An international quality assurance scheme for the quantitation of daidzein and genistein in food,urine and plasma

This quality assurance (proficiency testing) scheme was commissioned to enable the Food Standards Agency (FSA) to determine the quality of analytical results submitted by researchers measuring the concentrations of phytoestrogens in foods and biological fluids in FSA-funded research projects, and also, to demonstrate that FSA-funded laboratories are producing consistent and precise results. Non-FSA-funded laboratories from around the world were also invited to join in the scheme to increase the number participants. A secondary objective was to highlight the most successful methodologies used to analyse phytoestrogens.     

Postharvest dehydration of wine white grapes to increase genistein, daidzein and the main carotenoids

Wine white grape bunches of the Grechetto variety were dehydrated at 10, 20 and 30°C, RH 45% and forced air ventilation of 1.5m/s. Chemical and metabolic changes due to the effect of dehydration were studied at various stages of weight loss: 10%, 20%, 30% and 40%. Berry colour at 10 and 20°C tended to become greener with dehydration but at 30°C, at the final sampling, the colour darkened. Acidity decreased in all samples, while sugars increased. Total phenol content increased at 10°C until 30% weight loss was reached and then declined, while at 20 and 30°C the concentration decreased immediately. The contents of lutein and β-carotene (respectively 68 and 58mg/kg d.w.), representing the 80% of total carotenoids, did not change significantly until the 30% of weight loss, when at 30°C the value increased above all for lutein while at 10 and 20°C, the contents decreased significantly. Daidzein, at 10°C, rose significantly from about 150μg/kg d.w. to 1434μg/kg d.w. at 20% weight loss and then declined; at the same weight loss percentage, the genistein concentration began to increase. At 20°C both isoflavones rose until the end of the experiment, reaching values similar to the sample at 10°C. A temperature of 30°C was deleterious to grape isoflavones. A discussion on the changes in isoflavones related to temperature and time is reported.     

A survey of pork quality in relation to pre-slaughter conditions, slaughterhouse facilities, and quality assurance

A survey at five pigs' slaughterhouses was performed to investigate the effect of a quality assurance system, pre-slaughter conditions and slaughterhouse facilities on pork quality. Totally, 2246 pigs were included over four transports per slaughterhouse, i.e. two transports were produced according to a quality assurance system and the other two were conventional pigs. Meat quality was measured on 446 pigs. The pH in the longissimus dorsi muscle and the electrical conductivity in the semimembranosus muscle were measured 30min post-mortem. Twenty-four hours later pH and electrical conductivity in both the longissimus dorsi and the semimembranosus muscle were measured. Pigs managed according to a chain quality protocol showed an overall higher potential for improved meat quality. Differences in meat quality between the different slaughterhouses were also found, however they were dependent on muscle type and time of measuring. Influencing factors on pork quality seemed to be stocking density during transport, the handling during offloading the pigs from the truck, stocking density, and air temperature during lairage.     

婴儿配方粉中溶菌酶活力的测定及其校正系数的研究

在婴儿配方粉中添加溶菌酶已成为高档婴儿粉的一种趋势，因此奶粉中溶菌酶活力成为判定其质量的重要指标。根据经典的分光光度法探索出测定奶粉中溶菌活力的方法，并以纯酶液人对照初步得出校正系数r=1.2；本试验测得市售婴儿粉中每克粉中溶菌酶活力为25960u。     

乳铁蛋白的功能特性及其在婴儿配方奶粉中的应用

介绍了母乳中乳铁蛋白的功能特性，及其应用在婴儿配方奶粉中对非母乳喂养婴儿营养的重要性，国外有关乳铁蛋白在婴幼儿配方奶粉中的应用情况。探讨了乳铁蛋白在婴儿配方奶粉中应用的研究过程，包括有关配方设计依据、使用的原料、生产工艺流程、检验方法、检验结果等。     

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths. The organism is ranked by the International Commission for Microbiological Specifications for Foods (ICMSF) as a ‘Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration’. Present study aimed to isolate Cronobacter spp. from PIF and clinical samples, such as blood, stool and CSF collected from 93 neonates and child patients, age ranged from 0 to 24 months. We did not detect Cronobacter spp. in any of these samples. Later 32 PIF samples collected from retail markets in Bangladesh were tested for the presence of Cronobacter spp. Of these only one was found to be contaminated with Cronobacter sp. This is the first case of Cronobacter contaminated PIF found in Bangladesh to be reported. The organism was successfully identified based on its typical culture characteristics, producing blue-green colonies on chromogenic DFI agar and also by a standardized conventional PCR assay targeting the alpha glucosidase and 16 S rRNA gene sequence of Cronobacter sp. The 16 S rRNA gene was partially sequenced to provide for the phylogenetic analysis of this isolate (DA01) and found to cluster with some other Cronobacter isolates in the phylogram.     

