 4.1 and above 7.5, decrease in the viable cells was exponential with the number of freeze-thaw cycles. In meat, S. aureus was not killed at pH values 4.3–6.3; however, at pH 4.2, death resulted from the repeated stress. Repeated freeze-thaw cycling of foods should have little effect on the viability of S. aureus within the pH values of most foods implicated in S. aureus food poisoning.     

GROWTH OF STAPHYLOCOCCUS AUREUS AND ENTEROTOXIN A PRODUCTION IN FOODS CONTAINING POLYPHOSPHATES

Effects of pyro-, tripoly- and hexametaphosphates (0.5 and 1%, w/w) on growth of Staphylococcus aureus strain 196E and enterotoxin A (SEA) production were studied in cooked custard and beef at 22 and 30°C. No effect was observed in custard, where cell numbers/g increased from 10³ to 10⁸ and SEA reached 4.2 ng/g after 48 h at 22°C, irrespective of treatment. Cell numbers in cooked beef were ca. 10⁹/g after 48 h, and reduced numbers (by 1.5–2 log cycles) were found in samples containing 0.5 and 1% pyrophosphate during incubation at 22%, but not at 30°C. SEA concentrations in beef were 28 ng/g after 48 h at 22°C, and 93 and 184 ng/g after 24 and 48 h, respectively, at 30°C. SEA concentration correlated with amount of growth, and was nondetectable when cell numbers were ± 10⁶/g. Reduction of the meat pH by sodium acid pyrophosphate contributed to the observed inhibitory effect.     

PROTECTIVE EFFECT OF LYSOSTAPHIN FROM STAPHYLOCOCCUS SIMULANS AGAINST GROWTH OF STAPHYLOCOCCUS AUREUS IN MILK AND SOME OTHER FOOD PRODUCTS

The effect of lysostaphin from Staphylococcus simulans expressed in Escherichia coli TOP10 strain on Staphylococcus aureus used for inoculation of milk, ground pork and mayonnaise salad was investigated. The populations of this pathogen in ultrahigh‐temperature milk preserved at 4C by lysostaphin added up to concentrations of 1.5 or 3.0 µg/mL were reduced by 0.73 and 0.92 log(cfu/mL) in control samples without enzyme addition. The protective influence of lysostaphin was diminished in case of milk storage (20C) prolonged up to 24 h. Furthermore, a final reduction level by 0.92 log(cfu/mL) was achieved after 24 h of pork storage. The smaller and more dependent on enzyme concentration inactivation of S. aureus was observed in the case of the mayonnaise salad, and it led to the conclusion that some food components or proteolytic enzymes originating from other bacteria caused lysostaphin inactivation.     

ELECTRON MICROSCOPY OF HEAT-INJURED AND REPAIRED STAPHYLOCOCCUS AUREUS

Changes in the ultrastructure of Staphylococcus aureus 196E during heating at 50°C in 0.1 M phosphate buffer, and during repair in nutrient medium were studied using transmission electron microscopy. Injury was assessed by differential plating on tryptic soy agar (TSA) + 1% pyruvate and on TSA + 7% NaCl. Injury was > 99% in the first 15 min. Mild aldehyde fixation or osmium tetroxide fixation gave good cellular preservation. The observation of ribosome-free areas in all heated cells at all times (≥15 min) of injury is consistent with published reports that rRNA destruction is a primary locus of injury in heated bacteria. Cells heated 45 min or longer, as well as cells starved 1–2 h in buffer at 35°C, generated a variety of internal membranes, typically near the DNA region. At 90 and 120 min, extreme alterations of structure were apparent indicators of cell death. Cells heated 90 and 120 min had virtually no ribosomes, exaggerated internal membranes, and surface blebs. During repair of cells injured 30 min, the population assumed normal appearance in 4 h, although some cells clearly were incapable of repair. After 6 h, the number of cells undergoing division increased. The reappearance of normal ultrastructure paralleled regaining of salt tolerance in the culture.     

