The effect of homogenization and milk fat fractions on the functionality of Mozzarella cheese.

Mozzarella cheese was manufactured from milk containing either a low (olein) or a high (stearin) melting point fraction of milk fat or anhydrous milk fat. The fat was dispersed into skim milk by homogenization at 2.6 MPa before being manufactured into cheese. The melting point of the milk fat did not affect the size or shape of the fat globules, nor was there any effect of homogenization on the polymorphic state of the milk fat. There were no changes in milk fat globule size and shape concomitant with the amount of free oil formed. The polymorphic state of the milk fat did affect the amount of free oil formed and the apparent viscosity of the cheese. The lower melting point fraction yielded a larger amount of free oil. The higher melting point fraction yielded a higher viscosity of melted cheese at 60 degrees C. Mozzarella cheese was also manufactured from homogenized milk, nonhomogenized milk, and a 1:1 ratio of the two, without altering the milk fat composition. Increasing the proportion of homogenized milk yielded a lower free oil content and higher viscosity of the cheese.

The effect of compression, stretching, and cooking temperature on free oil formation in mozzarella curd.

The effect of the extent and rate of compression and stretching on free oil formation in Mozzarella cheese curd was investigated at 55, 65, and 75 degrees C. Confocal laser scanning microscopy was used to determine the maximum cross-sectional diameter, cross-sectional area, elongation factor (maximum divided by minimum cross-sectional diameter), and circularity of fat globules in the cheese curd at the different temperatures, and after stretching or compression. Free oil was not significantly affected by the rate of biaxial compression from 50 to 2000 mm/min at 65 degrees C, the rate of tensile stretching from 1000 to 2500 mm/min at 60 degrees C, or the extent of biaxial compression from 40 to 80% of the original height at 1000 mm/min and 65 degrees C. Increasing the rate of stretching from 1000 to 2500 mm/min increased the elongation factor from 1.91 to 2.61. Cross-sectional area, maximum diameter, and circularity were not affected by the rate of biaxial compression. The extent of curd compression had no effect on the milk fat globule size and shape. Increasing the extent of stretching at 60 degrees C and at 1000 mm/min increased the free oil content (on a fat basis) from 23.8% (curd stretched by 1.4x) to 32.3% (stretched by 4.6x) and the elongation factor of the globules, but did not affect any of the other globule parameters. Increasing the temperature of the cooking-stretching water increased the cross-sectional area, diameter of the globules, and free oil content from 24.1% at 55 degrees C to 34.5% at 75 degrees C for curd compressed to 50% height at 1000 mm/min.

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems.

The effects of 80% oxygen-20% carbon dioxide (O2-CO2) and 80% nitrogen-20% carbon dioxide (N2-CO2) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at -1 and 4 degrees C. In both N2-CO2 and O2-CO2 atmospheres, lactobacilli were predominant over Brochothrix, pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O2-CO2 atmospheres did not yield total viable counts in excess of 10(5) cfu cm-2 on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N2-CO2 atmospheres yielded maximum total viable counts of approximately 10(7) cfu cm-2 and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at -1 degree C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O2 on rancidity than by the high levels of lactobacilli.

Bacterial colonization and biofilm development on minimally processed vegetables.

Bacterial biofilms have been observed and reported on food and food-processing surfaces and can contribute to increased risks for product quality and food safety. The colonization of fruit and vegetables by pectynolitic bacteria like Pseudonomas fluorescens attributable to conditions such as soft rot, can also manifest as biofilms. A developed biofilm structure can provide a protective environment for pathogens such as Listeria monocytogenes reducing the effectiveness of sanitisers and other inhibitory agents. Understanding the colonization of bacteria on leaf surfaces is essential to the development of a better understanding of the leaf ecology of vegetable products. Studies of microbial colonization of leaf surfaces have been conducted using SEM and more recently using confocal microsocpy techniques. In the current study, a Leica TCS NT laser scanning confocal microscope was used to investigate biofilm formation using vital fluorescence staining on intact vegetable leaves. Reflection contrast and fluorescence three-dimensional imaging successfully delineated bacterial and biofilm morphology without disturbing the bacterial or leaf surface structure. The results demonstrate the presence and development of biofilm on the surface of lettuce. The biofilms appeared to originate on the cuticle in distinct micro-environments such as in the natural depression of the stomata, or in the intercellular junction. Bacteria also adhered to and developed biofilm colonies within an hour of contact and with clean stainless steel surfaces. Our study investigates the progression of biofilm formation from leaf colonization, and will assist in characterising the critical mechanisms of plant/host interaction and facilitate the development of improved preservation, sanitising and packaging strategies for minimally processed vegetable products.

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin.

A total of 663,533 colonies from 72 dairy and meat sources showed a detection rate of 0.2% for bacteriocin producers using direct plating techniques. A further 83,000 colonies from 40 fish and vegetable sources showed a detection rate of 3.4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus, Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.

Incorporation of nisin in micro-particles of calcium alginate.