Microwave-assisted extraction and determination of dicyandiamide residue in infant formula samples by liquid chromatography-tandem mass spectrometry

A simple, precise, accurate, and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of dicyandiamide residue in infant formula samples. Following microwave-assisted extraction with 5% formic acid and clean-up on a Sep-Pak AC-2 SPE cartridge, samples were separated on a ZIC-HILIC HPLC column (150 × 2.1 mm i.d., 5-µm film thickness; Merck KGaA, Darmstadt, Germany) with 20 mM ammonium acetate solution-acetonitrile as mobile phase at a flow rate of 0.25 mL/min. A linear calibration curve was obtained in the concentration range from 1.0 to 50 ng/mL. Infant formula samples were fortified with dicyandiamide at 3 levels, producing average recovery yields of 83.6 to 95.7%. The limits of detection and quantification of dicyandiamide were 3 and 10 μg/kg, respectively. Due to its simplicity and accuracy, the straightforward method is particularly suitable for routine dicyandiamide detection.     

Carrageenan analysis. Part 1: Characterisation of the carrageenan test material and stability in swine-adapted infant formula

A method was developed and validated in support of a 28-day feeding study of swine-adapted infant formula stabilised with carrageenan administered to neonatal piglets. Carrageenan concentrations in the test formulations were 0, 300, 1000 and 2250 mg kg^–1 formula. Extraction of carrageenan from swine-adapted infant formula was achieved by breaking carrageenan–protein cross-linkages using saturated sodium chloride, followed by separation of the non-gelling carrageenan fraction via centrifugation. The extraction of carrageenan from formula was successful with respect to consistent recovery of the non-gelling carrageenan fraction from both test and control formula samples. Molecular weight analysis (Mw) of the recovered carrageenan fractions from the test and control formula samples confirmed that the carrageenan used to manufacture the formula was not degraded during the infant formula production process and subsequent storage for 4 months covering the 28-day piglet dietary feeding study. Carrageenan has excellent stability in infant formulations.     

Determination of vanillin,ethyl vanillin,and coumarin in infant formula by liquid chromatography-quadrupole linear ion trap mass spectrometry

A simple, precise, accurate, and validated liquid chromatography-quadrupole linear ion trap mass spectrometry method was developed for the determination of vanillin, ethyl vanillin, and coumarin in infant formula samples. Following ultrasonic extraction with methanol/water (1:1, vol/vol), and clean-up on an HLB solid-phase extraction cartridge (Waters Corp., Milford, MA), samples were separated on a Waters XSelect HSS T3 column (150 × 2.1-mm i.d., 5-μm film thickness; Waters Corp.), with 0.1% formic acid solution-acetonitrile as mobile phase at a flow rate of 0.25 mL/min. Quanti?cation of the target was performed by the internal standard approach, using isotopically labeled compounds for each chemical group, to correct matrix effects. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring 2 multiple reaction monitoring transitions to ensure an accurate identi?cation of target compounds in the samples. Additional identi?cation and con?rmation of target compounds were performed using the enhanced product ion modus of the linear ion trap. The novel liquid chromatography-quadrupole linear ion trap mass spectrometry platform offers the best sensitivity and speci?city for characterization and quantitative determination of vanillin, ethyl vanillin, and coumarin in infant formula and fulfills the quality criteria for routine laboratory application.     

婴幼儿配方奶粉中添加的核苷酸钠盐与游离酸的理化性质研究

阐述了核苷酸对婴幼儿生长发育的作用,讨论了其重要的营养价值;列举了两种类型的核苷酸,即五钠型和两酸三钠型核苷酸。通过研究它们理化性质的差异,比较了它们作为婴幼儿配方乳粉添加剂的优劣性。结果表明,五钠型核苷酸和两酸三钠型核苷酸溶于水后pH值分别为7.1和4.7,则前者不会改变以婴幼儿奶粉的酸碱度。溶于水后,五钠型核苷酸的透光度明显优于两酸三钠型核苷酸,则前者不会改变婴幼儿奶粉的溶解性和感官性能。经65℃保温30 min处理后,前者可以保持很好的稳定性,很好的保持了它的功能性。以此得出结论,五钠型核苷酸是一种更理想的婴幼儿配方奶粉核苷酸添加剂。     

