Microbial spoilage of pre-cooked potato-topped pies

The ecological succession of bacteria which developed in pre-cooked potato-topped pies stored at two different temperatures was examined. Bacillus, Streptococcus and Staphylococcus-Micrococcus spp. were the predominant organisms isolated from freshly prepared pies and those stored at 4 degrees and 37 degrees C. None of these groups of bacteria caused significant biodeterioration of pies held at 4 degrees C, but all groups grew well in pies stored at 37 degrees C and achieved counts of ca 10(8)/g of sample. Bacillus spp. were the first group to grow, followed by Streptococcus and Staphylococcus-Micrococcus spp. Growth which occurred at 37 degrees C did so at the expense of glucose, lactate accumulated and the pH of pie components decreased. Amylase activity detected in all pie components during storage was associated with the growth of Bacillus spp. and probably supplemented glucose already present in pies, by hydrolytic cleavage of potato, flour or binder starches. Spoilage caused by growth and activity of the bacteria isolated was not associated with visual signs of biodeterioration, nor production of 'off' odour usually associated with spoilage of meats. These results suggest that pre-cooked potato-topped pies held at inappropriate temperatures represent a potential public health risk.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Nitrite loss and spoilage microflora development in chub-packed luncheon meat

Residual nitrite was lost from chub-packed luncheon meat during storage through both chemical breakdown and microbial consumption. The relative importance of these mechanisms in this pasteurized product was determined by the speed of development of the spoilage microflora, which is influenced by storage conditions. The nitrite half-life due to chemical loss was 13 d at 25°C and 36 d at 10°C. When microbial growth occurred these half-lives were reduced to 2.6 d and 21 d, respectively. Qualitative differences in the microflora that developed at these two temperatures (denitrifying Bacillus spp. at 25°C and non-denitrifying Streptococcus spp. at 10°C) account for the large temperature effect. Growth of Streptococcus spp. increased the rate of chemical nitrite loss in chubs by reducing the pH value. Nitrite did not inhibit the aerobic growth of either Bacillus or Streptococcus species associated with spoilage but did inhibit the anaerobic growth of Bacillus spp. This bacteriostatic effect of residual nitrite in anaerobic conditions will decrease during storage as nitrite level falls and oxygen penetrates the chub pack. Nitrite-mediated bacteriostasis does not obviate the need for refrigerated storage but does afford a real, if ephemeral, safeguard against spoilage occurring during short periods of temperature abuse.     

The microflora of soak water during natural fermentation of coarsely ground chickpea (Cicer arietinum) seeds

K. KATSABOXAKIS AND K. MALLIDIS. 1996. The microflora of soak water was studied during natural fermentation of coarsely ground chickpea seeds at 32°, 37° and 42°C. The soak water was slightly salted with NaCl 0.5% (w/v) and its proportion to sample was 10 : 1 (v/w). A gas-producing Clostridium species isolated from Reinforced Clostridium Agar (RCA) plates incubated anaerobically, was found to dominate this fermentation system particularly at the higher temperatures. Gram-negative bacteria and yeasts were not found. Bacillus species were isolated from Plate Count Agar (PCA) plates and Lactobacillus, Corynebacterium, Micrococcus and Pediococcus spp. from RCA plates were incubated aerobically. Butyric was the main acid produced at 37° and 42°C and CO₂ with hydrogen were identified as main fermentation gases.     

Examination of bovine faeces for the isolation and identification of Clostridium species