生乳中金黄色葡萄球菌产肠毒素特性及耐药性分析

了解生乳中金黄色葡萄球菌污染状况、产肠毒素特性及耐药性，为乳及乳制品的生产及制定HACCP以保证食品安全提供依据。采集省内20个奶牛场共209份生乳样品进行金黄色葡萄球菌的分离、产肠毒素（SEA—SEE）试验及耐药性试验。共检出金黄色葡萄球菌34株，检出率为16．27％，21株产肠毒素试验阳性，阳性率为61．76％，对青霉素、缸霉素、四环素、苯唑西林凝固酶阴性耐药性分别达88．24％、41．48％、35．29％、35．29％，并出现21种耐药谱。生乳中金黄色葡萄球菌的污染率及产肠毒素率较高，耐药性结果对人及畜的安全用药有指导性作用。控制生乳中金黄色葡萄球菌的污染对奶及奶制品食用安全有意义。     

INACTIVATION OF STAPHYLOCOCCUS AUREUS EXPOSED TO DENSE-PHASE CARBON DIOXIDE IN A BATCH SYSTEM

ABSTRACT

The inactivation of Staphylococcus aureus exposed to dense‐phase carbon dioxide (DPCD) was investigated, and the kinetics of come‐up time (CUT) in pressurization was monitored with come‐down time (CDT) and temperature fluctuation in depressurization. CUT was about 2.5, 3.5, 4.0 and 4.0 min; CDT was 3.4, 3.7, 4.5 and 4.5 min; lowest temperature of samples in depressurization was 4, ?1, ?15 and ?22C, corresponding to 10, 20, 30 and 40 MPa at 37C. The inactivation behavior of S. aureus was closely related to the variables of process pressure, holding‐pressure time (HPT), process temperature and process cycling. The log reduction of S. aureus at 40 MPa for 30‐min HPT was significantly greater (P < 0.05), but the inactivation effect at 10, 20 and 30 MPa was similar. The log reduction of S. aureus at 30 and 40 MPa for 60‐min HPT was similar and significantly greater (P < 0.05), while the inactivation effect at 10 and 20 MPa was similar. The inactivation of S. aureus against HPT conformed to a fast–slow biphase kinetics; the two stages were well fitted to a first‐order model with higher regression coefficients R² = 1.000 and 0.9238; their respective D values (decimal reduction time) were 16.52 and 70.42 min. As the process temperature increased, the log reduction of S. aureus increased significantly (P < 0.05); the inactivation kinetics of S. aureus versus process temperature was characterized with a fast inactivation rate from 32 to 45C and a slow inactivation rate from 45 to 55C. As compared to one‐process cycling for a total of 60‐min HPT, four‐process cycling resulted in a significant reduction of S. aureus, and its maximal reduction was near to 5 log cycles, indicating that more process cycling caused more inactivation of S. aureus under identical pressure and temperature with equal HPT. However, the maximal reduction was 0.09 and 0.12 log cycles for two‐ and four‐process cyclings with 0‐min HPT, indicating that pressurization and depressurization had a lesser effect on the inactivation of S. aureus, while HPT was significant in DPCD to inactivate S. aureus.

PRACTICAL APPLICATIONS

Dense‐phase carbon dioxide (DPCD) is a novel technology to achieve cold pasteurization and/or sterilization of liquid and solid materials, and is likely to replace or partially substitute currently and widely applied thermal processes. This study showed that DPCD effectively inactivated Staphylococcus aureus inoculated in 7.5% sodium chloride broth, and the inactivation behavior of S. aureus was closely related to the pressure, holding‐pressure time, temperature and process cycling. Based on this observation, the technology of DPCD can be applied in the pasteurization of foods such as milk and various fruit juices, especially thermal‐sensitive materials.

