Analysis of milk adulteration by means of a potentiometric electronic tongue.

The adulteration of milk presents significant challenges in the food industry, promoting the need for efficient detection methods. This study introduces a potentiometric electronic tongue for rapid and accurate milk adulteration detection. Utilizing polymeric membranes integrated with various additives, the electronic tongue distinguishes between different milk types and detects common adulterants. Experimental results demonstrate its effectiveness in discriminating raw, pasteurized, and medicated cow milk, as well as goat milk. Moreover, it successfully identifies adulterants like water and bovine milk in goat milk samples. Chemometric analyses, including Principal Component Analysis and Partial Least Squares regression, correlate sensor responses with traditional milk parameters such as fat, protein, and lactose content with up to a 0.97 R2 on the validation step. Strong correlations validate the electronic tongue's potential for rapid milk quality assessment. This innovative approach offers a cost-effective, reliable solution for detecting milk adulteration in contrast with current techniques that require numerous, time consuming experiments.

Goat milk as the connecting link in dengue and brucella coinfection.

In areas with widespread prevalence of myth of goat milk as a platelet booster, the goat milk can be connecting link in dengue and brucella coinfection.

Investigating the impact of milk protein, inulin, and honey on quality attributes of goat milk yoghurt.

In this present study, a three-factor Box-Behnken, response surface methodology (RSM) design was employed to optimize the skimmed milk powder (SMP)/whey protein concentrate (WPC) ratio (0.25-0.75%w/v) as a source of milk protein, inulin (1-2%w/v), and honey (4-6%w/v) for production of high-quality goat milk yoghurt (GMY). The resulting ANOVA and response surface equations revealed the significant effect (p < 0.05) of these variables on the various attributes such as total solid (%), pH, titratable acidity [(LA) % by weight], syneresis (%), DPPH (% inhibition), viscosity (m.Pa⋅s), whiteness index (WI), and overall acceptability (OA). The coefficient of determination (R2) for all response variables ranged from 0.88 to 0.99. Lack-of-fit tests resulted in non-significant F-values. The optimal conditions were determined as SMP/WPC at 0.36%w/v, inulin at 1.00%w/v, and honey at 6.00%w/v. The optimum values for total solid, pH, titratable acidity, syneresis, DPPH, viscosity, WI, and OA were 22.03, 4.46, 0.77, 6.34, 25.20, 182.30, 76.29 and 8.37, respectively with desirability value of 0.95.

Comparative analysis of the fatty acid profiles in goat milk during different lactation periods and their interactions with volatile compounds and metabolites.

This study aimed to compare the composition of fatty acids in goat milk during lactation with human milk, as well as analyze the differences in their interaction with odor and metabolites. Polyunsaturated fatty acids content was higher in human milk, while odd-chain, branched-chain, and monounsaturated fatty acids content were higher in goat milk with a decreasing trend during lactation. PUFAs in human milk undergo auto-oxidation to produce aldehydes (hexanal), giving it a mild aroma. Butyric acid in goat colostrum mediates the synthesis and auto-oxidation of PUFA, while taurine mediated the hydrolysis of amino acids. They produce a furanone compound (2(5H)-furanone) with a buttery flavor. The presence of butyric acid in goat transitional milk had an impact on flavor and metabolites. The medium chain fatty acid composition of the goat mature milk was affected by nucleic acid compounds, which then oxidized to produce methyl ketone (2-nonanone), giving it an unpleasant flavor.

Revealing the mechanism of flavor improvement of fermented goat milk based on lipid changes.