Freshly cooked luncheon meat in a plastic (PVDC) casing had an aerobic plate count of about 10²/g. The flora was composed of approximately equal numbers of Bacillus and Micrococcus spp. Storage at 10°C for 42 d produced little increase in bacterial numbers, or changes in pH value or glucose content at either the surface or core of the luncheon meat. Storage at 25°C allowed Bacillus spp. to proliferate at the surface. The inhibitory effect of salt and nitrite on the growth of heated Bacillus spores at low redox potentials probably accounts for the absence of growth within the product. Growth at the surface was accompanied by a fall in pH (6.8 to 6.2) and an increase in glucose (1.6 to 3.6 mg/g) and L(+)-lactic acid (1.2 to 2.3 mg/g). By day 14 the Bacillus spp. had been displaced by a Streptococcus sp. (10⁷/g) which remained the dominant organism until the experiment ended on day 28. The pH continued to fall from 5.7 on day 14 to 5.2 on day 28, the L(+)-lactic acid rose to 6.1 mg/g, but the glucose remained constant at the day 7 level (3.6 mg/g). This indicates that glucose converted to lactic acid was largely replaced by hydrolysis of the starch portion of the luncheon meat mediated by amylases produced by the Bacillus microflora. It appears that growth of the Streptococcus is dependent upon the denitrifying activities of the initial Bacillus flora reducing the concentration of nitrite ion to non-inhibitory levels.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Injury and repair in biocide-treated spores of Bacillus subtilis

Abstract Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37°C but not in broth at 22°C, sterile filtered water at 4, 22 or 37°C or germination medium at 37°C. Repair appeared to occur primarily during outgrowth and was initiated soonest for iodine-treated spores and latest for glutaraldehyde-treated ones.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

A note on Aeromonas spp. from chickens as possible food-borne pathogens

The possible role of Aeromonas spp. as potential food-borne psychrotrophic pathogens was investigated by examining organisms isolated from processed raw chicken for their biochemical characteristics, ability to produce exotoxins and to grow at chill temperatures. These strains, in particular A. sobria , with identical characteristics to human diarrhoea-associated aeromonads were readily found. Chicken, and human and environmental (water) strains characterized in a previous study, were investigated for their ability to grow at refrigeration temperatures (5 ± 2°C) and, for selected strains, the theoretical minimum temperature for growth ( T _min) was determined from the growth pattern in a temperature gradient incubator. All enterotoxigenic chicken strains tested were typical mesophiles, with an optimal growth temperature of ˜37°C and T _min values ˜4.5°C. They were rapidly outgrown by a psychrotrophic Pseudomonas sp. typical of spoilage biota found on food. Enterotoxin was not produced below 15°C by any of the toxigenic food strains tested. The Aeromonas strains isolated from chickens in this study seem unlikely therefore to be a significant health risk, provided the chickens are properly stored and cooked. This would appear to be substantiated by the lack of reports of food-associated outbreaks of illness from these sources.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

The Development of the Anaerobic Spoilage Flora of Meat Stored at Chill Temperatures

In meat juice medium under anaerobic conditions, spoilage bacteria utilized the following substrates: Microbacterium thermosphactum , glucose; Enterobacter , glucose and glucose-6-phosphate; Lactobacillus , glucose and arginine. On meat stored anaerobically. Lactobacillus grew faster than the other species at all temperatures between 2° and 15 °C. Enterobacter had a greater affinity for glucose than M. thermosphactum. As a result, high numbers of Enterobacter inhibited growth of M. thermosphactum under the glucose limiting conditions at the meat surface. High numbers of Lactobacillus inhibited growth of both competing species, apparently by producing an inhibitory substance.     

Isolation and properties of psychrotrophic and psychrophilic, pectolytic strains of Clostridium

Fourteen strains of pectolytic clostridia have been isolated that were capable of growth at 5–10°C in 7 d; two strains were psychrophiles and failed to grow at 20°C in 14 d and the remainder were psychrotrophs. The bacteria formed pectate lyase enzymes and were capable of degrading potato tissue; they are therefore a potential cause of soft rot of potatoes stored at low temperatures. Doubling times for representative strains were 15–19 h at 10°C and 21–53 h at 5°C. Twelve strains were classified with Group I Clostridium species and two strains with Group II. In the case of one strain the mature spores were not released from the sporangium. Electron microscopy of ultrathin sections of this strain showed the presence of disorganized lamellar structures associated with the spore coat.     