     

INHIBITION OF AEROBIC AND ANAEROBIC GROWTH OF STAPHYLOCOCCUS AUREUS IN A MODEL SAUSAGE SYSTEM

Sodium nitrite, potassium sorbate, and glycerol monolaurate all inhibited anaerobic growth of Staphylococcus aureus more than its aerobic growth in an agar-meat model sausage system, and all were more inhibitory when lactic acid was added. Whereas anaerobic growth of S. aureus was inhibited by concentrations of 100, 2500, and 2500 ppm, respectively, of nitrite, potassium sorbate, and glycerol monolaurate, corresponding concentrations of 150, 5000, and 5000 ppm were required to inhibit aerobic growth ((20 meq lactic acid added).
Sorbic acid, a 3:1 mixture of sorbic acid and glycerol monolaurate by weight, and butylated hydroxyanisole (BHA), however, did not show differential inhibitory effects toward aerobic and anaerobic growth of S. aureus and also were more effective inhibitors with addition of lactic acid. Sorbic acid suppressed staphylococcal growth in the model sausage system at 500 ppm, the mixture of sorbic acid and glycerol monolaurate at 750 ppm, and BHA at 10,000 ppm (20 meq lactic acid added).     

SURVIVAL OF ESCHERICHIA COLI, STAPHYLOCOCCUS AUREUS AND SALMONELLA ENTERITIDIS IN FROZEN CHICKEN HAMBURGER

The survival of food pathogens during frozen storage of chicken hamburgers was evaluated. Hamburgers were contaminated with Escherichia coli (ECHC), Staphylococcus aureus (SAFH) and Salmonella Enteritidis (SE86), separately, and stored at −18C for 28 days. Results demonstrated reductions of 0.63 log₁₀ cfu/g, 0.73 log₁₀ cfu/g and 0.27 log₁₀ most probable number/g in the populations of ECHC, SAFH and SE86, respectively. These microorganisms were also stored at −18C in 0.1% peptone water and presented significantly different reductions ( P <  0.05) when compared with frozen hamburgers, suggesting that components of chicken hamburger may exert a cryoprotective effect on microbial cells.

PRACTICAL APPLICATIONS

Even though Brazilian poultry meat industries present high levels of quality control, the presence of bacterial pathogens in hamburgers may occur. In Brazil, chicken hamburgers are frequently commercialized frozen, and it is well established that frozen temperatures are able to avoid microbial growth. However, frozen temperatures can also be able to inactivate part of the bacterial population, and the magnitude of its effect will vary according to the type of food and the process that it was submitted. This study evaluated the survival of Escherichia coli , Staphylococcus aureus , and Salmonella Enteritidis in frozen hamburgers, aiming to verify if this process could provide an additional margin of safety to this product.     

ENTEROTOXIN BIOSYNTHESIS BY PROGENY OF REPAIRED HEAT-INJURED CELLS OF STAPHYLOCOCCUS AUREUS

By using a method which permitted the selection of repaired cells from a population of heat-injured and noninjured cells of Staphylococcus aureus 196E, we were able to determine that the progeny of repaired cells retained the ability to produce enterotoxin A (SEA). There were large variations in the amount of SEA produced by the progeny of individual colony forming units (CFU) before and after heating. The average amount of SEA produced by the progeny of noninjured and repaired staphylococci were similar and not significantly different.     

超声波平板法检测金黄色葡萄球菌生物被膜的研究

针对生物被膜具有普遍存在、难发现和难去除等特点，对食品工业有现时的和潜在的危害，并会引起食品安全问题。本文以金黄色葡萄球菌为研究对象，对定量检测生物被膜的超声波平板法进行条件优化，对优化的超声波平板法与平板擦拭法进行比较，并采用银染法进行验证。结果表明：优化的超声波平板法的最佳条件为：超声波功率135W，处理时间14min，处理温度35℃，优于平板擦拭法。     

