2-Heptanone, 2-nonanone, and 2-undecanone confer oxidation off-flavor in cow milk storage.

Flavor sensation is one of the most prevalent characteristics of food industries and an important consumer preference regulator of dairy products. So far, many volatile compounds have been identified, and their molecular mechanisms conferring overall flavor formation have been reported extensively. However, little is known about the critical flavor compound of a specific sensory experience in terms of oxidized off-flavor perception. Therefore, the present study aimed to compare the variation in sensory qualities and volatile flavors in full-fat UHT milk (FFM) and low-fat UHT milk (LFM) samples under different natural storage conditions (0, 4, 18, 25, 30, or 37°C for 15 and 30 d) and determine the main component causing flavor deterioration in the FFM and LFM samples using sensory evaluation, electronic nose, and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). In addition, the Pearson correlation between the volatile flavor components and oxidative off-flavors was analyzed and validated by sensory reconstitution studies. Compared with the LFM samples, the FFM samples showed a higher degree of quality deterioration with increased storage temperature. Methyl ketones of odd carbon chains (i.e., 2-heptanone, 2-nonanone, 2-undecanone, 2-tridecanone, and 2-pentadecanone) reached a maximum content in the FFM37 samples over 30 d storage. The combined results of the Pearson correlation and sensory recombination study indicated that 2-heptanone, 2-nonanone, and 2-undecanone conferred off-flavor perception. Overall, the present study results provide potential target components for detecting and developing high-quality dairy products and lay a foundation for specific sensory flavor compound exploration in the food industry.

Influence of storage time on protein composition and simulated digestion of UHT milk and centrifugation presterilized UHT milk in vitro.

The centrifugation presterilizing UHT (C-UHT) sterilization method removes 90% of the microorganism and somatic cells from raw milk using high-speed centrifugation following UHT treatment. This study aimed to study the changes in protein composition and plasmin in the UHT and C-UHT milk. The digestive characteristics, composition, and peptide spectrum of milk protein sterilized with the 2 technologies were studied using a dynamic digestive system of a simulated human stomach. The Pierce bicinchoninic acid assay, laser scanning confocal microscope, liquid chromatography-tandem mass spectrometry, and AA analysis were used to study the digestive fluid at different time points of gastric digestion in vitro. The results demonstrated that C-UHT milk had considerably higher protein degradation than UHT milk. Different processes resulted during the cleavage of milk proteins at different sites during digestion, resulting in different derived peptides. The results showed there was no significant effect of UHT and C-UHT on the peptide spectrum of milk proteins, but C-UHT could release relatively more bioactive peptides and free AA.

Colloidal stability of milk: reinterpretation of alcohol test results by digital microscopy.

In this research communication we propose a new approach by portable digital microscopy with a 200× objective to improve the visualization of microparticles of pasteurized milk submitted to the alcohol test. Not only did the method reduce the subjectivity of the readings, but also generated high resolution images of the microparticles, which allows the creation of a specific image pattern for each type of final product. In comparison to a control pasteurized milk treatment, the results confirmed the effect and the specificity of added salts (sodium citrate, disodium phosphate or their combination) on the stability of the milk to the alcohol test. Finally, the mixture of stabilizing salts of citrate/phosphate provided the highest degree of stability to pasteurized milk among the treatments studied.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin.

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

Identification of Pseudomonas jessenii and Pseudomonas gessardii as the most proteolytic Pseudomonas isolates in Iranian raw milk and their impact on stability of sterilized milk during storage.

Identification of the most proteolytic Pseudomonas strains that can produce heat-resistant proteases and contribute to the Ultra High Temperature (UHT) milk destabilization is of great interest. In the present study, among the 146 Pseudomonas isolates that encoded the aprX gene, five isolates with the highest proteolytic activity were selected and identified based on 16S rRNA, rpoD and gyrB gene sequences data. The identification results were confirmed by phylogenetic analysis based on multilocus sequence analysis and identified the representative isolates as P. jessenii (two isolates) and P. gessardii (three isolates). Casein zymography demonstrated the ability of these species to produce heat-resistant enzymes, AprX, with molecular mass of about 48 kDa during storage at 7° C for 72 h. In sterilized milk samples, the residual activity of AprX caused a considerable enhancement in the degree of protein hydrolysis, non-protein nitrogen and non-casein nitrogen contents of the samples during a two-month storage. This enhancement was slightly higher in samples containing enzyme produced by P. jessenii compared to P. gessardii ones, resulting in earlier onset of sterilized milk destabilization. Hence, this study revealed that P. jessenii and P. gessardii can play a considerable role in deterioration of Iranian commercial long-life milk.

