UHT Milk Research Articles

Interaction of Sambiloto (Andrographis paniculata) Bioactive Compound with Milk Protein (Whey and Casein) Through Molecular Docking and Molecular Dynamics Simulation as a Basis for Encapsulation

This research aims for developing immune-boosting products necessary. The active ingredient in Andrographis paniculate (AP) acts as an immunostimulant which can improve the work of the immune system. The first stage of research was a collection of bioactive compounds from KnapSack database of Kanaya, Dr. Duke's Phytochemical and Ethnobotanical were compiled and selected based on the online pass results of each bioactive compound as an immunomodulator and 10 active compounds were obtained which will be continued. The second stage of research was a docking molecular between whey proteins (β-lactoglobulin and α-lactalbumin) with active compounds from AP and casein (α-Casein, β-Casein, and κ-Casein). The highest binding affinity was obtained for α-Casein with Neoandrographolide at -9.2 Kcal/mol. The results of the complex α-Casein with Neoandrographolide (CC) and α-Casein with Neoandrographolide ultraheat (CCT) support the research, namely to function α-Casein as an encapsulant well as a transporter or drug delivery of Neoandrographolide without changing the conformation of casein and disturbing its function. However, the conformation of casein will change drastically during ultraheat treatment to maintain the conformation and binding with the Neoandrographolide ligand. In addition, it supports the simulation results of single α-Casein at ultraheat temperatures which show conformational stability that is not much different from single α-casein and complexes at physiological temperatures

Use of distinct projective techniques in investigating participants' perception of a clean label dairy product: A study on the presence of additives/stabilizers in Ultra high temperature processing milk's label in Brazil

AbstractUsing an online questionnaire, the perception of additives/stabilizers in ultra‐high temperature (UHT) milk was investigated using different projective techniques. Clustering data on the Food Choice Questionnaire (FCQ) identified two groups of participants, Moderates and Judicious, with the latter assigning significantly higher agreement scores for all FCQ items. In the Word Association task, clean label UHT milk was more associated with “Nutrients and Constituents” by the Moderates and less by the Judicious, suggesting that for part of the interviewed participants, the absence of stabilizers increases the general quality of the product. In the Product Personality Profiling task, the same product was related to “young people” and a “balanced diet” by the Moderates. At the same time, the Judicious was designed for a stereotype of healthy living. Finally, the methodologies used provide valuable insights into the dairy sector, holistically highlighting the nuances in consumer preferences and expectations, offering a significant strategic opportunity for developing and promoting new clean‐label products in the segment.Practical ApplicationInvestigating consumer perceptions regarding the absence of additives offers valuable insights for marketing strategies and product development aimed at different consumer market segments, especially the dairy sector, and contributes to consumer psychology and behavioral research. At the same time, the food industry can use these findings to improve attribute communication, build consumer confidence, and adapt products to identified preferences, reflecting a practical intersection between academia and the industry.

Acceptance Analysis and Nutritional Content of Kluwih Seed Milk in Fulfilling Elderly Nutrition Intake

Kluwih seeds (Artocarpus communis) are a local food source that is still not widely used as a ready-to-eat food preparation. Kluwih seeds contain nutritional components such as protein, carbohydrates, fat, minerals and vitamins so that they can be used as a source of nutrients. The elderly are a group of people aged 60 years or over who currently need attention because the biological, physical and psychological aging that occurs in the elderly is feared to trigger inadequate food intake for the elderly so that adequate nutritional sources are needed. Kluwih seeds are a new inspiration to be processed into milk as a ready-to-eat food to meet the nutritional needs of the elderly. The aim of this research is to determine the acceptance of elderly people for kluwih seed milk and the percentage of nutritional content contained in kluwih seed milk. The research method used in this research is experimental. The samples in this study consisted of kluwih seed milk (P1), UHT milk (P2) and soy milk (P3). Acceptability analysis was carried out using scoring and hedonic test methods for taste, color and aroma parameters. Nutritional content analysis was carried out on the carbohydrate, protein and fat components of kluwih seed milk. Information on the acceptability of the elderly to kluwih seed milk was obtained from research subjects, namely 50 elderly panelists in 3 sample areas, namely Banjar Delod Padonan and Banjar Pengembungan, Pererenan Village and Banjar Negara, Sading Village, Mengwi District, Badung Regency, Bali. Based on the results of the hedonic test, elderly panelists in the sample area preferred kluwih seed milk (P1) over UHT milk (P2) and soy milk (P3), with the preferred characteristics of kluwih seed milk being yellowish white in color, very distinctive aroma, delicious and savory taste. The results of the nutritional content test show that kluwih seed milk contains 55.95% carbohydrates, 13.61% protein and 7.77% fat with a better nutritional ratio than UHT milk and soy milk.

