Evaluation of tilapia skin gelatin as a mammalian gelatin replacer in acid milk gels and low-fat stirred yogurt.

Tilapia skin gelatin (TSG) was studied in a 3-stage process (cooling, annealing, and heating) for pure gelatin gels and in a 4-stage process (acidification, cooling, annealing, and heating) for acid milk gels and cultured yogurt. The aim was to evaluate the use of TSG as a replacement for mammalian gelatin in yogurt. In pure TSG gels, stronger gels with higher melting temperatures were formed with increasing TSG concentrations. Compared with bovine gelatin (BG), which gelled at a concentration of 2.5%, TSG gels had lower gelling (14.1°C) and melting (24°C) temperatures but comparable storage moduli during annealing. In acid milk gels, addition of TSG increased the firmness of the gels with increasing concentration. Gelling and melting points of TSG in milk gels were observed at sufficient concentrations during cooling and heating. Strands and sheets were observed in the electron micrographs of milk gels with 1% TSG and a very dense structure was observed with 2.5% TSG. Yogurt with 0.4% TSG had similar viscosity, consistency, pseudoplasticity, and thixotropy as yogurt containing 0.4% BG; no difference was perceived by sensory panelists according to a triangle test. Addition of 0.4% TSG completely prevented whey separation from the acid milk gel and yogurt. The results suggest that TSG could be a suitable replacement for mammalian gelatin in low-fat stirred yogurt.

Blocked Lysine in Dairy Products: Formation, Occurrence, Analysis, and Nutritional Implications.

Lysine residues in milk proteins become "blocked" when they react with reducing sugars, particularly lactose, in the Maillard reaction. The blocked or glycated lysines reduce the biological availability of the lysine to metabolic processes and also hinder hydrolysis of the parent protein by digestive enzymes. Heating and storage of milk and milk products are the major promotants of the Maillard reaction and resulting chemical damage to the proteins. Several methods have been proposed to estimate the extent of this protein damage. Two major compounds, furosine, a product of acid hydrolysis of lactulosyl-lysine, the 1st stable product of the Maillard reaction, and carboxymethyl-lysine are used for assessing the early and advanced stages of the Maillard reaction, respectively. In addition, several methods are used for assessing the bioavailability of lysine in a protein; these include chemical, enzymic, and animal-based methods. This review discusses the Maillard reaction and its significance in milk and dairy products, methods of assessing the extent of the reaction and of the bioavailability of lysine, and the nutritional significance of blocked lysines and associated Maillard reaction products in milk proteins.

Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis strains.

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

Reduction of aggregation of ß-lactoglobulin during heating by dihydrolipoic acid.

Prevention of the heat-induced aggregation of ß-lactoglobulin (ß-Lg) would improve the heat stability of whey proteins. The effects of lipoic acid (LA, or thioctic acid), in both its oxidised and reduced form (dihydrolipoic acid, DHLA), on heat-induced unfolding and aggregation of ß-Lg were investigated. LA/DHLA was added to native ß-Lg and the mixture was heated at 70, 75, 80 or 85 °C for up to 30 min at pH 6·8. The samples were analysed by Polyacrylamide Gel Electrophoresis (PAGE) and Size-exclusion HPLC (SE-HPLC). LA was not as effective as DHLA in reducing the formation of aggregates of heated ß-Lg. Heating ß-Lg with DHLA resulted in formation of more ß-Lg monomers (due to dissociation of native dimers) and significantly less ß-Lg aggregates, compared with heating ß-Lg alone. The aggregates formed in the presence of DHLA were both covalently linked, via disulphide bonds, and non-covalently (hydrophobically) linked, but the amount of covalently linked aggregates was much less than when ß-Lg was heated alone. The results suggest that DHLA was able to partially trap the reactive ß-Lg monomer containing a free sulphydryl (-SH) group, by forming a 'modified monomer', and to prevent some sulphydryl-sulphydryl and sulphydryl-disulphide interactions that lead to the formation of covalently linked protein aggregates. The effects of DHLA were similar to those of N-ethylmaleimide (NEM) and dithio(bis)-p-nitrobenzoate (DTNB). However, the advantage of using DHLA over NEM and DTNB to lessen aggregation of ß-Lg is that it is a food-grade compound which occurs naturally in milk.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

Effects of nisin, EDTA and salts of organic acids on Listeria monocytogenes, Salmonella and native microflora on fresh vacuum packaged shrimps stored at 4 °C.