Assessment of physico-chemical changes in UHT milk during storage at different temperatures.

This research communication describes enzymatic and physico-chemical changes during storage of UHT milk. The UHT milk sample was stored at 5 and 30°C for 4 months and analyzed regularly at intervals of one month. During storage of UHT milk, there was a significant (P < 0.001) increase in non-protein nitrogen, non-casein nitrogen, soluble calcium, soluble magnesium and proteolysis, while a significant (P < 0.001) decrease in pH was observed. There was a slight change in the particle size and zeta potential of casein micelles. Changes were more pronounced in milk samples stored at 30°C than in those stored at 5°C. During storage, there occurred changes in pH, viscosity, salt balance and nitrogenous components which adversely affected quality. It was concluded that the proteolysis led to the acidification which had a destabilizing effect on the milk.

The combined use of 1 H and 2D NMR-based metabolomics and chemometrics for non-targeted screening of biomarkers and identification of reconstituted milk.

BACKGROUND: The illegal undeclared addition of reconstituted milk powder to ultra-heat treated (UHT) milk to lower production costs is an example of economically motivated adulteration. This activity not only defrauds consumers but also places honest traders at a disadvantage, which could damage the reputation of milk producers and reduce the integrity of the markets. In this research, a non-targeted analytical strategy that combines proton (1 H) nuclear magnetic resonance (NMR) spectroscopy with a chemometrics data mining tool was developed for the authentication of bovine UHT milk. RESULTS: Unsupervised principal component analysis was used to distinguish UHT and tap-water-reconstituted powdered milk. Partial least squares-discriminant analysis (PLS-DA) with R2 (Y) and Q2 equal to 0.859 and 0.748, respectively, was used to differentiate UHT and reconstituted milk samples. Three compounds were selected as biomarkers to distinguish UHT and reconstituted milk and identified according to the standard NMR-spectra database. Finally, a PLS-DA model was established, according to the characteristic spectral bands, to identify UHT milk and reconstituted milk. CONCLUSION: This procedure demonstrated the feasibility of using non-targeted NMR profiling combined with chemometric analysis to combat mislabeling and fraudulent practices in milk production. © 2019 Society of Chemical Industry.

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review.

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra-high-temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat-resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf-life.

Age Gelation, Sedimentation, and Creaming in UHT Milk: A Review.

Demand for ultra-high-temperature (UHT) milk and milk protein-based beverages is growing. UHT milk is microbiologically stable. However, on storage, a number of chemical and physical changes occur and these can reduce the quality of the milk. These changes can be sufficiently undesirable so as to limit acceptance or shelf life of the milk. The most severe changes in UHT milk during storage are age gelation, with an irreversible three-dimensional protein network forming throughout, excessive sedimentation with a compact layer of protein-enriched material forming rapidly at the bottom of the pack, and creaming with excessive fat accumulating at the top. For age gelation, it is known that at least two mechanisms can lead to gelation during storage. One mechanism involves proteolytic degradation of the proteins through heat-stable indigenous or exogenous enzymes, destabilizing milk and ultimately forming a gel. The other mechanism is referred to as a physico-chemical mechanism. Several factors are known to affect the physico-chemical age gelation, such as milk/protein concentration, heat load during processing (direct compared with indirect UHT processes), and milk composition. Similar factors to age gelation are known to affect sedimentation. There are relatively few studies on the creaming of UHT milk during storage, suggesting that this defect is less common or less detrimental compared with gelation and sedimentation. This review focuses on the current state of knowledge of age gelation, sedimentation, and creaming of UHT milks during storage, providing a critical evaluation of the available literature and, based on this, mechanisms for age gelation and sedimentation are proposed.

Lipid compositional changes and oxidation status of ultra-high temperature treated Milk.