Nisin was successfully incorporated into a matrix of calcium alginate and ground into micro-particles smaller than 150 microns. Formation of micro-particles and incorporation of nisin was verified by scanning electron microscopy and by the reduction in the inactivation of nisin activity with proteolytic enzymes. Incorporation efficiency was 87-93% and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in MRS broth and reconstituted skim milk.

Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter.

The effect of bacteriocin, piscicolin 126, on the growth of Listeria monocytogenes and cheese starter bacteria was investigated in milk and in Camembert cheese manufactured from milk challenged with 10(2) cfu ml(-1) L. monocytogenes. In milk incubated at 30 degrees C, piscicolin 126 added in the range of 512-2,048 AU ml(-1) effectively inhibited growth of L. monocytogenes for more than 20 d when challenged with approximately 10(2) cfu ml(-1) L. monocytogenes. At higher challenge levels (10(4) and 10(6) cfu ml(-1)), piscicolin 126 reduced the viable count of L. monocytogenes by 4-5 log units immediately after addition of the bacteriocin; however, growth of Listeria occurred within 24 h. The minimum inhibitory concentration (MIC) of piscicolin 126 against lactic acid cheese starter bacteria was generally greater than 204,800 AU ml(-1) , and the viable count and acid production of these starter cultures in milk were not affected by the addition of 2,048 AU ml(-1) piscicolin 126. Camembert cheeses made from milk challenged with L. monocytogenes and with added piscicolin 126 showed a viable count of L. monocytogenes 3-4 log units lower than those without piscicolin 126. Inactivation of piscicolin 126 by proteolytic enzymes from cheese starter bacteria and mould together with the emergence of piscicolin 126-resistant isolates was responsible for the recovery of L. monocytogenes in the cheeses during ripening.

Adsorption of bacteriocins by ingestible silica compounds.

Bacteriocins including nisin, pediocin PO2, brevicin 286 and piscicolin 126 were adsorbed from culture supernates by various food-grade porous silica anti-caking agents and the food colourant, titanium dioxide. All the porous silica (calcium silicate or silicon dioxide) materials showed substantial capacity in adsorbing bacteriocin activities from the culture supernate and biological activity was recovered in the adsorbents. In contrast, the food colourant titanium dioxide adsorbed most of the bacteriocin activity from the supernate, with minimal biological activity retained in the adsorbent. Experiments with piscicolin 126 showed that optimum adsorption could be achieved with Micro-Cel E within 30 min, independent of the supernate pH (2.0-10.0). Piscicolin activity of up to 5 x 10(7) AU g(-1) of Micro-Cel E was obtained after adsorption from culture supernates and the adsorbed piscicolin demonstrated substantial biological activity against Listeria monocytogenes in both broth and a milk growth medium.

Characterization of the chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced by Carnobacterium piscicola JG126.

A novel peptide bacteriocin produced by the lactic acid bacterium Carnobacterium piscicola JG126 isolated from spoiled ham was purified and characterized. This bacteriocin, designated piscicolin 126, inhibited the growth of several gram-positive bacteria, especially the food-borne pathogen Listeria monocytogenes, but had no effect on the growth of a number of yeasts and gram-negative bacteria. Bactericidal activity was not destroyed by exposure to elevated temperatures at low pH values; however, bactericidal activity was lost at high pH values, especially when high pH values were combined with an elevated temperature. Piscicolin 126 activity was not affected by catalase, lipase, or lysozyme but was destroyed by exposure to a range of proteolytic enzymes. Piscicolin 126 was purified to homogeneity and was found to be a peptide having a molecular weight of 4,416.6 +/- 1.9. A sequence analysis revealed that this compound is a cystibiotic (class IIa) bacteriocin containing 44 amino acid residues and one intrapeptide disulfide ring. Piscicolin 126 has regions of homology with some other bacteriocins obtained from lactic acid bacteria and is most closely related to sakacin P and pediocin PA-1 (levels of identity, 75 and 55%, respectively). Addition of piscicolin 126 to a devilled ham paste test food system inhibited the growth of L. monocytogenes for at least 14 days. Piscicolin 126 was more effective than two commercially available bacteriocin preparations tested in the same system.

A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate.

Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification.

Production of brevicin 286 by Lactobacillus brevis VB286 and partial characterization.

Brevicin 286 was produced by Lactobacillus brevis VB286 isolated from vacuum-packaged meat and was partially purified by ammonium sulphate precipitation, gel filtration and dialysis. The bacteriocin was susceptible to proteolytic enzymes, stable to heating at 100 degrees C particularly under acidic against Listeria sp. Production of brevicin 286 was optimal during exponential growth at 20 degrees C. Higher rates of cell growth occurred between 30 and 37 degrees C but with little or no expression of brevicin 286. A food-grade formulation consisting of 4% yeast extract and 1% glucose was found to be adequate for optimal brevicin 286 production and the bacteriocin-containing culture supernate was successfully spray dried with full recovery of antibacterial activity in the resultant powder.