The mechanism of goat milk (GM) flavor improvement based on lipid changes requires understanding. According to sensory evaluation results, the texture, taste, appearance, aroma, and overall acceptability score of Guishan fermented goat milk (GMF) were higher than those of GM. In total, 779 lipid molecules and 121 volatile compounds were formed from the metabolite-lipid level in the GM and GMF, as determined through lipidomics and gas chromatography-mass spectrometry. The key volatile flavor compounds in the GMF were (E,E)-2,4-decadienal, ethyl acetate, acetoin, 2,3-pentanedione, acetic acid, and 2,3-butanedione. Of them, 60 lipids significantly contributed to the flavor profiles of the GMF, based on the correlation analysis. The triacylglycerides (TAGs) 12:0_14:0_16:0 and 13:0_13:0_18:2 contributed to aroma retention, while TAG and phosphatidylethanolamine were identified as key substrates for flavor compound formation during fermentation. Lipids associated with glycerophospholipid and linoleic acid metabolism pathways significantly affected volatile compound formation in the GMF. This study provides an in-depth understanding of the lipids and flavors of the GMF, and this information will be useful for the development of specific GMF products.

Differences in proteomic profiles and immunomodulatory activity of goat and cow milk fat globule membrane.

This study aimed to clarify the composition and bioactivity differences between goat and cow milk fat globule membrane (MFGM) protein by proteomic, and the immunomodulatory activity of MFGM proteins was further evaluated by using mouse splenic lymphocytes in vitro. A total of 257 MFGM proteins showed significant differences between goat and cow milk. The upregulated and unique MFGM proteins in goat milk were significantly enriched in the positive regulation of immune response, negative regulation of Interleukin-5 (IL-5) secretion, and involved in nucleotide-binding oligomerization domain (NOD)-like receptor signaling. The contents of IL-2 and Interferon-γ in the supernatant of spleen lymphocytes treated with goat MFGM proteins were much higher than those of IL-4 and IL-5, suggesting a Th1-skewed immune response. These results revealed that goat MFGM proteins could possess better immunomodulatory effects as compared to cow milk. Our findings may provide new insights to elucidate the physiological functions and nutritional of goat milk.

Machine learning methods for unveiling the potential of antioxidant short peptides in goat milk-derived proteins during in vitro gastrointestinal digestion.

Milk serves as an important dietary source of bioactive peptides, offering notable benefits to individuals. Among the antioxidant short peptides (di- and tripeptides) generated from gastrointestinal digestion are characterized by enhanced bioavailability and bioaccessibility, while assessing them individually presents a labor-intensive and expensive challenge. Based on 4 distinct types of amino acid descriptors (physicochemical, 3D structural, quantum, and topological attributes) and genetic algorithms for feature selection, 1 and 4 machine learning predicted models separately for di- and tripeptides with ABTS radical scavenging capacity exhibited excellent fitting and prediction ability with random forest regression as machine learning algorithm. Intriguingly, the electronic properties of N-terminal amino acid were considered as only factor affecting the antioxidant capacity of dipeptides containing both tyrosine and tryptophan. Four peptides from the potential di- and tripeptides exhibited highly predicted values by the constructed predicted models. Subsequently, a total of 45 dipeptides and 52 tripeptides were screened by a customized workflow in goat milk during in vitro simulated digestion. In addition to 5 known antioxidant dipeptides, 9 peptides were quantified during digestion, falling within the range of 0.04 to 1.78 mg L-1. Particularly noteworthy was the promising in vivo functionality of antioxidant dipeptides with N-terminal tyrosine, supported by in silico assays. Overall, this investigation explored crucial molecular properties influencing antioxidant short peptides and high-throughput screening potential peptides with antioxidant activity from goat milk aided by machine learning, thereby facilitating the identification of novel bioactive peptides from milk-derived proteins and paving the way for understanding their metabolites during digestion.

Comparison of the Lipid Composition of Milk Fat Globules in Goat (Capra hircus) Milk during Different Lactations and Human Milk.

Goat milk is considered the optimal substitute for human milk and is characterized by variations in the lipid composition of its fat globules across lactation phases. Therefore, the objective of this study was to thoroughly analyze the differences between goat milk during different lactations and human milk, aiming to offer scientific guidance for the production of functional dairy products. Compared with transitional and mature milk, the findings indicated that the total membrane protein content in goat colostrum exhibited greater similarity to that found in human milk. Additionally, goat milk exhibited higher milk fat globule size, as well as a higher total lipid and protein content than human milk. A total of 1461 lipid molecules across 61 subclasses were identified in goat milk and human milk. The contents of glycerides and glycerophospholipids were higher in goat colostrum, whereas sphingolipids and fatty acids were more abundant in human milk. Meanwhile, the compositions of lipid subclasses were inconsistent. There were 584 differentially expressed lipids identified between human and goat milk, including 47 subclasses that were primarily involved in the metabolism of glycerophospholipids, sphingolipids, and triglycerides. In summary, for both the membrane protein and the lipid composition, there were differences between the milk of different goat lactations and human milk.