Differences in development of volatiles and bitter peptides between pre- and post-hydrolysed lactose-free UHT milk during storage

Lactose-hydrolysed UHT milk (LHM) has a limited shelf life, especially due to early browning and off-flavour development. This study aimed to directly compare the development in colour, volatile and bitter peptide formation in pre- (lactase addition prior to UHT treatment) and post-hydrolysed (lactase addition after UHT treatment) milk using three different lactases and stored at 25 or 35 °C for up to one year. Pre-hydrolysed milk exhibited more pronounced browning than post-hydrolysed milk. Furthermore, level of Maillard related compounds like acetic acid and furfural were higher in pre-hydrolysed milk compared to post-hydrolysed milk, whereas post-hydrolysed milk was higher in Strecker aldehyde related volatile organic compounds (VOCs). After storage, level of bitter peptides was higher in post-hydrolysed milk using more impure lactases. Generally, lower VOC concentrations and bitter peptides were observed for LHM produced by more pure lactases suggesting higher purity lactases can aid in the development of ambient long-life LHM.

Bovine dairy production and diversity of regional dairy products sold in Presidente Médici markets - RO, Western Amazon

This study investigates bovine dairy production in Rondônia, as well as the availability of bovine dairy products produced in the state available in markets in the urban area of ​​Presidente Médici - RO. To this end, the SIDRA/IBGE Municipal Livestock Survey database was used, from 2015 to 2022; To assess the availability of dairy products, visits to markets were carried out. For the period analyzed, Rondônia emerges as the largest producer in the North Region, presenting its peak in 2018, with 1,160,198 liters, the state's contribution at national level is approximately 1.9%, the municipalities with the highest milk production were: Jaru, Machadinho do Oeste and Ouro Preto, respectively; the municipality of Presidente Médici ranked 11th with production of 212,913 liters. Regarding the commercialization of dairy products produced in the state, these were found in 100% of the markets, with cheese having the greatest diversity of types and manufacturers, followed by butter, yogurt, type C milk, UHT milk and dulce de leche, respectively. Dairy production in Rondônia and the municipality of Presidente Médici may be related to the need for genetic improvement of the herd, low technology and greater technical assistance to producers. With regard to dairy products available in the markets, it is clear that entrepreneurship through agroindustrialization is one of the economic strategies that integrate several stages of the production chain, adding value to the product, increasing the use of local labor, which contributes to reducing the rural exodus and increases the well-being and comfort of the families and communities involved.

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.

Identification of aroma-active compounds in milk by 2-dimensional gas chromatography-olfactometry-time-of-flight mass spectrometry combined with check-all-that-apply questions

Understanding consumers' sensory preferences for dairy products is essential. This study employed sensory analysis and instrumental techniques to analyze the flavor of pasteurized milk and UHT milk. There were 6 milk samples with similar fat content (4.0-4.6 g/100 mL) and protein content (3.2-3.8 g/100 mL). Sensory data from consumer tests was collected using check-all-that-apply (n = 100) and 9-point hedonic preferences. Research showed that Chinese consumers could distinguish the flavor of the 2 types of milk, and UHT milk showed a higher preference score, which may be due to the more pronounced milky flavor and sweet taste of UHT milk. A total of 48 aroma-active compounds were sniffed through 2-dimensional gas chromatography olfactometry time-of-flight MS, among which 11 were determined as key aroma-active compounds. Correlation analysis showed that milky odor, sweetness, and aftertaste-milky were positively correlated with γ-dodecalactone and γ-nonanolactone. Cooked and oxidized taste were positively correlated with 1-octen-3-ol and E-2-octenal. This study is important for developing Chinese dairy products and exporting dairy products to China by multinational companies.