BACKGROUND: Milk fat is one of the complex fat and most sensitive biochemical compounds towards auto-oxidation. To enhance the shelf life, milk is subjected to Ultra-high Temperature (UHT) treatment followed by aseptic packaging. During the storage, several chemical and biochemical changes take place in lipid fraction of UHT milk. In current investigation, the effect of UHT treatment and storage was determined by making a comparison in fatty acid profile, triglyceride composition, organic acids and lipid oxidation of the thermally treated and stored milk with raw milk, which was not reported in earlier investigations. METHODS: Raw milk samples were collected from the bulk storage facility of a dairy industry. The same milk was routed to UHT treatment and aseptically packaged samples were collected. The fatty acid profile, triglyceride composition, organic acids and lipid oxidation was determined in raw and UHT treated milk at 0, 30, 60 and 90 days. Fatty acid and triglyceride profile was determined on GC-MS while organic acids were determined by HPLC. For the measurement of induction period, professional Rancimat was used. Lipid oxidation was characterized through free fatty acids, peroxide value, anisidine value and conjugated dienes. RESULTS: Compositional attributes of milk remain unchanged during the entire length of storage. Concentrations of short-chain fatty acids in raw and UHT milk were 10.49% and 9.62%. UHT treatment resulted in 8.3% loss of short-chain fatty acids. Up to 30 days, storage did not have any significant effect on fatty acid profile of UHT milk. Concentration of medium-chain fatty acids in raw and UHT treated milk was 54.98% and 51.87%. After 30, 60 and 90 days of storage, concentration of medium chain fatty acids was found 51.23%, 47.23% and 42.82%, respectively. Concentration of C18:1 and C18:2 in raw and UHT milk was 26.86% and 25.43%, respectively. The loss of C18:1 and C18:2 in UHT treatment was 5.32%. After 30, 60 and 90 days of storage, the concentrations of C18:1 and C18:2 were 24.6%, 21.06% and 18.66%, respectively. Storage period of 30 days was found non-significant, while noticeable variations were found in triglyceride profile of 60 and 90 days old samples of UHT milk. UHT treatment and storage period significantly affected the concentration of organic acids in milk. After UHT treatment, concentration of lactic acid, acetic acid, citric acid, pyruvic acid, formic acid, succinic acid and oxalic acid increased by 3.45, 0.66, 3.57, 0.68, 2.24, 2.16 and 1.63 mg/100 g. Effect of storage period on the production of organic acids in UHT milk was non-significant up to 30 days. After 60 days of storage period, the increase in concentration of lactic acid, acetic acid, citric acid, pyruvic acid, formic acid, succinic acid and oxalic acid was 3.79, 0.75, 4.69, 0.78, 2.83, 3.03 and 2.38 mg/100 g. After 90 days of storage period, the increase in concentration of lactic acid, acetic acid, citric acid, pyruvic acid, formic acid, succinic acid and oxalic acid was 7.3, 2.18, 9.96, 3.58, 11.37, 5.22 and 5.96%. Free fatty acids content of raw, UHT treated and 90 days old milk were 0.08%, 0.11% and 0.19%. UHT treated version of milk showed similar peroxide value. While, the storage remarkably affected the peroxide value. After 30, 60 and 90 days, peroxide value was 0.42, 0.62 and 1.18 (MeqO2/kg). Induction period of raw, UHT and stored milk was strongly correlated with peroxide value and fatty acid profile. Mean value of lipase activity in raw milk was 0.73 ± 0.06 µmoles/ml. UHT treatment significantly decreased the lipase activity. The lipase activity of milk immediately after the UHT treatment was 0.18 ± 0.02 µmoles/ml. Lipase activity of UHT milk after 30, 60 and 90 days of room temperature storage was 0.44 ± 0.03, 0.95 ± 0.07 and 1.14 ± 0.09 µmoles/ml. Color, flavor and smell score decreased through the storage of UHT milk for 90 days. CONCLUSION: The results of this investigation revealed that fatty acid and triglyceride profile changed after 60 and 90 days of storage. Production of organic acids led to the drop of pH and sensory characteristics in UHT milk during the long-term storage. Induction period can be successfully used for the determination of anticipatory shelf life of UHT milk.

Effect of moderate inlet temperatures in ultra-high-pressure homogenization treatments on physicochemical and sensory characteristics of milk.

The effect of ultra-high-pressure homogenization (UHPH) on raw whole milk (3.5% fat) was evaluated to obtain processing conditions for the sterilization of milk. Ultra-high-pressure homogenization treatments of 200 and 300 MPa at inlet temperatures (Ti) of 55, 65, 75, and 85 °C were compared with a UHT treatment (138 °C for 4s) in terms of microbial inactivation, particle size and microstructure, viscosity, color, buffering capacity, ethanol stability, propensity to proteolysis, and sensory evaluation. The UHPH-treated milks presented a high level of microbial reduction, under the detection limit, for treatments at 300 MPa with Ti of 55, 65, 75, and 85 °C, and at 200 MPa with Ti = 85 °C, and few survivors in milks treated at 200 MPa with Ti of 55, 65, and 75 °C. Furthermore, UHPH treatments performed at 300 MPa with Ti = 75 and 85 °C produced sterile milk after sample incubation (30 and 45 °C), obtaining similar or better characteristics than UHT milk in color, particle size, viscosity, buffer capacity, ethanol stability, propensity to protein hydrolysis, and lower scores in sensory evaluation for cooked flavor.