A Dual and Rapid RPA-CRISPR/Cas12a Method for Simultaneous Detection of Cattle and Soybean-Derived Adulteration in Goat Milk Powder.

The adulteration of goat milk powder occurs frequently; cattle-derived and soybean-derived ingredients are common adulterants in goat milk powder. However, simultaneously and rapidly detecting cattle-derived and soybean-derived components is still a challenge. An efficient, high-throughput screening method for adulteration detection is needed. In this study, a rapid method was developed to detect the adulteration of common cattle-derived and soybean-derived components simultaneously in goat milk powder by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). A dual DNA extraction method was employed. Primers and crRNA for dual detection were designed and screened, and a series of condition optimizations were carried out in this experiment. The optimized assay rapidly detected cattle-derived and soybean-derived components in 40 min. The detection limits of both cattle-derived and soybean-derived components were 1% (w/w) for the mixed adulteration models. The established method was applied to a blind survey of 55 commercially available goat milk powder products. The results revealed that 36.36% of the samples contained cattle-derived or soybean-derived ingredients, which revealed the noticeable adulteration situation in the goat milk powder market. This study realized a fast flow of dual extraction, dual amplification, and dual detection of cattle-derived and soybean-derived components in goat milk powder for the first time. The method developed can be used for high-throughput and high-efficiency on-site primary screening of goat milk powder adulterants, and provides a technical reference for combating food adulteration.

Integrated analysis of genomics and transcriptomics revealed the genetic basis for goaty flavor formation in goat milk.

Goat milk exhibits a robust and distinctive "goaty" flavor. However, the underlying genetic basis of goaty flavor remains elusive and requires further elucidation at the genomic level. Through comparative genomics analysis, we identified divergent signatures of certain proteins in goat, sheep, and cow. MMUT has undergone a goat-specific mutation in the B12 binding domain. We observed the goat FASN exhibits nonsynonymous mutations in the acyltransferase domain. Structural variations in these key proteins may enhance the capacity for synthesizing goaty flavor compounds in goat. Integrated omics analysis revealed the catabolism of branched-chain amino acids contributed to the goat milk flavor. Furthermore, we uncovered a regulatory mechanism in which the transcription factor ZNF281 suppresses the expression of the ECHDC1 gene may play a pivotal role in the accumulation of flavor substances in goat milk. These findings provide insights into the genetic basis underlying the formation of goaty flavor in goat milk. STATEMENT OF SIGNIFICANCE: Branched-chain fatty acids (BCFAs) play a crucial role in generating the distinctive "goaty" flavor of goat milk. Whether there is an underlying genetic basis associated with goaty flavor is unknown. To begin deciphering mechanisms of goat milk flavor development, we collected transcriptomic data from mammary tissue of goat, sheep, cow, and buffalo at peak lactation for cross-species transcriptome analysis and downloaded nine publicly available genomes for comparative genomic analysis. Our data indicate that the catabolic pathway of branched-chain amino acids (BCAAs) is under positive selection in the goat genome, and most genes involved in this pathway exhibit significantly higher expression levels in goat mammary tissue compared to other species, which contributes to the development of flavor in goat milk. Furthermore, we have elucidated the regulatory mechanism by which the transcription factor ZNF281 suppresses ECHDC1 gene expression, thereby exerting an important influence on the accumulation of flavor compounds in goat milk. These findings provide insights into the genetic mechanisms underlying flavor formation in goat milk and suggest further research to manipulate the flavor of animal products.

