The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum-based products.

Pasteurisation of raw milk, colostrum, dairy or colostrum-based products must be achieved using at least 72°C for 15 s, at least 63°C for 30 min or any equivalent combination, such that the alkaline phosphatase (ALP) test immediately after such treatment gives a negative result. For cows' milk, a negative result is when the measured activity is ≤ 350 milliunits of enzyme activity per litre (mU/L) using the ISO standard 11816-1. The use and limitations of an ALP test and possible alternative methods for verifying pasteurisation of those products from other animal species (in particular sheep and goats) were evaluated. The current limitations of ALP testing of bovine products also apply. ALP activity in raw ovine milk appears to be about three times higher and in caprine milk about five times lower than in bovine milk and is highly variable between breeds. It is influenced by season, lactation stage and fat content. Assuming a similar pathogen inactivation rate to cows' milk and based on the available data, there is 95-99% probability (extremely likely) that pasteurised goat milk and pasteurised sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively. The main alternative methods currently used are temperature monitoring using data loggers (which cannot detect other process failures such as cracked or leaking plates) and the enumeration of Enterobacteriaceae (which is not suitable for pasteurisation verification but is relevant for hygiene monitoring). The inactivation of certain enzymes other than ALP may be more suitable for the verification of pasteurisation but requires further study. Secondary products of heat treatment are not suitable as pasteurisation markers due to the high temperatures needed for their production. More research is needed to facilitate a definitive conclusion on the applicability of changes in native whey proteins as pasteurisation markers.

Kinetics of drosopterin release as indicator pigment for heat-induced color changes of brown shrimp (Crangon crangon).

Heat-induced color changes of crustaceans are commonly described as the release of astaxanthin. In this study on Crangon crangon, it was found that astaxanthin plays a minor role in the (dis)coloration. By LC-HRMS, two polar, process dependent pigments were found. One pigment was identified as riboflavin and one as drosopterin (level-2 certainty). Thermal treatments had highest effect on drosopterin concentration changes and were chosen as indicator for a kinetic study of heat-induced color changes. The kinetic data fitted a consecutive step model (r2 = 0.971), including a first step in which drosopterin was released (kd,85°C = 0.95 ±â€¯0.09 min-1; Ead = 105 ±â€¯4 kJ/mol) and a second step where drosopterin is degraded (kb,85°C = 0.02 ±â€¯0.002 min-1; Eab = 190 ±â€¯15 kJ/mol). The kinetic model shows that shrimp should be heated at lower temperatures (<80 °C) than the heating temperatures used by fishermen (86-101 °C), creating opportunities for quality optimization. Therefore, this study delivers essential information needed in a comprehensive quality optimization study of the cooked brown shrimp.

The Biodiversity of the Microbiota Producing Heat-Resistant Enzymes Responsible for Spoilage in Processed Bovine Milk and Dairy Products.

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk.

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52 kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Thermal inactivation kinetics of proteases and polyphenoloxidase in brown shrimp (Crangon crangon).

To optimize product quality of the cooked brown shrimp (Crangon crangon), quantitative data on the influence of all relevant process parameters (treatment time and temperature) on several quality attributes is required. Surprisingly, kinetic data and models on heat induced inactivation of important endogenous spoilage enzymes of the brown shrimp are not available today. In this study the thermal inactivation kinetics of the most important spoilage enzymes, proteases and polyphenoloxidase (PPO), were determined from isothermal heat treatments of enzyme extracts of the cephalothorax. For both enzymes, inactivation kinetics showed first order decay(s). Proteases showed two distinct stability fractions. A labile fraction, representing 42±2% of the total activity with kl,60°C=0.94±0.14 min(-1) and Ea,l=178±8.5 kJ/mol, and a stable fraction, representing 58±2%, with ks,60°C=0.020±0.002 min(-1) and Ea,s=155±7.0 kJ/mol. PPO showed a single fraction with k60°C=1.58±0.02 min(-1) and Ea=161±2.2 kJ/mol. Based on these results, the proteolytic activity, in particular the thermostable fraction, should be considered as a target in thermal processing of brown shrimp in relation to enzyme induced product quality changes during storage.

