Characterization of Pseudomonas spp. contamination and in situ spoilage potential in pasteurized milk production process.

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.

Influence of flash heating and aspergillopepsin I supplementation on must and wine attributes of aromatic varieties.

The protein instability with haze formation represents one of the main faults occurring in white and rosé wines. Among the various solutions industrially proposed, aspergillopepsin I (AP-I) supplementation coupled with must heating (60-75 °C) has been recently approved by OIV and the European Commission for ensuring protein stability of wines. This study investigates the impact of AP-I either applied independently or in combination with flash pasteurization on the chemical composition of grape must and wines derived from Sauvignon Blanc and Gewürztraminer. The efficacy on protein stability of a complete treatment combining heat (70 °C) and AP-I (HP) was confirmed through heat test and bentonite requirement, although no differences were observed between must heating and HP treatments. However, high-performance liquid chromatography analysis of unstable pathogenesis-related proteins revealed that AP-I supplementation reduced chitinases and thaumatin-like proteins compared to the non-enzymed samples, with and without must heating. Amino acid increase was reported only in HP musts, particularly in Sauvignon Blanc. The concentration of yeast-derived aroma compounds in Gewürztraminer wines was increased by must heating; compared to controls, flash pasteurization rose the overall acetate esters content of 85 % and HP of 43 %, mostly due to isoamyl acetate. However, heat treatments -with or without AP-I- reduced terpenes up to 68 %. Despite the different aroma profiles, no differences were observed for any descriptor for both varieties in wine tasting, and only a slight decrease trend was observed for the floral intensity and the typicality descriptors in heated wines.

Formation of Key Aroma Compounds During 30 Weeks of Ripening in Gouda-Type Cheese Produced from Pasteurized and Raw Milk.

Gouda-type cheeses were produced on a pilot-scale from raw milk (RM-G) and pasteurized milk (PM-G). Sixteen key aroma compounds previously characterized by the sensomics approach were quantitated in the unripened cheeses and at five different ripening stages (4, 7, 11, 19, and 30 weeks) by means of stable isotope dilution assays. Different trends were observed in the formation of the key aroma compounds. Short-chain free fatty acids and ethyl butanoate as well as ethyl hexanoate continuously increased during ripening but to a greater extent in RM-G. Branched-chain fatty acids such as 3-methylbutanoic acid were also continuously formed and reached a 60-fold concentration after 30 weeks, in particular in PM-G. 3-Methylbutanal and butane-2,3-dione reached a maximum concentration after 7 weeks and decreased with longer ripening. Lactones were high in the unripened cheeses and increased only slightly during ripening. Recent results have shown that free amino acids were released during ripening. The aroma compounds 3-methylbutanal, 3-methyl-1-butanol, and 3-methylbutanoic acid are suggested to be formed by microbial enzymes degrading the amino acid l-leucine following the Ehrlich pathway. To gain insight into the quantitative formation of each of the three aroma compounds, the conversion of the labeled precursors (13C6)-l-leucine and (2H3)-2-keto-4-methylpentanoic acid into the isotopically labeled aroma compounds was studied. By applying the CAMOLA approach (defined mixture of labeled and unlabeled precursor), l-leucine was confirmed as the only precursor of the three aroma compounds in the cheese with the preferential formation of 3-methylbutanoic acid.

Influence of Milk Pasteurization on the Key Aroma Compounds in a 30 Weeks Ripened Pilot-Scale Gouda Cheese Elucidated by the Sensomics Approach.

Gouda cheese was produced from pasteurized milk and ripened for 30 weeks (PM-G). By application of gas chromatography/olfactometry and an aroma extract dilution analysis on the volatiles isolated by extraction/SAFE distillation, 25 odor-active compounds in the flavor dilution (FD) factor range from 16 to 4096 were identified. Butanoic acid, 2- and 3-methylbutanoic acid, and acetic acid showed the highest FD factors, and 2-phenylethanol, δ-decalactone, and δ-dodecalactone were most odor-active in the neutral-basic fraction. Quantitations by stable isotope dilution assays followed by a calculation of odor activity values (OAVs) revealed acetic acid, 3-methylbutanoic acid, butanoic acid, and butane-2,3-dione with the highest OAVs. Finally, an aroma recombinate prepared based on the quantitative data well agreed with the aroma profile of the PM-G. In Gouda cheese produced from raw (nonpasteurized) milk (RM-G), qualitatively the same set of odor-active compounds was identified. However, higher OAVs of butanoic acid, hexanoic acid, and their corresponding ethyl esters were found. On the other hand, in the PM-G, higher OAVs for 3-methylbutanoic acid, 3-methylbutanol, 3-methylbutanal, and butane-2,3-dione were determined. The different rankings of these key aroma compounds clearly reflect the aroma differences of the two Gouda-type cheeses. A higher activity of lipase in the RM-G and higher amounts of free l-leucine in PM-G on the other side were responsible for the differences in the concentrations of some key aroma compounds.