Nisin (500 IU ml⁻¹), EDTA (0.02 M), potassium sorbate (PS) (3%, w/v), sodium benzoate (SB) (3%, w/v) or sodium diacetate (SD) (3%, w/v); alone or in combination were used to dip uninoculated shrimps and shrimps inoculated with Listeria monocytogenes or Salmonella (∼4.0-5.0 log CFU g⁻¹). Shrimps were then drip-dried, vacuum packaged and stored at 4 °C for 7 days. Untreated shrimps were used as a control. Numbers of L. monocytogenes, Salmonella and native background microflora were determined on uninoculated and inoculated shrimps on days 0, 3 and 7. Nisin-EDTA-PS and nisin-EDTA-SD significantly reduced (p < 0.05) L. monocytogenes numbers by 1.07-1.27 and 1.32-1.36 log CFU g⁻¹, respectively, on day 0 and 3. However, all treatments failed to significantly reduce (p > 0.05) Salmonella counts on shrimps throughout storage. On day 7, numbers of aerobic bacteria, psychrotrophic bacteria and Pseudomonas on combined nisin-EDTA-salt of organic acids treated shrimps were significantly lower (p < 0.05) by 4.40-4.60, 3.50-4.01, and 3.84-3.99 log CFU g⁻¹ respectively, as compared to the control. Dipping in organic acids solutions followed by vacuum packaging and chilled storage can help reduce L. monocytogenes and native microflora, but not Salmonella, on fresh shrimps.

Kinetics of enthalpy relaxation of milk protein concentrate powder upon ageing and its effect on solubility.

Kinetics of enthalpy relaxation of milk protein concentrate (MPC) powder upon short-term (up to 67 h) storage at 25 °C and aw 0.85, and long-term (up to 48 days) storage at 25 °C and a range of aw values (0-0.85) were studied by differential scanning calorimetry (DSC). The short-term study showed a rapid recovery of enthalpy for the first 48 h, followed by a slower steady increase with time. The non-exponential ß parameter was calculated using the Kohlrausch-Williams-Watts function and found to be 0.39. Long-term storage showed that enthalpy relaxation depends on both storage period and water activity. The enthalpy value was much less for lower moisture content (mc) (aw ≤ 0.23, mc ≤ 5.5%) than for higher mc (aw ≥ 0.45, mc ≥ 8%) samples for a particular storage period. The results suggest that the presence of more water molecules, in close proximity to the protein surface facilitates kinetic unfreezing and subsequent motion of molecular segments of protein molecules towards thermodynamic equilibrium. Although de-ageing of stored samples did not reverse storage-induced solubility losses, the timescale of enthalpy relaxation was similar to that of solubility loss. It is suggested that enthalpy relaxation within stored samples allows structural rearrangements that are responsible for subsequent solubility decreases.

Ageing-induced solubility loss in milk protein concentrate powder: effect of protein conformational modifications and interactions with water.

BACKGROUND: Protein conformational modifications and water-protein interactions are two major factors believed to induce instability of protein and eventually affect the solubility of milk protein concentrate (MPC) powder. To test these hypotheses, MPC was stored at different water activities (a(w) 0.0-0.85) and temperatures (25 and 45 °C) for up to 12 weeks. Samples were examined periodically to determine solubility, change in protein conformation by Fourier transform infrared (FTIR) spectroscopy and water status (interaction of water with the protein molecule/surface) by measuring the transverse relaxation time (T(2) ) with proton nuclear magnetic resonance ((1) H NMR). RESULTS: The solubility of MPC decreased significantly with ageing and this process was enhanced by increasing water activity (a(w) ) and temperature. Minor changes in protein secondary structure were observed with FTIR which indicated some degree of unfolding of protein molecules. The NMR T(2) results indicated the presence of three distinct populations of water molecules and the proton signal intensity and T(2) values of proton fractions varied with storage condition (humidity) and ageing. CONCLUSION: Results suggest that protein/protein interactions may be initiated by unfolding of protein molecules that eventually affects solubility.

Storage induced changes to high protein powders: influence on surface properties and solubility.