Milk serum peptidomics revealed the age gelation of direct UHT milk.

Age gelation is undesirable for direct UHT (dUHT) milk, which is closely related to protein hydrolysis. However, little information is available for the role of serum peptides during the age gelation. In this study, the composition and protein morphology of serum phase were characterized by RP-HPLC, ICP-MS and TEM. The results showed significant increases in soluble proteins, free amino acids, calcium, and phosphorus from casein micelles, indicating protein hydrolysis and peptide release into the serum phase. 23,466 peptides derived from caseins and other proteins were identified in serum phase by peptidomics. The serum peptide profiles of age gelation milk changed dramatically. Peptide fingerprinting revealed that plasmin and cathepsin contributed to the protein hydrolysis during age gelation, with a significant increase in their activity observed. 23 characteristic peptides were ultimately selected as potential indicators for age gelation. These findings provide new insights into the age gelation of UHT milk.

Exploration of Novel Plasmin Inhibitor from ß-Lactoglobulin for Enhancing the Storage Stability of UHT Milk.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. ß-Lactoglobulin (ß-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in ß-Lg. Herein, plasmin inhibitory peptides were explored from ß-Lg using experimental and computational techniques. The results revealed that increased denaturation of ß-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the ß-Lg/plasmin complex. Denatured ß-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to ß-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from ß-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Characterization of the flavor profile of UHT milk during shelf-life via volatile metabolomics fingerprinting combined with chemometrics.

Ultra-high temperature (UHT) milk is popular among consumers. However, its flavor and texture change in its shelf life. Flavor is highly determinative for the success of dairy products and for consumers' willingness to buy. It is important to milk producers to ensure the optimal flavor of their products in the shelf life. In order to be able to control and predict the flavor quality of UHT milk during the shelf life, this study compared the variations in sensory quality, volatile aroma release and backbone flavor factors and developed a discriminant model to assess flavor quality based on flavouromics data of five competing milk sample during storage. Using partial least squares discriminant analysis (PLS-DA) with Electronic-nose (E-nose) data excellent classification sensitivity and specificity were achieved compared to models based on gas chromatography-mass spectrometry (GC-MS) data. The PLS-DA model using E-nose data exhibited a 100% correct classification rate for the storage period, and a 92% correct rate based on the eight variable importance in the projection (VIP) elements screened for volatile components from different groups. The discriminative model developed herein based on E-nose combined with chemometrics demonstrated advantages such as speed, efficiency, and environmental friendliness. This method shows promise as a precise tool for analyzing aroma changes in UHT milk during its shelf life, and provide support for controlling the flavor substances and milk product development.

Immunogold Labeling of Milk Proteins at Transmission Electron Microscopy.

Milk intended for human consumption is subjected to technological treatments to ensure its safety and storage stability. These treatments deeply modify some of its structural and nutritional characteristics. Principal modifications involve proteins that partly adsorb onto the membrane of milk fat globules upon homogenization or whey proteins that undergo denaturation and aggregation during thermal treatments. Transmission electron microscopy is a powerful approach to investigate milk ultrastructure, due to its high-resolution power. Immunogold labeling of ß-lactoglobulin and ß-casein proteins represents a sophisticated approach to examine their structure and localization following technological processes such as homogenization and UHT treatment. However, liquid milk is a very challenging matrix because of its complex multiphasic nature. To preserve both ultrastructure and antigenicity, and to obtain an efficient labelling in liquid milk samples, some precautions shall be adopted in fixation, embedding, and labeling steps as here reported.

Bisphenol determination in UHT milk and packaging by paper spray ionization mass spectrometry.

This study evaluates the use of paper spray ionization mass spectrometry (PSI-MS) for rapid determination of bisphenol A (BPA) and bisphenol S (BPS) in UHT milk and milk packaging. The packages were analyzed by cutting the cartons into triangular shapes and submitting them to PSI-MS analysis. The milk samples were subjected to a simple liquid-liquid extraction and the supernatant was deposited onto a triangular paper that was subsequently used for PSI-MS analysis. In milk, BPS and BPA levels ranged from 60.0 to 150.8 ng mL-1. The LOD and LOQ values were 1.5 and 4.8 ng mL-1 for BPA, and 4.8 and 16.0 ng mL-1 for BPS, respectively. Linearity was R2 > 0.98 for both compounds. Precision values were below 20%, and recoveries close to 100%. The PSI-MS can be used as a simple, rapid, and accurate methodology to determine bisphenols in milk and milk packaging.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Effect of Thermostable Enzymes Produced by Psychrotrophic Bacteria in Raw Milk on the Quality of Ultra-High Temperature Sterilized Milk.