Harnessing Nature's Defence: The Antimicrobial Efficacy of Pasteurised Cattle Milk-Derived Extracellular Vesicles on Staphylococcus aureus ATCC 25923.

Increasing antimicrobial resistance (AMR) challenges conventional antibiotics, prompting the search for alternatives. Extracellular vesicles (EVs) from pasteurised cattle milk offer promise, due to their unique properties. This study investigates their efficacy against five pathogenic bacteria, including Staphylococcus aureus ATCC 25923, aiming to combat AMR and to develop new therapies. EVs were characterised and tested using various methods. Co-culture experiments with S. aureus showed significant growth inhibition, with colony-forming units decreasing from 2.4 × 105 CFU/mL (single dose) to 7.4 × 104 CFU/mL (triple doses) after 12 h. Milk EVs extended lag time (6 to 9 h) and increased generation time (2.8 to 4.8 h) dose-dependently, compared to controls. In conclusion, milk EVs exhibit dose-dependent inhibition against S. aureus, prolonging lag and generation times. Despite limitations, this suggests their potential in addressing AMR.

Use of Botanical Ingredients: Nice Opportunities to Avoid Premature Oxidation of NABLABs by Increasing Their ORAC Values Strongly Impacted by Dealcoholization or Pasteurization.

Even when fresh, non-alcoholic, and low-alcoholic beers (NABLABs) exhibit significant staling defects due to premature oxidation. In this study, the antioxidant power of eleven fresh commercial NABLABs was assessed by means of three different assays: the oxygen radical absorbance capacity (ORAC), the linoleic acid-induced oxidation (TINH), and the indicator time test (ITT). Only the first two assays, both involving radicalar degradations initiated by AAPH, were found to correlate with each other. NABLABs displayed lower ORAC values than conventional beers (on average, 6127 µmol eq. Trolox/L), except for three samples made with special-colored malts or dry-hopped. Dealcoholization was the step with the greatest impact on the ORAC value (up to a 95% loss) and on flavan-3-ols, sotolon, and polyfunctional thiols, while pasteurization strongly affected color, TBA, and Strecker aldehydes. ORAC assays applied to hop, alternative cereals, and various botanical ingredients indicated that mashing with red sorghum, dry hopping/spicing, and wood maturation could bring the antioxidant power of a NABLAB close to those of conventional beers. With an ORAC value not reached by any other tested botanical ingredient (5234 µmol eq. Trolox/g), African Vernonia amygdalina leaves (traditionally used for Rwandan Ikigage beers) emerged here as the best candidate.

Optimized Ultraviolet-C Processing Inactivates Pathogenic and Spoilage-Associated Bacteria while Preserving Bioactive Proteins, Vitamins, and Lipids in Human Milk.

Holder pasteurization (HoP) enhances donor human milk microbiological safety but damages many bioactive milk proteins. Though ultraviolet-C irradiation (UV-C) can enhance safety while better preserving some milk proteins, it has not been optimized for dose or effect on a larger array of bioactive proteins. We determined the minimal UV-C parameters that provide >5-log reductions of relevant bacteria in human milk and how these treatments affect an array of bioactive proteins, vitamin E, and lipid oxidation. Treatment at 6000 and 12 000 J/L of UV-C resulted in >5-log reductions of all vegetative bacteria and bacterial spores, respectively. Both dosages improved retention of immunoglobulin A (IgA), IgG, IgM, lactoferrin, cathepsin D, and elastase and activities of bile-salt-stimulated lipase and lysozyme compared with HoP. These UV-C doses caused minor reductions in α-tocopherol but not γ-tocopherol and no increases in lipid oxidation products. UV-C treatment is a promising approach for donor human milk processing.

