Powdered Infant Formula Research Articles

Profile Analysis of Iodine in Powdered Infant Formula and Infant Complementary Foods by Inductively Coupled Plasma Mass Spectrometry

Profile Analysis of Iodine in Powdered Infant Formula and Infant Complementary Foods by Inductively Coupled Plasma Mass Spectrometry

Toz bebek mamalarının mikrobiyolojik kalitelerinin ve mama hazırlama önerilerine uygunluğunun araştırılması

The present study aimed to investigate the microbiological quality of powdered infant formulas on sale in Turkey and to assess the compliance of the preparation instructions recommended by the formula companies with the guidelines of the World Health Organization (WHO). In the study, 36 powdered infant formulas belonging to 10 different companies were analysed regarding the presence of Cronobacter sakazakii, Bacillus cereus, Staphylococcus aureus, coliform and faecal coliform, mould-yeast, total mesophilic aerobic bacteria counts. C. sakazakii was detected in two of the examined formula samples. At various levels, B. cereus in nine, S. auerus in four, coliform bacteria in 10 and faecal coliform bacteria in eight of the formula samples were found and these identified values were discovered above the legal limits. When the compliance of the preparation instructions recommended by the formula companies with the WHO guidelines was evaluated, the instruction requiring the water temperature to be >70°C, which is critical in the control of C. sakazakii, was not included in the preparation instructions of any formula companies. Although the microbiological quality of the formulas was determined by the legal limits, it was observed that the targeted microbiological quality could not be reached in some of the examined formula samples and the instructions prepared by the companies may not be adequate for the food safety.

Visual detection of Cronobacter sakazakii on a microfluidic chip fabricated by a 3D molding method.

Cronobacter sakazakii (C. sakazakii) is a pathogenic bacterium associated with life-threatening neonatal infections that have been linked to contaminated powdered infant formula (PIF). Most C. sakazakii testing is still limited in microbiology laboratories due to the need for sophisticated equipment and professional technicians. Microfluidic chips combined with isothermal amplification analysis are shown to be one of the most attractive microbiological on-site detection platforms. In this study, PDMS microfluidic chips were fabricated by a simple 3D molding method and sealed with "PDMS glue". The chip consisted of an inlet, a microchannel, six reaction wells, and six vent holes. And based on the 16S rRNA and ITS genes of C. sakazakii, we have successfully proposed a multiplex competitive annealing mediated isothermal amplification (mCAMP) assay on the microfluidic chip for the visual detection of C. sakazakii in PIF samples. The primers were fixed in the reaction wells of the chip before detection, which can be preserved for 60 days at 4 °C. The results showed that the established mCAMP assay had high specificity, and the limit of detection was 2.2 × 103 CFU g-1. With enrichment culture, even if the initial inoculation level is 1 CFU g-1, the mCAMP assay can still detect the presence of C. sakazakii in spiked PIF samples. The test results are visible to the naked eye, which is suitable for rapid analysis in resource-limited settings.

An investigation into the preparation of powdered infant formula in households in Ireland

An investigation into the preparation of powdered infant formula in households in Ireland

Better Particularizing Solutions to Defuse the Infant Formula Powder Keg

Better Particularizing Solutions to Defuse the Infant Formula Powder Keg

Rapid and Visual Determination of Cronobacter sakazakii in Powdered Infant Formula Using Competitive Annealing Mediated Isothermal Amplification (CAMP)

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen that causes serious infections, particularly in infants. Neonatal infections with this pathogen have an 80% mortality rate. One mechanism that C. sakazakii infects infants is through contaminated powdered infant formula (PIF). Here, based on the 16S rRNA gene, competitive annealing-mediated isothermal amplification (CAMP) is reported for the determination of C. sakazakii to reduce the risk of infection. The established CAMP method was able to accurately identify all target bacterial strains and did not yield false positive results with non-target bacterial strains. Furthermore, accelerating primers were designed and included in the CAMP reaction to enhance the amplification efficiency. The sensitivity of the assay for C. sakazakii in spiked PIF was 1.9 × 103 CFU/g. The CAMP assay with enrichment accurately detected the presence of C. sakazakii in PIF even at an initial inoculation level of 1 CFU/g. Additionally, to make the procedure simpler, hydroxynaphthol blue was employed as a visual indicator. For positive results, the reaction solution turns blue instead of violet, while the negative samples remain violet, which is suitable for rapid analysis in resource-limited settings.