Partial characterisation of pediocin PO2 and comparison with nisin for biopreservation of meat products.

A plasmid associated bacteriocin (pediocin PO2) was isolated by ammonium sulphate precipitation from cell-free growth media and subsequent studies showed that the partially purified pediocin PO2 was most likely identical (molecular mass approximately 3200 daltons in size by SDS-PAGE, stable to low pH and heat at 121 degrees C for 15 min, inactivated by various proteolytic enzymes and resistant to treatment with a range of solvents, except 10% formaldehyde) to other pediocins (PA-1 and AcH) previously reported. The antagonistic spectrum of activity of pediocin PO2 was compared with nisin and showed a narrower host-range, but a much greater activity against Listeria species including strains of Listeria monocytogences, than did nisin. A rapid method of reflectance colorimetry was used to quantitate growth and acid production (as determined by the colour change in bromcresol purple) of Lactobacillus curvatus, added to a meat product model system. The combined effects of refrigeration temperature, microbial load and bacteriocin concentration were determined in the model over 15 days storage. Both nisin and pediocin demonstrated inhibitory activity against Lactobacillus curvatus in the model system. However, when bacteriocins were incorporated into a manufactured cooked meat product only low nisin activity and no pediocin activity was detected, after challenge of vacuum packaged slices of product with Lactobacillus curvatus, over a 21 day storage trial under refrigeration temperatures.

Controlling microbial contamination on beef and lamb meat during processing.

The microbiological quality of carcases, meat and environmental surfaces was evaluated in commercial boning rooms processing beef and lamb. There was considerable variation in the level of microbial contamination on both carcases and meat, with counts ranging from less than 20 to 10(8)/cm2 on carcases and to 2 x 10(7)/cm2 on meat. The level of microbial contamination on meat was influenced by the level of carcase contamination at boning and by the boning process itself. Carcase contamination was the major determinant of microbiological quality, as more than 70% of carcase had microbial counts greater than 10(3)/cm2. Cutting boards were a major source for microbial dissemination during boning, particularly when carcase counts were less than 10(3)/cm2. If carcases were heavily contaminated, the contamination of processing surfaces was irrelevant in determining microbial loads on meat. Where carcase contamination was at low to moderate levels, the contribution of the boning process to the contamination on meat assumed increased significance. Under these conditions, improved sanitation of cutting surfaces in the boning room resulted in a significant reduction in microbial contamination on the surface of meat. These results can form the basis for ensuring that improvements made in carcase management before boning, to improve microbiological quality, will be preserved through attention to cutting board hygiene during boning.

The effect of spices and manganese on meat starter culture activity.

Three species, two proprietary spice blends and six starter preparations used in commercial salami manufacture were analysed for manganese and magnesium content. A mettwurst spices blend showed the highest levels of manganese (0·77 ppm expressed as effective product level assuming a 1% spice content) while mild and hot paprika and milano blend contained levels of manganese 1 4 - 1 3 lower. Magnesium levels for spices ranged from 3·14 to 25·81 ppm. Only two of the six meat starter cultures showed high levels of manganese (7·77 and 16·12 ppm as effective product level based on inoculation rate) while magnesium levels for all starter cultures did not exceed 0·37 ppm. The pH of salami products made with starter cultures containing no added manganese lagged behind that of products made with added mangenese (5 ppm) by 0·2 pH units at 48 h. The effect of manganese ions on the fermentation rate of starter bacteria was studied further in a salami model system, in the absence and presence of added spices. The mettwurst blend produced greatest stimulation and the milano the least. A level of 1·2 ppm of added manganese was sufficient to achieve an optimal (< 4·9 pH units within 48 h) fermentation in the presence of all five spices tested in the salami model system.

Growth characteristics of meat starter cultures.

The Australian Code of Practice for manufacture of dry and semi-dry sausage (salami) states that fermentation temperatures must not exceed 25°C and that a pH of 5·2 must be achieved in the product within 48 h. In order to select the most appropriate starter cultures for fermentation, Lactobacillus plantarum, Pediococcus pentosaceus and Staphylococcus carnosus were characterised with respect to growth and acid production determined at constant pH (4·7, 5·5 and 6·3). L. plantarum and P. pentosaceus showed similar characteristics over the pH range studied while S. carnosus was sensitive to lower pH. Also, P. pentosaceus showed greater psychrotrophic growth without pH control than L. plantarum or S. carnosus. Salami made with P. pentosaceus maintained higher viable numbers in the product over 8 days than did L. plantarum. Growth of S. carnosus in salami could not be detected in the presence of the more pH-tolerant organisms. The growth of L. plantarum and P. pentosaceus did not prevent the development of high levels of non-starter flora, a factor that can be important in determining salami quality. However, the more psychrotrophic P. pentosaceus ensured a greater dominance of starter over non-starter flora.

Transport and metabolism of lactose, glucose, and galactose in homofermentative lactobacilli.

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained beta-galactosidase activity, and no significant phospho-beta-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.

Metabolism of pyruvate and citrate in lactobacilli.

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.