The entwined circles of quality improvement & advocacy.

Health policy and quality improvement initiatives exist symbiotically. Quality projects can be spurred by policy decisions, such as the creation of financial incentives for high-value care. Then, advocacy can streamline high-value care, offering opportunities for quality improvement scholars to create projects consistent with evidenced-based care. Thirdly, as pediatrics and neonatology reconcile with value-based payment structures, successful quality initiatives may serve as demonstration projects, illustrating to policy-makers how best to allocate and incentivize resources that optimize newborn health. And finally, quality improvement (QI) can provide an essential link between broad reaching advocacy principles and boots-on-the-ground local or regional efforts to implement good ideas in ways that work practically in particular environments. In this paper, we provide examples of how national legislation elevated the importance of QI, by penalizing hospitals for low quality care. Using Medicaid coverage of pasteurized human donor milk as an example, we discuss how advocacy improved cost-effectiveness of treatments used as tools for quality projects related to reduction of necrotizing enterocolitis and improved growth. We discuss how the future of QI work will assist in informing the agenda as neonatology transitions to value-based care. Finally, we consider how important local and regional QI work is in bringing good ideas to the bedside and the community.

Dynamics of microbiota and antimicrobial resistance in on-farm dairy processing plants using metagenomic and culture-dependent approaches.

On-farm dairy processing plants, which are situated close to farms and larger dairy processing facilities, face unique challenges in maintaining environmental hygiene. This can impact various stages of dairy processing. These plants operate on smaller scales and use Low-Temperature-Long-Time (LTLT) pasteurization, making them more susceptible to microbial contamination through direct and indirect contact. Antimicrobial-resistant bacteria found on dairy farms pose risks to human health by potentially transferring resistance via dairy products. Our study aimed to investigate microbial distribution and antimicrobial resistance at four key stages: the farm, pre-pasteurization, post-pasteurization, and processing environments. We assessed microbial distribution by quantifying indicator bacteria and conducting metagenomic analysis. Antimicrobial resistance was examined by identifying resistance phenotypes and detecting resistance genes in bacterial isolates and metagenomes. Our results showed that the indicator bacteria were detected at all stages of on-farm dairy processing. We observed a significant reduction in aerobic microbes and coliforms post-pasteurization. However, contamination of the final dairy products increased, suggesting potential cross-contamination during post-pasteurization. Metagenomic analysis revealed that Pseudomonas, a representative psychrotrophic bacterium, was predominant in both the farm (24.1 %) and pre-pasteurization (65.9 %) stages, indicating microbial transfer from the farms to the processing plants. Post-pasteurization, Pseudomonas and other psychrotrophs like Acinetobacter and Enterobacteriaceae remained dominant. Core microbiota analysis identified 74 genera in total, including 13 psychrotrophic bacteria, across all stages. Of the 59 strains isolated from these plants, 49 were psychrotrophic. Antimicrobial resistance analysis showed that 74.6 % (44/59) of isolates were resistant to at least one antibiotic, with cefoxitin-, ampicillin-, amoxicillin-, and ticarcillin-resistant bacteria present at all stages. Identical antimicrobial resistance patterns were observed in isolates from serial stages of the same farm and season, suggesting bacterial transmission across stages. Additionally, 27.1 % (16/59) of isolates carried plasmid-mediated resistance genes, which were also detected in the metagenomes of non-isolated samples, indicating potential antimicrobial resistance gene transmission and their presence in uncultured bacteria. These findings reveal the persistence of antimicrobial-resistant psychrotrophic bacteria in on-farm dairy processing plants, which pose potential health risks via dairy consumption. Our study underscores the importance of both culture-dependent and culture-independent methods to fully understand their distribution and impact.

Effects of feeding pasteurized waste milk or saleable milk to calves on weight, health and fecal Escherichia coli antimicrobial resistance.