Selection of marker peptides from casein phosphopeptide and application for quantification in infant formula

ABSTRACT

Casein phosphopeptides (CPPs) have been used worldwide as a nutritional supplement. However, the peptide components have been unknown; as a consequence, few quantification methods of CPP in infant formula were reported. This study introduced a quantification method based on peptide marker and corresponding peptide selection strategy using a simplified model with four commercial types of CPP. The peptides from four commercial CPPs were first identified. Due to the great variety of CPPs, two marker selection strategies were adopted: on one hand, universal marker peptide VLPVPQK can be used for the quantification of all four commercial CPPs, if the CPP can be obtained as a standard. On the other hand, the specific marker peptide LYQEPVLGPV can be used for identification and quantification of commercial CPP type K content in infant formula with a fixed calculation factor. In the simplified model, the combination use of the two markers can meet most of the requirements of CPP analysis in infant formula. The method validation revealed that this was suitable for the routine analysis laboratories without proteomics backgrounds. This selection strategy was suggested for the large-scale marker peptide selection with all commercial CPPs, which can give a comprehensive solution of CPP quantification in infant formula.

Abbreviations: CPP: Casein phosphopeptides; LC: Liquid chromatography; TQMS: Triple quadrupole mass spectrometry; MRM: Multiple reaction monitoring; RSD: Relative standard deviation; L*: [¹³C₆, ¹⁵N]-leucine; SSSEE: Peptides sequence of serine-serine-serine-glutamic acid-glutamic acid     

Microbial community structure and distribution in the air of a powdered infant formula factory based on cultivation and high-throughput sequence methods

The air in a powdered infant formula (PIF) factory is a potential transfer medium for microorganisms. In this study, air samples from 6 main processing areas, almost covering the whole PIF processing line and 1 outdoor location, were collected from a PIF manufacturing plant during the winter and summer periods. A cultivation-based and an Illumina (San Diego, CA) high-throughput 16S rRNA sequencing method was used to investigate the community structures and distributions of bacteria in the air. High microbial diversity (25 genera, 56 species), with a dominant community including Staphylococcus, Bacillus, Acinetobacter, and Kocuria, was found by the cultivation-based method. Moreover, 104 genera were obtained from all samples by high-throughput sequencing methods. Lactococcus (32.3%), Bacillus (29.6%), and Staphylococcus (14.0%) were the preponderant genera. The indices from high-throughput sequencing results indicated that the bacterial community of the air samples was highly diverse. Significant differences in the diversity and distribution at 6 sampling locations were revealed using the 2 methods. In particular, the packaging process contained the highest proportion (39.4%) of isolated strains. The highest diversity in bacterial community structure was found in the outdoor location. More bacterial isolates and higher community diversity were observed in the summer samples compared with the winter samples. In addition, some pathogens, such as Acinetobacter baumannii, Bacillus cereus, and Staphylococcus cohnii, were mainly found in the large bag filling process, can filling, and packaging process areas. The present study provides greater insight into the microbial community and identifies potential sources of air contamination in PIF production environments and can serve as a guide to reduce the risk of microbial contamination in the production of PIF.     

Method development and validation for low-level propineb and propylenethiourea analysis in baby food,infant formula and related matrices using liquid chromatography-tandem mass spectrometry

ABSTRACT

Two simple, selective and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for determination of propineb and propylenethiourea (PTU) in infant formula, fruit-based and cereal-based baby food and raw materials used in production of infant formula, including carbohydrates, protein isolates, vegetable oils and emulsifiers. The sample preparation procedure for propineb analysis was based on streamlined derivatisation to form and stabilise the target analyte (propylenebisdithiocarbamate-dimethyl), followed by extraction using a modified QuEChERS procedure with a dispersive solid phase extraction (d-SPE). The PTU determination employed an aqueous extraction with optimised protein precipitation and single-step SPE clean-up. To achieve maximum sensitivity, electrospray ionisation and atmospheric-pressure chemical ionisation were employed for LC-MS/MS analysis of propineb and PTU, respectively. Validation of the developed methods was performed in accordance with Document SANTE/11813/2017. Mean recoveries were in the range of 86–120% for propineb and PTU, respectively, with interday and intraday repeatabilities below 13%. A limit of quantification (LOQ) of 0.003 mg kg^–1 was validated for most of the evaluated analyte/sample matrix combinations with the exception of PTU in soy protein isolate and soybean oil, for which an LOQ of 0.01 mg kg^–1 was obtained. This is the first report that provides validated methods for monitoring propineb and PTU in infant formula and baby foods at concentrations compliant with the maximum residue levels established in the EU legislation.     