BACKGROUND: MPC 80 is a high-protein (80%) milk powder commonly used in the food industry as a functional ingredient and valued for its nutritional quality. However, its rehydration properties decline during storage, causing more time to be required for rehydration of the powder by the end user. It is thought that changes at the surface of the powder particles contribute to this reduced solubility during storage. RESULTS: Surface composition and structural changes in milk protein concentrate (MPC) were observed during 90 days of storage at temperatures of 25 and 40 °C and relative humidities of 44, 66 and 84%. No significant changes to the surface composition (fat, protein and lactose) of the MPC powder samples occurred during storage; however, some changes in the microstructure of the powders were observed. Scanning electron microscopy analysis of the powder particles during dissolution showed the formation of a crust, consisting of a thin layer of fused casein micelles, on the surface of the stored powders. An increase in the hydrophobicity at the surface of the particles was evident by X-ray photoelectron spectroscopy analysis of the bonding state of the elements at or near the surface and by atomic force microscopy measurements of the adherence of particles to the surface of a material. CONCLUSION: The development of this 'crust' is thought to contribute to the decrease in the solubility of the powder particles during storage. The increase in the hydrophobicity at the surface and the casein micelle interactions resulting in the surface crust formation appear to contribute to the decrease in the solubility of MPC during storage.

The effects of temperature, chlorine and acids on the survival of Listeria and Salmonella strains associated with uncooked shrimp carapace and cooked shrimp flesh.

The purpose of this study was to assess the influence of the association of Listeria and Salmonella with shrimp surfaces on the effects of temperature, chlorine and acids on their survival. Planktonic, attached and colonized cells of Listeria monocytogenes Scott A, L. monocytogenes V7, Salmonella Senftenberg 1734b and S. Typhimurium ATCC 14028 were challenged with high (50 degrees , 60 degrees and 70 degrees C) and low (4 degrees C) temperature, 100 ppm sodium hypochlorite solution, and acetic, hydrochloric and lactic acids (pH 4.0). Attached and colonized Listeria and Salmonella showed significantly greater (p < 0.05) resistance to heat ( approximately 1.3-2.6 fold increase in D-values), hypochlorite ( approximately 6.6 >or= 40.0 fold) and acids ( approximately 4.0-9.0 fold) than their planktonic counterparts. There were no significant differences (p > 0.05) in the survival of planktonic, attached or colonized cells of Listeria and Salmonella stored under refrigerated conditions. The association of Listeria and Salmonella with shrimp surfaces enhances their resistance to heat, chlorine and acids. Both attachment to, and subsequent colonization of, shrimp surfaces by pathogens may reduce the efficacy of methods used in their control. Strategies to reduce attachment of these pathogens to shrimp are required to assure safety of this product.

Processing and Technology of Dairy Products: A Special Issue.

When this Special Issue was launched, we cast the net widely in terms of the subject matter we considered suitable for the papers. We stated that papers on "well-established unit operations such as heat treatments and membrane separation in addition to emerging technologies" would be welcomed. The seven papers accepted do, indeed, cover a range of topics including UHT milk, proteolytic digestion, membrane technologies, cheese and yogurt. Three papers [...].

Biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation--a review.

Biogenic amines are non-volatile amines formed by decarboxylation of amino acids. Although many biogenic amines have been found in fish, only histamine, cadaverine, and putrescine have been found to be significant in fish safety and quality determination. Despite a widely reported association between histamine and scombroid food poisoning, histamine alone appears to be insufficient to cause food toxicity. Putrescine and cadaverine have been suggested to potentiate histamine toxicity. With respect to spoilage on the other hand, only cadaverine has been found to be a useful index of the initial stage of fish decomposition. The relationship between biogenic amines, sensory evaluation, and trimethylamine during spoilage are influenced by bacterial composition and free amino acid content. A mesophilic bacterial count of log 6-7 cfu/g has been found to be associated with 5 mg histamine/100 g fish, the Food and Drug Administration (FDA) maximum allowable histamine level. In vitro studies have shown the involvement of cadaverine and putrescine in the formation of nitrosamines, nitrosopiperidine (NPIP), and nitrosopyrrolidine (NPYR), respectively. In addition, impure salt, high temperature, and low pH enhance nitrosamine formation, whereas pure sodium chloride inhibits their formation. Understanding the relationships between biogenic amines and their involvement in the formation of nitrosamines could explain the mechanism of scombroid poisoning and assure the safety of many fish products.

Relationship between the physicochemical properties of nonchitinolytic Listeria and Salmonella and their attachment to shrimp carapace.