The aim of this study was to compare the effects of feeding pasteurized waste milk or saleable milk to calves on weight, health and emergence of antimicrobial resistance in Escherichia coli strains isolated from those calves. An experimental study under field conditions on a commercial pasture-based Argentinian dairy farm was carried out. Forty Holstein calves were assigned randomly to either pasteurized waste milk (PWM) or non-pasteurized saleable milk (SM). The antimicrobial agents (AM) used on the farm, both to treat or prevent diseases, were recorded. The passive immunity level, calf live weight, AM presence in milk, clinical examination of calves, and E. coli isolation and identification, were performed. A total of 258 E. coli strains were isolated from fecal samples (132 isolates from SM calves and 126 from PWM calves at six sampling times). All E. coli isolated were used to perform AM susceptibility tests (disc diffusion and agar dilution). No differences were observed between groups in health parameters, average daily gain or prevalence of resistant E. coli strains to any AM evaluated throughout the study. Peaks of trimethoprim, sulfamethoxazole and enrofloxacin minimum inhibitory concentration (MIC) were observed at 30 d in E. coli from both groups of calves, whilst additional peaks to tetracyclin and ampicillin were observed only in SM calves. All MIC apart from gentamicin decreased at 75 and 90 d of age (during the weaning period). Gentamicin MIC behaved differently, having no peaks and increasing at 90 d only in PWM group. In conclusion, we found no evidence that emergence of antibiotic resistance is related to the consumption of pasteurized waste milk.

Implementation of hazard analysis and critical control point (HACCP) in yogurt production.

This study aimed to review hazard analysis and critical control points (HACCP) in the dairy industry for the production of yogurt. The food safety management system (FSMS) was implemented over the last several decades with several amendments. The need for practical and proactive procedures in the dairy industry was identified so that HACCP implementation could ensure that consumers would always have safe food. The concept of HACCP is a systemic and science-based method that can result in safe dairy products such as yogurt based on the complete analysis of manufacturing processes, recognition of hazards potentially present at all stages of production, and risk prevention. In yogurt production, raw milk receipt, pasteurization, packaging, and storage are the steps most susceptible to contamination and were considered critical control points. Further steps also need to be implemented to achieve other related control measures, and these will be discussed.

Protein aggregation caused by pasteurization processing affects the foam performance of liquid egg white.

Pasteurization is necessary during the production of liquid egg whites (LEW), but the thermal effects in pasteurization could cause an unavoidable loss of foaming properties of LEW. This study intended to investigate the mechanism of pasteurization processing affects the foam performance of LEW. The foaming capacity (FC) of LEW deteriorated significantly (ΔFCmax = 72.33 %) and foaming stability (FS) increased slightly (ΔFSmax = 3.64 %) under different temperature-time combinations of pasteurization conditions (P < 0.05). The increased turbidity and the decreased solubility together with the decreased absolute value of Zeta potential indicated the generation of thermally induced aggregates and the instability of the protein particles, Rheological characterization demonstrated improved viscoelasticity in pasteurization liquid egg whites (PLEW), explaining enhanced FS. The study revealed that loss in foaming properties of PLEW resulted from thermal-induced protein structural changes and aggregation, particularly affecting FC. This provided a theoretical reference for the production and processing of LEW products.

The impact of heat treatment of bovine milk on gastric emptying and nutrient appearance in peripheral circulation in healthy females: a randomized controlled trial comparing pasteurized and ultra-high temperature milk.

BACKGROUND: Heat treatments of dairy, including pasteurization and ultra-high temperature (UHT) processing, alter milk macromolecular structures, and ultimately affect digestion. In vitro, animal, and human studies show faster nutrient release or circulating appearance after consuming UHT milk (UHT-M) compared with pasteurized milk (PAST-M), with a faster gastric emptying (GE) rate proposed as a possible mechanism. OBJECTIVES: To investigate the impact of milk heat treatment on GE as a mechanism of faster nutrient appearance in blood. We hypothesized that GE and circulating nutrient delivery following consumption would be faster for UHT-M than PAST-M. METHODS: In this double-blind randomized controlled cross-over trial, healthy female (n = 20; 27.3 ± 1.4 y, mean ± SD) habitual dairy consumers, consumed 500 mL of either homogenized bovine UHT-M or PAST-M (1340 compared with 1320 kJ). Gastric content volume (GCV) emptying half-time (T50) was assessed over 3 h by magnetic resonance imaging subjective digestive symptoms, plasma amino acid, lipid and B vitamin concentrations, and gastric myoelectrical activity were measured over 5 h. RESULTS: Although GCV T50 did not differ (102 ± 7 min compared with 89 ± 8 min, mean ± SEM, UHT-M and PAST-M, respectively; P = 0.051), GCV time to emptying 25% of the volume was 31% longer following UHT-M compared with PAST-M (42 ± 2 compared with 32 ± 4 min, P = 0.004). Although GCV remained larger for a longer duration following UHT-M (treatment × time interaction, P = 0.002), plasma essential amino acid AUC was greater following UHT-M than PAST-M (55,324 ± 3809 compared with 36,598 ± 5673 µmol·min·L-1, P = 0.006). Heat treatment did not impact gastric myoelectrical activity, plasma appetite hormone markers or subjective appetite scores. CONCLUSIONS: Contrary to expectations, GE was slower with UHT-M, yet, as anticipated, aminoacidemia was greater. The larger GCV following UHT-M suggests that gastric volume may poorly predict circulating nutrient appearance from complex food matrices. Dairy heat treatment may be an effective tool to modify nutrient release by impacting digestion kinetics. CLINICAL TRIAL REGISTRY: www.anzctr.org.au (ACTRN12620000172909).