Direct application of supercritical carbon dioxide for the reduction of Cronobacter spp. (Enterobacter sakazakii) in end products of dehydrated powdered infant formula

The objective of this study was to develop a viable new method for inactivation of Cronobacter spp. that could be applied directly to dehydrated powdered infant formula (PIF) using supercritical carbon dioxide (SC-CO₂). Samples inoculated with Cronobacter spp. were subjected to SC-CO₂ treatment under various conditions (temperature: 63, 68, and 73°C; pressure: 15, 20, and 25 MPa; time: 10, 20, and 30 min). The survival of Cronobacter spp. was assayed, as were any changes in the quality of the treated PIF. Inactivation of Cronobacter spp. by SC-CO₂ was enhanced as temperature and pressure conditions increased (>6.32 log₁₀ cfu/g). In a validation assay using low-level inoculation (3.21 log₁₀ cfu/g), treatment at 73°C and 15 MPa for 30 min, 20 MPa for 20 and 30 min, or 25 MPa for 20 and 30 min reduced Cronobacter spp. to undetectable levels, with no recovery of cell viability. There was no significant change in water activity, pH, and color of the treated PIF. Overall, the optimum conditions for elimination of Cronobacter spp. were determined to be 73°C and 20 MPa for 20 min. These parameters for effective SC-CO₂ treatment are feasibly applicable to end product of dehydrated PIF. The results of our study may contribute to the development of an efficient method for improving the microbiological safety of PIF.     

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18 mg/L) and infant formulas (∼9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.     

Comparison of methods for the microbiological identification and typing of Cronobacter species in infant formula

Cronobacter species represent an emerging opportunistic foodborne pathogen associated with meningitis and necrotizing enterocolitis in infants. Current evidence indicates that powdered infant formula (PIF) is the main source of Cronobacter contamination. A total of 75 strains of Cronobacter spp. from different geographic regions, as well as from PIF processing environments, were identified and typed with different methods, including biochemical profiling by the API 20E system (bioMérieux, Marcy l’Etoile, France), protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and genotypic profiling by ribotype. Analysis by MALDI-TOF MS and biochemical identification was more accurate compared with ribotype analysis. However, MALDI-TOF MS typing and ribotype analysis showed more discriminatory ability compared with biochemical phenotyping. In conclusion, MALDI-TOF MS is a rapid and reliable tool to identify Cronobacter spp. in PIF and has the potential to trace dissemination of Chronobacter along the production chain.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

Improvement of accuracy for the determination of vitamin A in infant formula by isotope dilution-liquid chromatography/tandem mass spectrometry

Analysis of vitamin A in food has been an intricate subject due to its labile properties. In this study, an isotope dilution-liquid chromatography/mass spectrometric (ID-LC/MS) method was established as a candidate reference method for the accurate determination of vitamin A in infant formula. We found that the oxidative degradation of vitamin A in sample preparation processes is a key fact that hampers reliability of measurement results. Therefore, sample preparation processes were carefully designed to minimise oxidative degradation of vitamin A by preventing sample from exposure to air. Similar cautions were applied for the preparation of calibration solutions. Both within-day repeatability and among-day reproducibility tests of the ID-LC/MS method showed less than 1.0% of relative standard deviation, and the measurement uncertainty by this method is estimated to be less than 1.0%. The newly designed method showed greatly improved performance compared to methods reported in other literatures.     

Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model’s applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.     

高效液相色谱-串联质谱法同时测定婴幼儿配方食品中香兰素、甲基香兰素和乙基香兰素

目的建立了高效液相色谱-串联质谱法同时测定婴幼儿食品中香兰素、甲基香兰素和乙基香兰素的方法。方法 样品经Tris溶液和乙腈振荡提取,离心后提取液用正己烷脱脂过滤后上机测定,外标法定量。结果 3种化合物在5.0~200μg/L范围内线性良好,在液态奶中测定低限均为25μg/kg,在奶粉中测定低限均为50μg/kg,方法加标收率在89.5%~115%之间,相对标准偏差(RSD)小于15.6%。结论 该方法简便、快速,准确,灵敏度及精密度高,适用于婴幼儿食品中香兰素、甲基香兰素和乙基香兰素的测定。