Lactiplantibacillusplantarum JS19-adjunctly fermented goat milk alleviates D-galactose-induced aging by modulating oxidative stress and intestinal microbiota in mice.

Oxidative stress is a crucial factor in the age-related decline in physiological, genomic, metabolic, and immunological functions. We screened Lactiplantibacillus plantarum JS19 (L. plantarum JS19), which has been shown to possess therapeutic properties in mice with ulcerative colitis. In this study, L. plantarum JS19-adjunctly fermented goat milk (LAF) was employed to alleviate D-galactose-induced aging and regulate intestinal flora in an aging mouse model. The oral administration of LAF effectively improved the health of spleen and kidney in mice, while mitigating the hepatocyte and oxidative damage induced by D-galactose. Additionally, LAF alleviated D-galactose-induced dysbiosis of the intestinal flora by reducing the abundance of harmful bacteria Desulfovibrio and Helicobacter, while greatly promoting the growth of beneficial Rikenellaceae_RC9_gut_group and Eubacterium. Biomarker 5-hydroxyindole-3-acetic acid was found to be positively linked with those harmful bacteria, while bio-active metabolites were strongly correlated with the beneficial genus. These observations suggest that LAF possesses the capability to mitigate the effects of D-galactose-induced aging in a mouse model through the regulation of oxidative stress, the gut microbiota composition, and levels of fecal metabolites. Consequently, these findings shed light on the potential of LAF as a functional food with anti-aging properties.

Quality characteristics of goat milk powder produced by freeze drying followed by UV-C radiation sterilization.

Goat milk was directly freeze-dried into milk powder after freezing and then sterilized using UV-C radiation to produce low-dose, medium-dose and high-dose UV-C radiation sterilized freeze-dried goat milk powder (LGP, MGP and HGP). UV-C sterilization effectively reduced the total bacteria count and coliform bacteria in the goat milk powder while preserving the active proteins, and maintaining the color unchanged. Additionally, LGP, MGP, and HGP all exhibited a moisture content below 5 g/100 g and water activity below 0.5. Upon reconstitution, the milk powder formed uniform and stable emulsion. During accelerated storage tests, the increased Aw did not compromise the microbial quality of milk powder, and there were no significant changes in active proteins as confirmed via SDS-PAGE results. Furthermore, the color parameters (a*, b* and ΔE) showed a strong correlation with hydroxymethyl furfural levels.

Selection of starter lactic acid bacteria capable of forming biofilms on wooden vat prototypes for their future application in traditional Sicilian goat's milk cheese making.

In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.

Fortification of goat milk yogurts with date palm (Phoenix dactylifera L.) coproducts: Impact on their quality during cold storage.

The aim of this study was to investigate the impact of different concentrations (3% and 6%) of two ingredients (paste and flour) obtained from the valorization of date fruit coproducts on the nutritional (proximate composition and mineral profile), technological (coagulation curve, pH, acidity, sugar and organic acid content and syneresis), physicochemical (color, water activity and texture), microbiological and sensory properties of goat's yogurt during 21 days of refrigerated storage. Both ingredients enhanced the growth and stability of the yogurt starter culture, thereby improving the probiotic potential of date-added yogurts. Physicochemically, the addition of date flour (at both concentrations) induces stronger modifications (texture, color and syneresis) in yogurts than the date paste. During storage, date paste reduced the syneresis and hence maintained yogurts' physical quality. Consumers preferred the yogurts with date paste (3% and 6%) rather than with date flour, because its addition led to a more brownish color and granular texture.

Comprehensive analysis of differences in the physiochemical composition of foremilk and hindmilk of grazing indigenous and crossbred dairy goats; implications for growth, milk production and industry.