Ultra-high temperature sterilized milk (UHT) is a popular dairy product known for its long shelf life and convenience. However, protein gel aging and fat quality defects like creaming and flavor deterioration may arise during storage. These problems are primarily caused by thermostable enzymes produced by psychrotrophic bacteria. In this study, four representative psychrotrophic bacteria strains which can produce thermostable enzymes were selected to contaminate UHT milk artificially. After 11, 11, 13, and 17 weeks of storage, the milk samples, which were contaminated with Pseudomonas fluorescens, Chryseobacterium carnipullorum, Lactococcus raffinolactis and Acinetobacter guillouiae, respectively, demonstrated notable whey separation. The investigation included analyzing the protein and fat content in the upper and bottom layers of the milk, as well as examining the particle size, Zeta potential, and pH in four sample groups, indicating that the stability of UHT milk decreases over time. Moreover, the spoiled milk samples exhibited a bitter taste, with the dominant odor being attributed to ketones and acids. The metabolomics analysis revealed that three key metabolic pathways, namely ABC transporters, butanoate metabolism, and alanine, aspartate, and glutamate metabolism, were found to be involved in the production of thermostable enzymes by psychrotrophic bacteria. These enzymes greatly impact the taste and nutrient content of UHT milk. This finding provides a theoretical basis for further investigation into the mechanism of spoilage.

The effects of packaging type and storage temperature on some of UHT milk quality indexes.

The objective of the present study was to illustrate the changes in physicochemical properties in ultra-high temperature (UHT) milk packed into a pouch and Tetra Brik during storage. UHT milk samples were kept at 5 and 25 °C for 3 months and regularly analyzed monthly. During storage, significant increases (p < 0.05) in titratable acidity (TA), water-soluble nitrogen (WSN), and non-protein nitrogen (NPN) when UHT milk was packed into pouch versus Tetra Brik and stored at 25 versus 5 °C. Neither type of packaging nor storage temperature affect pH values during storage. Spore-forming bacterial (SFB) count was always higher in UHT milk packed into pouch versus Tetra Brik. Refrigerated storage kept UHT milk without detectable SFB compared to UHT milk held at 25 °C. Pouch packages were responsible for the migration of phthalate derivatives [dimethyl phthalate "DMP", diethyl phthalate "DEP", dibutyl phthalate "DBP", and di-(2-Ethylhexyl) phthalate "DEHP"] into milk with significantly greater levels than milk filled into Tetra Brik. The total sensory scores were decreased significantly during storage, which was more pronounced in UHT milk filled into pouch versus Tetra Brik or stored at 25 °C versus 5 °C. It is concluded that UHT milk filled into Tetra Brik stored at 5 and 25 °C is better in terms of quality and safety indexes than such filled into a pouch.

Phylogenetic characterization and biodiversity of spore-forming bacteria isolated from Brazilian UHT milk.

Among the milk contaminating microorganisms, those which are able to form heat-resistant spores are concerning, especially for dairy companies that use ultra-high temperature (UHT) technology. These spores, throughout storage, can germinate and produce hydrolytic enzymes that compromise the quality of the final product. This study evaluated 184 UHT milk samples from different batches collected from seven Brazilian dairy companies with a possible microbial contamination problem. The bacteria were isolated, phenotypically characterized, clustered by REP-PCR, and identified through 16S rDNA sequencing. The presence of Bacillus sporothermodurans was verified using biochemical tests (Gram staining, catalase and oxidase test, glucose fermentation, esculin hydrolysis, nitrate reduction, and urease test). According to these tests, none of the isolates presented typical characteristics of B. sporothermodurans. In sequence, the isolates, that presented rod-shapes, were submitted to molecular analyses in order to determine the microbial biodiversity existing among them. The isolates obtained were grouped into 16 clusters, four of which were composed of only one individual. A phylogenetic tree was constructed using the sequences obtained from the 16S rDNA sequencing and some reference strains of species close to those found using BLAST search in the NCBI nucleotide database. Through this tree, it was possible to verify the division of the isolates into two large groups, the Bacillus subtilis and the Bacillus cereus groups. Furthermore, most isolates are phylogenetically closely related, which makes it even more difficult to identify them at the species level. In conclusion, it was possible to assess, in general, the groups of sporulated contaminants in Brazilian UHT milk produced in the regions evaluated. In addition, it was also possible to determine the biodiversity of spore-forming bacteria found in UHT milk samples, thus opening up a range of possible research topics regarding the effects of the presence of these microorganisms on milk quality.