INHIBITORY EFFECTS OF FLAVOR COMPOUNDS ON STAPHYLOCOCCUS AUREUS WRRC B1241

Acetanisole (4′-methoxyacetophenone) (AC), benzaldehyde (BE), cinna-maldehyde (CI), diacetyl (DI), phenylpropionaldehyde (PH), and pyruvaldehyde (PY) were tested against growth of S. aureus WRRC B124 in brain heart infusion broth. Activity was assessed in the presence and absence of oxygen at 12, 19 and 37C, and in combination with mild (20 min at 50 or 60C) heat treatments. The carbonyl compounds limited S. aureus growth at minimal inhibitory concentrations (MIC) of 0.5–8.0 mM. After 4 h at 37C, a 2 to 3-log₁₀ CFU/ml population reduction was observed with cultures containing 8.0 mM PH, PY, DI or CI. Activity was O₂-tension independent, with CI (0.5 mM), DI(2.0 mM) and PY(2.0 mM) being most active. The MIC for CI was temperature independent, while PY was most effective at 19C, and PH and DI at 12C. Mild heat treatment of carbonyl-supplemented samples reduced previously observed MICs. At 60C, for example, the MICs for AC and BE, 4.0 and 8.0 mM respectively, were both reduced to 0.5 mM. The decimal reduction times for S. aureus exposed to both UV-light and 8 mM flavor compounds were 3.3 and 4.3 s for CI and DI, respectively. However, the other compounds were not as effective in the presence of UV since the decimal reduction times ranged from 7.7 to 9.0s. The carbonyl compounds tested were effective antistaphylococcal agents and their use in combination with thermal processing may serve as a new approach to control S. aureus growth and other gram-positive foodborne pathogens.     

DETECTION OF STAPHYLOCOCCUS AUREUS PRODUCTS IN FOODS USING ENZYME LINKED IMMUNOSORBENT ASSAY AND SPECTROPHOTOMETRIC THERMONUCLEASE ASSAY

The comparative sensitivity of an enzyme linked immunosorbent assay (ELISA) using four different antistaphylococcal antisera and a spectrophotometric assay for thermonuclease were determined using cheese and ravioli samples seeded with strains of Staphylococcus aureus and S. epidermidis. The ELISA used antisera to enterotoxin A, enterotoxin B, S. aureus strains 14609 (human), and UNH-570 (bovine). The 570 ELISA and spectrophotometric thermonuclease assay were of comparable sensitivity and detected seeded culture in concentrations as low as 2 × 10⁷ CFU/g of cheese. A simple two hour method for extracting thermonuclease from foods was 50% efficient when as little as 50 ng of purified enzyme was seeded per g of cheese. Analyses of 43 commercial cheeses for viable S. aureus found five (12%) positive with 3 × 10⁴ CFU/g of cheese being the highest counts detected. All samples were negative by ELISA and thermonuclease assay. A simple screening procedure for demonstration of S. aureus contamination of foods is discussed.     

EFFECT OF WATER ACTIVITY ON THE GROWTH OF STAPHYLOCOCCUS AUREUS AT MEAT-CHEESE INTERFACES

There is increased marketing of ready-to-eat nonrefrigerated snack foods which consist of meat or sausage products with low or intermediate moisture levels combined with high moisture food products, i.e., cheese products. Packaging the intermediate moisture meat in direct contact with a high moisture food might change the water activity (a_w) of the products sufficiently to support growth of Staphylococcus aureus at contaminated interfaces. To evaluate this possibility, sterile sausage slices (a_w= 0.60 to 0.82) were surface inoculated with log 2-3 CFU/g of S. aureus, interfaced with processed cheese slices (a_w= 0.94), vacuum packaged, and incubated at 19, 28, 37C and at cyclic temperature of 19–37–19C. S. aureus levels and water activities were determined weekly for 0 to 9 weeks. The a_w at the interface changed rapidly and reached an a_w that supported S. aureus growth. Growth of S. aureus occurred under all test conditions when the samples were stored at 28 and 37C. At 19C storage S. aureus remained viable for the length of the study.