This study was conducted on 90 grazing Hair, Alpine × Hair F1 (AHF1), and Saanen × Hair F1 (SHF1) crossbred goats in three farms located around the Taurus Mountains in Konya, Türkiye. The study investigated variation in milk production, physico-chemical traits of milk fractions (foremilk, hindmilk and total milk), and growth traits. Genotype, parity, offspring sex, birth type, and flock factors significantly influenced milk production and quality traits of milk fractions (P < 0.05 to P < 0.01). Does with male/single offspring produced less milk, but with higher nutrient density (P < 0.05). Hindmilk was 272%, 31% and 61% richer in fat, total solids and energy content than foremilk, respectively (P < 0.001). However, the protein, lactose and solids-non-fat content of hindmilk was on average 7% lower than that of foremilk (P < 0.001). Physico-chemical quality traits of foremilk, hindmilk and total milk had a strong negative correlation with daily milk yield (P < 0.05 to P < 0.001). Live weight and average daily gains (ADG) of kids were influenced by maternal parity, flock, offspring sex and birth type (P < 0.05). The overall Kleiber ratios (KR) from birth to 2 months, birth to 3 months, birth to 6 months and 3 to 6 months of age were 21.0 ± 0.22, 17.1 ± 0.11, 10.5 ± 0.06 and 8.5 ± 0.21 g/kg of metabolic weight, respectively. It was concluded that these findings are critical for milk sampling protocols, offspring growth strategies, product development and precision livestock management.

Impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response post weaning time.

Milk is a whitish liquid that is secreted from mammary glands; and considered as the primary source of nutrition for newborns since they are not able to digest solid food. However, it contains primary nutrients, as well as growth and immune factors. Early weaning is a critical issue that face women and their babies in developing countries. To avoid infant malnutrition, they tend to use other milk types instead of baby formula. Therefore, the present study aimed to evaluate the impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response in male and female Sprague Dawley rats post weaning time. The amino acids, fatty acids, minerals and vitamins in the tested milk types were evaluated. Animals were divided into 5 groups (control, cow, buffalo, goat and camel milk administrated groups) (10 rats/group); each animal was administrated by 3.4 ml/day. Rats were administered with milk for 6 weeks; at the end of the 5th week, five animals of each group were isolated and the remaining five animals were immunized with sheep red blood cells (SRBCs) and kept for another week to mount immune response. The effect of different milk types on rats' immune response towards SRBCs was evaluated through pro-inflammatory cytokines, antioxidants, ESR and CRP measurement; together, with the histopathological examination of spleen samples and hemagglutination assay. Camel milk consumption reduced oxidative stress and inflammation in spleen that resulted from SRBCs immunization; in addition to, B cell stimulation that was apparent from the high level of anti-SRBCs antibodies. Camel milk is recommended for newborn consumption, due to its high-water content, unsaturated fatty acids, and vitamin C, as well as low lactose and fat content.

Linking variation in the casein fraction and salt composition to casein micelle size in milk of Dutch dairy goats.

The casein (CN) composition, salt composition and micelle size varies largely between milk samples of individual animals. In goats, the link between those casein characteristics are unknown and could provide useful insights into goat casein micelle structure. In this study, the casein- and salt composition of 42 individual Dutch goats from 17 farms was studied and linked to casein micelle size. Micelle size, proportions of individual caseins, and protein content were associated with each other. Milk with smaller casein micelles was higher in protein content, salt content, and proportion of αs1-CN, but lower in αs2-CN and ß-CN. The higher salt content in milk with small casein micelles was mainly attributed to a higher protein content, but changes in casein composition might additionally contribute to differences in mineralization. The non-sedimentable casein content in goat milk correlated with non-sedimentable fractions of ß-CN and κ-CN and was independent of micelle size. Between large and small casein micelles, goat casein micelles showed more differences in casein and salt composition than bovine micelles, indicating differences in internal structure. Nevertheless, the casein mineralization in goat milk was similar to casein mineralization in bovine milk, indicating that mineralization of casein micelles follows a general principle. These results can help to better understand how composition and micelle structure in goat milk are related to each other, which may be useful to improve processing and product properties of goat milk in the future.

Dynamic In Vitro Gastric Digestion Behaviour of Commercial Infant Formulae Made with Cow, Goat and Sheep Milk.