Pulsed electric field treatment for preservation of Chlorella suspensions and retention of gelling capacity.

Pulsed electric field (PEF) processing has emerged as an alternative to thermal pasteurization for the shelf-life extension of heat-sensitive liquids at industrial scale. It offers the advantage of minimal alteration in physicochemical characteristics and functional properties. In this study, a pilot-scale continuous PEF processing (Toutlet < 55 °C) was applied to microalgae Chlorella vulgaris (Cv) suspensions (pH = 6.5), which was proposed as a functional ingredient for plant-based foods. Cv suspensions were inoculated with three distinct food spoilage microorganisms (Pseudomonas guariconensis, Enterobacter soli and Lactococcus lactis), isolated from the Cv biomass. PEF treatments were applied with varying electric field strength Eel of 16 to 28 kV/cm, pulse repetition rate f of 100 to 140 Hz, with a pulse width τ of 20 µs and an inlet product temperature Tin of 30 °C. The aim was to evaluate the PEF-induced microbial reduction and monitor the microbial outgrowth during a 10-day cold storage period (10 °C). Maximum inactivation of 4.1, 3.7 and 3.6 logs was achieved (28 kV/cm and 120 Hz) for the investigated isolates, respectively. Under these conditions, the critical electric field strengths Ecrit, above which inactivation was observed, ranged from 22.6 to 24.6 kV/cm. Moreover, repeated PEF treatment resulted in similar inactivation efficiency, indicating its potential to enhance shelf-life further.

Ultraviolet irradiation as alternative non-thermal cold pasteurization to improve quality and microbiological parameters of mango juice during cold storage.

The objectives of this research were to study the effect of UV irradiation on quality characteristics of mango juice during cold storage. Mango juice exposed to UV radiation was also used to determine zero-order and first-order kinetic models of microbial (total plate count, yeast and mold count, and Escherichia coli) reduction. According to the microbiological results, UV light at 120 J/cm2 caused a 5.19 log reduction. It was found that microbial inactivation of all tested microorganisms followed first-order kinetic model. The treatments did not differ significantly in terms of the quality metrics. L*, b*, pH, total soluble solid, total phenolic compound, total flavonoid content, and antioxidant activity as measured by the DPPH and FRAP assay all tended to decline during storage at 4 °C, whereas a*, ∆E, titratable acidity, total plate count, yeast and mold count, as well as the total plate count, had an increasing trend. During storage at 4 °C, UV irradiation increased the shelf life of mango juice by about 14 days compared to the control sample. In conclusion, this study demonstrated the potential of UV treatment as an alternative to thermal pasteurization for preserving mango juice quality and safety while also prolonging shelf life.

Gut Microbiome and Metabolome Modulation by High-Hydrostatic-Pressure-Processed Tomato Juice.

High hydrostatic pressure (HHP) is a non-thermal pasteurization technology for the enhancement of food products' safety and quality. The components of tomato juice can be affected by HHP processing. Little is known about the effects of HHP-processed tomato juice on the gut microbiome and metabolism. Here, we performed high-throughput sequencing and metabolomics profiling to determine the critical differences in gut microbiota structure and metabolic profiles in mice administered with HHP-processed tomato juice. Tomato juice administration significantly increased the gut bacterial alpha diversity and the relative abundance of Bacteroides. The mice administered with HHP-processed tomato juice were characterized by the enrichment of Bacteroidetes, Alistieps, and Faecalibaculum compared with those administered with HTST-processed tomato juice. Moreover, HHP-processed tomato juice promoted SCFA levels, which were positively correlated with the enriched Alistieps. Our results show that HHP-processed tomato juice may drive healthy gut microbes and metabolites.