Listeria and Salmonella are important foodborne pathogens normally associated with the shrimp production chain. This study investigated the potential of Salmonella Typhimurium, Salmonella Senftenberg, and Listeria monocytogenes (Scott A and V7) to attach to and colonize shrimp carapace. Attachment and colonization of Listeria and Salmonella were demonstrated. Shrimp abdominal carapaces showed higher levels of bacterial attachment (P < 0.05) than did head carapaces. Listeria consistently exhibited greater attachment (P < 0.05) than did Salmonella on all surfaces. Chitinase activity of all strains was tested and found not to occur at the three temperatures (10, 25, and 37 degrees C) tested. The surface physicochemical properties of bacterial cells and shrimp carapace were studied to determine their role in attachment and colonization. Salmonella had significantly (P < 0.05) more positive (-3.9 and -6.0 mV) cell surface charge than Listeria (-18 and -22.8 mV) had. Both bacterial species were found to be hydrophilic (<35%) when measured by the bacterial adherence to hydrocarbon method and by contact angle (theta) measurements (Listeria, 21.3 and 24.8 degrees, and Salmonella, 14.5 and 18.9 degrees). The percentage of cells retained by Phenyl-Sepharose was lower for Salmonella (12.8 to 14.8%) than it was for Listeria (26.5 to 31.4%). The shrimp carapace was found to be hydrophobic (theta = 74.5 degrees), and a significant (P < 0.05) difference in surface roughness between carapace types was noted. There was a linear correlation between bacterial cell surface charge (r2 = 0.95) and hydrophobicity (r2 = 0.85) and initial attachment (P < 0.05) of Listeria and Salmonella to carapaces. However, the same properties could not be related to subsequent colonization.

Optimum Thermal Processing for Extended Shelf-Life (ESL) Milk.

Extended shelf-life (ESL) or ultra-pasteurized milk is produced by thermal processing using conditions between those used for traditional high-temperature, short-time (HTST) pasteurization and those used for ultra-high-temperature (UHT) sterilization. It should have a refrigerated shelf-life of more than 30 days. To achieve this, the thermal processing has to be quite intense. The challenge is to produce a product that has high bacteriological quality and safety but also very good organoleptic characteristics. Hence the two major aims in producing ESL milk are to inactivate all vegetative bacteria and spores of psychrotrophic bacteria, and to cause minimal chemical change that can result in cooked flavor development. The first aim is focused on inactivation of spores of psychrotrophic bacteria, especially Bacillus cereus because some strains of this organism are pathogenic, some can grow at ≤7 °C and cause spoilage of milk, and the spores of some strains are very heat-resistant. The second aim is minimizing denaturation of ß-lactoglobulin (ß-Lg) as the extent of denaturation is strongly correlated with the production of volatile sulfur compounds that cause cooked flavor. It is proposed that the heating should have a bactericidal effect, B* (inactivation of thermophilic spores), of >0.3 and cause ≤50% denaturation of ß-Lg. This can be best achieved by heating at high temperature for a short holding time using direct heating, and aseptically packaging the product.

Combining nonthermal technologies to control foodborne microorganisms.

Novel nonthermal processes, such as high hydrostatic pressure (HHP), pulsed electric fields (PEFs), ionizing radiation and ultrasonication, are able to inactivate microorganisms at ambient or sublethal temperatures. Many of these processes require very high treatment intensities, however, to achieve adequate microbial destruction in low-acid foods. Combining nonthermal processes with conventional preservation methods enhances their antimicrobial effect so that lower process intensities can be used. Combining two or more nonthermal processes can also enhance microbial inactivation and allow the use of lower individual treatment intensities. For conventional preservation treatments, optimal microbial control is achieved through the hurdle concept, with synergistic effects resulting from different components of the microbial cell being targeted simultaneously. The mechanisms of inactivation by nonthermal processes are still unclear; thus, the bases of synergistic combinations remain speculative. This paper reviews literature on the antimicrobial efficiencies of nonthermal processes combined with conventional and novel nonthermal technologies. Where possible, the proposed mechanisms of synergy is mentioned.

Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.

Effect of sulphydryl reagents on the heat stability of whey protein isolate.

The effects of sulphydryl (-SH) reagents on protein aggregation reactions in heated whey protein isolate (WPI) and pure α-lactalbumin (α-La) were investigated. In contrast to its previously reported effect with pure ß-Lg, dihydrolipoic acid (DHLA) markedly reduced the heat stability of WPI, especially the α-La component, which aggregated much more readily in the presence of DHLA than in WPI alone. Whilst pure α-La is quite stable to heat, it is much less stable in the presence of DHLA. An effect similar to DHLA was observed with reduced glutathione (GSH). N-ethylmaleimide (NEM), and to a lesser extent, dithio(bis)-p-nitrobenzoate (DTNB), improved the heat stability of WPI; these reagents had little effect on α-La.

Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring.

Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162Da (cleavage of galactose) and 216Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins.

Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.