Growth of Staphylococcus aureus MF 31 on the Top and Cut Surfaces of Southern Custard Pies

A Staphylococcus strain was inoculated on the top and cut surfaces of freshly baked Southern custard pies which were then packaged in a pasteboard carton and held at 30 C. Daily plate counts of surface sections 0.3 inch (0.76 cm) in thickness were made. The top surface inoculum showed a 24-hr lag time. This was due to the protective action of a top cakelike layer as shown by homogenization of the mix and coating of the surface. Substitution of all sweeteners with dextrose completely inhibited growth on the top surface. Further addition of dextrose to lower water activity (Aw) to 0.9 prevented growth on the cut surface as well, but such pies were organoleptically unacceptable. Growth on the top surface could also be prevented by 80 mug of undissociated sorbic acid per g in combination with 100 mug of undissociated propionic acid per g in the baked pie. Growth on the cakelike top surface was always retarded longer than on the cut surface provided the packaging allowed evaporation of surface moisture. Reducing the Aw of a different type of cream pie to 0.907 prevented top surface growth. It was concluded that baked cream pies with a cakelike top layer could be marketed with a "refrigerate after opening" label, provided the package maintains the moisture gradient caused by the surface skin and either a combination of 80 mug of undissociated sorbic acid per g and 100 mug undissociated propionic acid per g is present in the baked pie or the Aw of the baked pie is 0.920 or lower.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

A Diagnostic Key for Identifying the Lactic Acid Bacteria of Vacuum Packed Bacon

S ummary : The lactic acid bacteria isolated from stored vacuum packed sliced bacon cured in fresh brine were divided into nine groups according to a simple diagnostic key. The key was developed to help in recognizing genera which were often morphologically similar.
The genera were Streptococcus (group D), Leuconostoc, Pediococcus and Lactobacillus (both hetero- and homofermentative), and all could grow at temperatures down to 10°. A 'tetracoccus' and a group of motile lactobacilli were also found. Some homofermentative groups were atypical and differed from species at present recognized. Intermediate forms were found among the heterofermenters.     

Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4°C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus -type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacte-riostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62°C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes . Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.     

The role of oxygen in the microbial spoilage of luncheon meat cooked in a plastic casing

The shelf life at 25°C of chub-packed luncheon meat was inversely related to oxygen (O₂) availability within the casing. With fibrous casings that are freely permeable to O₂, shelf life was less than 3 d. With plastic casings of low O₂ permeability, the shelf life was 7 d when air was trapped in the emulsion during the casing filling process, 14 d when air was not trapped (normal vacuum-stuffing) and greater than 28 d when vacuum-stuffed chubs were stored in hydrogen (H₂). The initial spoilage bacteria, Bacillus spp., grew only at the surface unless air was trapped in the emulsion when growth occurred throughout the luncheon meat. Bacillus spp. failed to grow on luncheon meat stored under H₂. Oxygen availability within the casing determined both the site and rate of microbial spoilage of chub-packed luncheon meat.     

Properties of Pseudomonads Causing Spoilage of Vegetables Stored at Low Temperature

Twenty-four strains of pectolytic, fluorescent pseudomonads were isolated from soft rots of celery stored at 0.4-1°C and five strains were isolated from soft rots in cabbage stored at 1°C. When inoculated into the vegetable from which they were isolated these bacteria caused soft rot of wounded, but not of unwounded tissue. According to their biochemical reactions, the organisms were divided into three groups; Group 1 (15 strains) were identified with Pseudomonas fluorescens Biotype II (Doudoroff & Palleroni 1974) ( Ps. marginalis ); Group 2 (12 strains) and Group 3 (two strains) would be included in the 'Miscellaneous strains'of Ps. fluorescens described by the above authors. One strain biochemically representative of Group 1 showed a maximum growth rate at 27°C (doubling time, 0.88 h) and a doubling time at 0.2°C of 14.9 h. A strain representative of Group 2 showed a maximum growth rate at 29°C (doubling time 0.96 h) and a doubling time at 0.2°C of 16.6 h. Neither strain grew at 36°C. The temperature characteristics (calculated for the range 0.2-20.8°C) were 83 011 and 79 534 J/mol, respectively. The mean doubling time for the remaining Group 1 strains at 0.2°C was 17.6 h and for remaining Group 2 strains was 17.1 h.