Influence of pasteurization and spray drying on the fat digestion behavior of human milk fat analog emulsion: a simulated in vitro infant digestion study.

BACKGROUND: Human milk fat analog emulsion (HMFAE) is an emulsion that mimics the composition and structure of human milk (HM) fat globules. The application of HMFAE in infant formula requires a series of milk powder processing steps, such as pasteurization and spray drying. However, the effect of milk powder processing on fat digestion of HMFAE is still unclear. In this study, the influence of pasteurization and spray drying on the lipolysis behavior of HMFAE was studied and compared with HM using a simulated infant in vitro digestion model. RESULTS: Pasteurization and spray drying increased the flocculation and aggregation of lipid droplets in HMFAE during digestion. Spray drying destroyed the lipid droplet structure of HMFAE, and partial milk fat globule membrane-covered lipid droplets turned into protein-covered lipid droplets, which aggravated lipid-protein aggregation during gastric digestion and hindered fat digestion in the small intestine. The final lipolysis degree was in the order HM (64.55%) > HMFAE (63.41%) > pasteurized HMFAE (61.75%) > spray-dried HMFAE (60.57%). After complete gastrointestinal digestion, there were no significant differences in free fatty acid and sn-2 monoacylglycerol profile among the HMFAE, pasteurized HMFAE, and spray-dried HMFAE. CONCLUSION: Milk powder processing can reduce lipolysis by altering the lipid droplet structure of HMFAE and the degree of lipid droplet aggregation during digestion. © 2024 Society of Chemical Industry.

Effect of different sterilization methods on the appearance, composition, and flavor of sugarcane juice.

Cold-pressed sugarcane juice (SCJ) is a beverage rich in vitamins, carbohydrates, and antioxidants. Various sterilization methods impact fruit juice's appearance, nutrients, and flavor. Hence, this study aims to assess how different sterilization techniques affect the flavor, appearance, and nutritional value of SCJ. Freshly prepared SCJs were subjected to two sterilization methods: pasteurization (referred to as PTG) and autoclaving (referred to as HTHP). The pasteurization process was carried out at 63°C for 30 min, whereas the HTHP process was applied at 115°C for 30 min. The appearances, Brix value, colors, sugar, organic acid content, and aromatic compounds were determined. The Brix and pH values of the juice show little variation across different heat treatments. The color index of PTG was similar to that of the control group, whereas the L* value of HTHP increased about 21%, resulting in a significant color change. The glucose and fructose contents of HTHP were 7.03 and 5.41 mg/mL, which were much higher than those of PTG (3.26 and 2.33 mg/mL) and control group (3.33 and 2.48 mg/mL). A total of 77 aromatic compounds were identified in the SCJ after various heat treatments. Among them, pentanoic acid, octanal, and ß-damascenone were the most abundant substances contributing to the overall flavor in the control group, PTG, and HTHP. Pasteurization preserved the original flavor of the juice, whereas autoclaving triggered the Maillard reaction, forming pyrazine and furan-like compounds that altered the SCJ's flavor. In conclusion, pasteurization retained SCJ's original characteristics, whereas HTHP induces changes in nutrition and imparts a distinct flavor.

Troubleshooting high laboratory pasteurization counts in organic raw milk requires characterization of dominant thermoduric bacteria, which includes nonsporeformers as well as sporeformers.

Laboratory pasteurization count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. No regulatory limit has presently been set for LPC, but LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Although it is occasionally used as a proxy for levels of bacterial spores in raw milk, limited knowledge is available on the types of organisms that are enumerated by LPC in contemporary raw milk supplies. Although historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as gram-positive sporeformers, gram-positive nonsporeformers, and gram-negatives, respectively; 0.5% could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as gram-positive sporeformers were predominantly Bacillus (168/200), and gram-positive nonsporeformers were predominantly Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk, and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using MALDI-TOF mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. Although our data indicated agreement between 16S rDNA sequencing and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF MS, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.