Characterization of phospholipidome in milk, yogurt and cream, and phospholipid differences related to various dairy processing methods

Milk phospholipids have multiple health benefits, but the deficiency of detailed phospholipid profiles in dairy products brings obstacles to intake calculation and function evaluation of dairy phospholipids. In present study, 306 phospholipid molecular species were identified and quantified among 207 milk, yogurt and cream products using a HILIC-ESI-Q-TOF MS and a HILIC-ESI-QQQ MS. The phospholipid profiles of five mammals' milk show that camel milk contains the most abundant phosphatidylethanolamine, phosphatidylserine and sphingomyelin; cow, yak and goat milk have similar phospholipidomes, while buffalo milk contains abundant phosphatidylinositol. Fewer plasmalogens but more lyso-glycerolphospholipids were found in ultra-high-temperature (UHT) sterilized milk than in pasteurized milk, and higher proportions of lyso-glycerolphospholipid/total phospholipid were observed in both cream and skimmed/semi-skimmed milk than whole milk, indicating that UHT and skimming processes improve glycerolphospholipid degradation and phospholipid nutrition loss. Meanwhile, more diacyl-glycerolphospholipids and less of their degradation products make yogurt a better phospholipid resource than whole milk.

The Impact of Pulsed Electric Field Technology on Enzymes, Microbial and Nutritional Quality in Milk Processing - A Comprehensive Review

A pulsed electric field is a non-thermal technology used in the processing of foods, especially liquid foods such as milk, fruit juices, etc. The objective of this review is to provide an understanding of pulsed electric field technology and its application in milk processing, particularly in whole milk, skim milk, and UHT milk. This technology can be used as an alternative to traditional heat treatments such as pasteurizing, hot filling, blanching, and sterilization. In this technology, short high-intensity electric field is used at moderate temperatures. A pulsed electric field is an effective technology to inactivate microorganisms and enzymes with minimum changes in nutritional, functional, and organoleptic qualities compared to heat treatments. Pathogenic and spoilage bacteria can be reduced effectively when using pulsed electric field technology. However, there are variations between studies. As a recent advance, the pulsed electric field is used with mild heating. This shows a significant reduction in microbial count with less detrimental effects on the physico-chemical and organoleptic properties of food. It also has minimal impact on nutrients present in the milk such as proteins, milk fat globules, and vitamins. In this review, we describe in detail the introduction, the principle of pulsed electric field, the impact of pulsed electric field on microorganisms, enzymes, and nutrients, and the advantages and disadvantages of using PEF technology. Further studies can be conducted to investigate the combined approach of the pulsed electric field with mild heating treatment methods including pasteurization and sterilization.

Microbial Assessment of Biscuits, Chips, and UHT Milk in Hargeisa Somaliland

Microbial Assessment of Biscuits, Chips, and UHT Milk in Hargeisa Somaliland

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

Quantification of Adulterant Residues in UHT Milk Products using ATR-FTIR Spectroscopy Coupled with Multivariate Analysis

Quantification of Adulterant Residues in UHT Milk Products using ATR-FTIR Spectroscopy Coupled with Multivariate Analysis

Comparative study on microbiological, physicochemical and nutritional properties of whole cow milk by thermal and non-thermal processing technologies