Flexible tool for simulating the bulk optical properties of polydisperse spherical particles in an absorbing host: experimental validation.

In this study, a flexible tool to simulate the bulk optical properties of polydisperse spherical particles in an absorbing host medium is described. The generalized Mie solution for Maxwell's equations is consulted to simulate the optical properties for a spherical particle in an absorbing host, while polydispersity of the particle systems is supported by discretization of the provided particle size distributions. The number of intervals is optimized automatically in an efficient iterative procedure. The developed tool is validated by simulating the bulk optical properties for two aqueous nanoparticle systems and an oil-in-water emulsion in the visible and near-infrared wavelength range, taking into account the representative particle sizes and refractive indices. The simulated bulk optical properties matched closely (R2 ≥ 0.899) with those obtained by reference measurements.

Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Antimicrobial resistance in the food chain: a review.

Antimicrobial resistant zoonotic pathogens present on food constitute a direct risk to public health. Antimicrobial resistance genes in commensal or pathogenic strains form an indirect risk to public health, as they increase the gene pool from which pathogenic bacteria can pick up resistance traits. Food can be contaminated with antimicrobial resistant bacteria and/or antimicrobial resistance genes in several ways. A first way is the presence of antibiotic resistant bacteria on food selected by the use of antibiotics during agricultural production. A second route is the possible presence of resistance genes in bacteria that are intentionally added during the processing of food (starter cultures, probiotics, bioconserving microorganisms and bacteriophages). A last way is through cross-contamination with antimicrobial resistant bacteria during food processing. Raw food products can be consumed without having undergone prior processing or preservation and therefore hold a substantial risk for transfer of antimicrobial resistance to humans, as the eventually present resistant bacteria are not killed. As a consequence, transfer of antimicrobial resistance genes between bacteria after ingestion by humans may occur. Under minimal processing or preservation treatment conditions, sublethally damaged or stressed cells can be maintained in the food, inducing antimicrobial resistance build-up and enhancing the risk of resistance transfer. Food processes that kill bacteria in food products, decrease the risk of transmission of antimicrobial resistance.

Toxinogenic and spoilage potential of aerobic spore-formers isolated from raw milk.

The harmful effects on the quality and safety of dairy products caused by aerobic spore-forming isolates obtained from raw milk were characterized. Quantitative assessment showed strains of Bacillus subtilis, the Bacillus cereus group, Paenibacillus polymyxa and Bacillus amyloliquefaciens to be strongly proteolytic, along with Bacillus licheniformis, Bacillus pumilus and Lysinibacillus fusiformis to a lesser extent. Lipolytic activity could be demonstrated in strains of B. subtilis, B. pumilus and B. amyloliquefaciens. Qualitative screening for lecithinase activity also revealed that P. polymyxa strains produce this enzyme besides the B. cereus group that is well-known for causing a 'bitty cream' defect in pasteurized milk due to lecithinase activity. We found a strain of P. polymyxa to be capable of gas production during lactose fermentation. Strains belonging to the species B. amyloliquefaciens, Bacillus clausii, Lysinibacillus sphaericus, B. subtilis and P. polymyxa were able to reduce nitrate. A heat-stable cytotoxic component other than the emetic toxin was produced by strains of B. amyloliquefaciens and B. subtilis. Heat-labile cytotoxic substances were produced by strains identified as B. amyloliquefaciens, B. subtilis, B. pumilus and the B. cereus group. Variations in expression levels between strains from the same species were noticed for all tests. This study emphasizes the importance of aerobic spore-forming bacteria in raw milk as the species that are able to produce toxins and/or spoilage enzymes are all abundantly present in raw milk. Moreover, we demonstrated that some strains are capable of growing at room temperature and staying stable at refrigeration temperatures.

Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples.

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.

Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies.

To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.