There are a wide range of commercial infant formulae available on the market. These are made using milk from different species, such as goat, sheep, and cow. The different protein compositions of these milks and the process used during infant-formulae manufacture, such as heat treatment, may impact the digestion of nutrients. This study compared the effect of protein composition and heat treatment on the in vitro gastric digestion behaviour of commercial infant formulae made with cow, goat, and sheep milk using a dynamic infant human gastric simulator (IHGS). During the simulated dynamic gastric digestion, the goat milk infant formula (GIF) showed earlier signs of aggregate formation compared to cow milk infant formula (CIF) and sheep milk infant formula (SIF). In addition, the microstructures of GIF chyme showed fragmented and porous structures. On the contrary, CIF formed dense protein networks that trapped oil droplets, whereas SIF exhibited a microstructure of smooth oil droplets surrounded by fewer protein networks. The different aggregation behaviours and aggregate structures of the three infant-formulae chyme were related to their different protein compositions, especially the different casein compositions. Furthermore, the open fragile structure of GIF aggregates provided easier access to pepsin, allowing it to hydrolyse protein. The results from the present study provided some information to assist in understanding the coagulation and digestion behaviours of commercial infant formulae made from different species of milk.

Dynamic Gastrointestinal Digestion of Bovine, Caprine and Ovine Milk Reconstituted from Commercial Whole Milk Powders.

The global dairy market has been increasingly diversified with more dairy product offerings of milk products from different animal species. Meanwhile, milk powders remain the main exported dairy product format due to their ease of transportation. In this work, we studied the structural changes, protein hydrolysis and nutrient delivery during dynamic gastric digestion and small intestinal digestion of cow, goat and sheep milk reconstituted from commercial whole milk powders. The results show that the reconstituted milks digest similarly to processed fresh milk. The digestion behaviors of the three reconstituted ruminant milks are broadly similar (gastric coagulation, kinetics of gastric emptying of protein and fat and the high digestibility in the small intestine) with some differences, which are likely contributed by the processing history of the milk powders. The delivery of individual amino acids to the small intestine differed between the early and late stages of gastric digestion, which were primarily affected by the abundance of amino acids in caseins and whey proteins but also by the difference between milk types associated with their gastric coagulation behaviors. This work showed that powdered milk is similar to fresh processed milk in digestion behavior, and the inherent differences between ruminant milks can be modified by processing treatments.

Plasma Sphingomyelins and Carnitine Esters of Infants Consuming Whole Goat or Cow Milk-Based Infant Formulas or Human Milk.

BACKGROUND: Infant formulas are typically manufactured using skimmed milk, whey proteins, and vegetable oils, which excludes milk fat globule membranes (MFGM). MFGM contains polar lipids, including sphingomyelin (SM). OBJECTIVE: The objective of this study was comparison of infant plasma SM and acylcarnitine species between infants who are breastfed or receiving infant formulas with different fat sources. METHODS: In this explorative study, we focused on SM and acylcarnitine species concentrations measured in plasma samples from the TIGGA study (ACTRN12608000047392), where infants were randomly assigned to receive either a cow milk-based infant formula (CIF) with vegetable oils only or a goat milk-based infant formula (GIF) with a goat milk fat (including MFGM) and vegetable oil mixture to the age ≥4 mo. Breastfed infants were followed as a reference group. Using tandem mass spectrometry, SM species in the study formulas and SM and acylcarnitine species in plasma samples collected at the age of 4 mo were analyzed. RESULTS: Total SM concentrations (∼42 µmol/L) and patterns of SM species were similar in both formulas. The total plasma SM concentrations were not different between the formula groups but were 15 % (CIF) and 21% (GIF) lower in the formula groups than in the breastfed group. Between the formula groups, differences in SM species were statistically significant but small. Total carnitine and major (acyl) carnitine species were not different between the groups. CONCLUSIONS: The higher total SM concentration in breastfed than in formula-fed infants might be related to a higher SM content in human milk, differences in cholesterol metabolism, dietary fatty acid intake, or other factors not yet identified. SM and acylcarnitine species composition in plasma is not closely related to the formula fatty acid composition. This trial was registered at Australian New Zealand Clinical Trials Registry as ACTRN12608000047392.