The majority of the bioactive components in milk exhibit sensitivity to heat treatments. This research is the first systematically compare the effects of thermal (low-temperature long time pasteurization, LTLT; high-temperature short time pasteurization, HTST; ultra-high temperature, UHT; microwave-assisted heating; retort sterilization) and non-thermal (ultraviolet-C, UV-C; high hydrostatic pressure processing, HPP; filtered sterilization; high-intensity ultrasound, HIUS) treatments on the physicochemical properties and immune-active serum proteins of whole milk aliquots under the equivalent microbicidal efficacy. LTLT and HTST had little impact on whole milk. HPP at 400 MPa for 5 min was an optimal alternative to pasteurized milk in terms of equivalent microbicidal efficacy, higher nutrient retention (∼6% and 16.2% loss for IgG and lactoferrin) and fewer oxidation products (0.69 nmol/mg protein carbonyls). The serum protein denaturation of 600 MPa HPP-treated milk (79.4% and 31.36% loss for IgG and lactoferrin) was significantly higher than in pasteurized milk, but still lower than in UHT milk (97.16% and 70.85% loss for IgG and lactoferrin). Since photo-oxidation induced by high-dose irradiation was detrimental to the organoleptic properties, relying on UV-C as stand-alone replacement to pasteurization is not advisable. Combined with existing pasteurization methods, UV-C can achieve superior microbial inactivation at mild sterilization temperatures. HIUS, a promising novel tool, should be further optimized regarding the relevant standards to enable it to be used in industrial-scale processing and reproducibility. Compared with traditional thermal treatments, non-thermal strategies offer a significant reduction in the microbial load of raw milk while better preserving bioactive proteins, However, optimizing processing parameters remains essential to strike a balance between microbial inactivation and retention of active substances.

Effect of food environment on the ability of microorganisms to form biofilms

AbstractThe aim of this study is to understand the effect of the food product's, even in trace amounts, in the biofilm formation potential of Escherichia coli and Staphylococcus aureus. Bacterial adhesion was tested using three different food media, in which bacterial cells were adapted before determining how this would affect their adhesion score and biofilm formation potential: sterilized milk, mineral water, and pasteurized commercial orange juice. After characterization of the bacteria and the adhesion support by the contact angle, the adhesion score obtained by the theoretical mathematical model XDLVO developed by van Oss was compared to that obtained by the experimental approach, in order to examine the role of non‐specific (thermodynamic) and specific (biological) interactions in adhesion. Changes in the membrane composition of bacteria, as a function of acclimation conditions, were evaluated by infrared spectroscopy. Results reveal variability in the spectral region between 900 and 1200 cm−1 specific to capsular polysaccharides (CPS) or lipopolysaccharides (LPS), and also in the range between 2800 and 3000 cm−1 characteristic of membrane lipids and phospholipids. Biofilm formation by S. aureus and E. coli was affected by commercial UHT milk. This led to increased hydrophobicity and total adhesion energy within the environment of these strains. However, no correlation was observed between the adhesion scores obtained through experimental and theoretical approaches. This underscores the necessity of considering the specific characteristics of individual food products in understanding the phenomenon of biofilm formation. Understanding, the mechanism, the variations in bacterial surface characteristics, and the conditions that favor or disadvantage the formation of biofilms, may have important implications, in the development of preventive and curative strategies, to control of food‐borne infections.

Attenuation of biofilm formation of Listeria monocytogenes by bacteriocin producing Pediococcus pentosaceus DS1 on selected biotic and abiotic surfaces

The present study explores the antilisterial activities of potentially probiotic strain Pediococcus pentosaceus DS1 isolated from fermented bamboo shoot. 16s rRNA gene sequence analysis confirmed the identity of the strains. Class IIA bacteriocin pediocin expressing YGNGV motif was identified and its antilisterial activity was validated by well diffusion assay. When cocultured with P. pentosaceus DS1, in case of L. monocytogenes AMDK2, maximum biofilm exclusion (55.54 ± 3.44%) was observed which could be visually observed by Scanning electron micrographs. P. pentosaceus DS1 showed maximum adhesion inhibition to Caco-2 cell line in case of L. monocytogenes AMDK2 (91.8%) whereas maximum decrease in invasion was observed in case of L. monocytogenes MTCC 839 (52.9%). Coculture in UHT milk also significantly decreased microbial growth parameters of Listeria suggesting the efficiency of P. pentosaceus DS1 in the removal of Listeria contamination from food.

Destabilization of ultra-instantaneous ultra-high-temperature sterilized milk stored at different temperatures

Ultra-instantaneous UHT (UI-UHT, > 155°C, < 0.1 s) treated milk exhibits higher retention of active protein than regular UHT milk. However, UI-UHT products demonstrate increased susceptibility to destabilization during storage. This study aimed at monitoring the destabilizing process of UI-UHT milk across different storage temperatures and uncovering its potential mechanisms. Compared with regular UHT treatment, ultra-instantaneous treatment markedly accelerated the milk's destabilization process. Aged gel formation occurred after 45 d of storage at 25°C, while creaming and sedimentation were observed after 15 d at 37°C. To elucidate the instability mechanism, measurements of plasmin activity, protein hydrolysis levels, and proteomics of the aged gel were conducted. In UI-UHT milk, plasmin activity, and protein hydrolysis levels significantly increased during storage. Excessive protein hydrolysis at 37°C resulted in sedimentation, while moderate hydrolysis and an increase in protein particle size at 25°C resulted in aged gel formation. Proteomics analysis results indicated that the aged gel from UI-UHT milk contained intact caseins, major whey proteins, and their derived peptides. Furthermore, specific whey proteins including albumin, lactotransferrin, enterotoxin-binding glycoprotein PP20K, and MFGM proteins were identified in the gel. Additionally, MFGM proteins in UI-UHT milk experienced considerable hydrolysis during storage, contributing to fat instability. This study lays a theoretical foundation for optimizing UI-UHT milk storage conditions to enhance the quality of liquid milk products.

The impact of different temperatures on spore count and browning of UHT milk over a long storage period

One of the major microbiological challenges for the ultra-high temperature (UHT) milk industry is the presence of sporeformers in milk, which can survive pasteurization and the UHT process. The present study has aimed to investigate the spore populations in UHT milk stored at 20 °C and 35 °C for 14 months. Samples of bulk tank milk, pasteurized milk, and UHT milk were randomly assigned to different temperatures and storage times and were analyzed to study the mesophilic populations of aerobic spores and any color changes. The results showed that aerobic sporeformers can survive the UHT process. Bacillus licheniformis group and Bacillus pumilus group were the most isolated aerobic sporeformers. The b∗ value increased significantly in samples stored at 35 °C. These data provide information on the aerobic spore populations of bulk tank milk and UHT milk produced in different seasons and stored at different temperatures for 14 months.

Detection of Adulterant Residues in UHT Milk Products using ATR-FTIR Spectroscopy

Milk adulteration has been done to gain economic benefit by which the quality of milk is purposely compromised either by combining or substituting important ingredients of milk with adulterants. The objectives of this study were to determine the milk quality parameters of adulterated and unadulterated milk products through quality analysis and to investigate the ATR-FTIR spectroscopy technique in detection of adulterant residues in milk products. UHT milk samples were used with three common adulterants (melamine, formalin, anionic detergent) in five different concentrations (0.2%, 0.8%, 1.2%, 1.5% and 2.0%). Quality analysis was done on unadulterated milk and adulterated milk samples. Results showed that there were no significant differences between them. Thus, ATR-FTIR spectroscopic analysis was used for the qualification of adulterants in the wavenumber range of 4000 to 500. These results show the potential of FTIR spectroscopy as a rapid and sensitive technique for the detection of adulterant residues in UHT milk products.

Impact of hydrolysis method and lactase preparation on proteolysis and glycation in long-term stored lactose-hydrolysed UHT milk

Shorter shelf-life of lactose-hydrolysed UHT milk (LHM) can be linked to lactase purity both due to unwanted proteolytic activity and Maillard reactions. To study this, pre- and post-hydrolysed milk was added one of three types of lactase enzymes of increasing purity (A, B and C), and stored for up to 1 year at 25 or 35 °C. Proteolytic side-activities in post-hydrolysed milk depended on lactase purity, with lactase A causing the highest proteolysis. The lowest proteolytic activity occurred with lactase C in both pre- and post-hydrolysed milk. LHM with lactase A exhibited proteolysis in C-terminal regions of major milk proteins, particularly in post-hydrolysed milk, which also showed endopeptidase activity. Glycation initiated by UHT treatment was higher in pre-hydrolysed milk than in post-hydrolysed milk, but subsequent storage led to similar glycation levels in both. Therefore, using post-hydrolysis with highly purified lactases, proteolytic side-activities can be lowered and glycation can be delayed.

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

BackgroundHeat 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. ObjectivesTo 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. MethodsIn 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. ResultsAlthough 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. ConclusionsContrary 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 Registrywww.anzctr.org.au (ACTRN12620000172909).