"Multidimensional correlation analysis of temperature and contact time on eradication of biofilms of Cronobacter sakazakii on abiotic surfaces by combination of hypochlorite and malic acid".

In the present study, malic acid in combination with sodium hypochlorite is evaluated for eradication of biofilms formed by Cronobacter sakazakii strains individually and in a cocktail on different abiotic surfaces. The biofilm formation by five strains of C. sakazakii and their cocktail culture on different substrates was studied in Tryptone Soy Broth (TSB) and reconstituted Powdered Infant Formula (PIF). Further, the effect of temperature (4, 27, 37 and 50°C) and contact time (10, 20, 30, 40, 50 and 60min) on antibiofilm potential of test solution (0.0625mol l-1 malic acid and 0.00004mol l-1 sodium hypochlorite) against biofilm formed by C. sakazakii cocktail culture was investigated on these surfaces. The effect was evaluated in terms of viable cell count and biofilm texture using scanning electron microscopy (SEM). Principal Component Analysis (PCA) revealed that the maximum biofilm reduction was observed for stainless steel at 4°C after 60min of contact whereas at 25, 37 and 50°C, maximum biofilm reduction was observed for polycarbonate. For glass and polyurethane, maximum log reductions were observed at 50°C. The SEM images revealed cell surface deformation and disruption in biofilms after treatment with the test solution. The antibiofilm potential was observed to be greatly affected by contact time and temperature. These results indicated that the combination of malic acid NaOCl can effectively kill and remove C. sakazakii biofilms from food contact surfaces and enteral feeding tubes.

Risk assessment of Cronobacter sakazakii in infant formula powder

Objective: To assess the risk of foodborne diseases caused by Cronobacter sakazakii in infant formula powder from retail to feeding and provide formulate suggestions for safe feeding of infants at home. Methods: This study used the special monitoring and risk monitoring data of infant formula powder in Heilongjiang Province and combined data at home and abroad. The @RISK software was used to evaluate the disease risk caused by Cronobacter sakazakii in the process of infant formula powder from retail to feeding. Results: According to the results of this quantitative risk assessment, the risk of foodborne diseases caused by Cronobacter sakazakii at the current consumption pattern in Heilongjiang Province was 5.158×10-5 persons/million (40.0 ℃ and 50.0 ℃), 1.072×10-7 persons/million (60.0 ℃), 5.544×10-14 persons/million (70.0 ℃). When the feeding time of infant formula powder was adjusted to 0-2 h and 2-3 h respectively, the above prediction results did not change. When it was adjusted to 3-4 h, the risk increased. If it was adjusted to 4-24 h, the number of Cronobacter sakazakii increased by 14-24 orders of magnitude at room temperature. If the initial pollution concentration (after flushing) was adjusted to 1 MPN/ml, the average disease risk per meal was 805.7 persons/million (40.0 ℃ and 50.0 ℃), 1.7 persons/million (60.0 ℃) and 9.1 × 10-7 persons/million (70.0 ℃). The results of sensitivity analysis showed that the water temperature (70.0 ℃), initial pollution concentration, room storage time and temperature were important factors of risk. Conclusion: Controlling the contamination level of Cronobacter sakazakii in infant formula powder, controlling the feeding time within 3 h, storing in refrigerator and mixing with water with temperature not lower than 70.0 ℃ are effective measures to prevent infants from eating infant formula powder infected by Cronobacter sakazakii.

Factors Influencing Biofilm Formation by Salmonella enterica sv. Typhimurium, E. cloacae, E. hormaechei, Pantoea spp., and Bacillus spp. Isolated from Human Milk Determined by PCA Analysis.

Bacteria enter milk during poor hygiene practices and can form a biofilm on surfaces that come into contact with human milk. The presence of a biofilm increases the risk of infections among newborns as bacteria protected by biofilm are resistant to washing and disinfection processes. The formation of the biofilm depends on the microbial species, environmental conditions, and the specific materials colonized. The aim of this study is to analyze the effects of factors such as temperature, incubation time, and initial cell concentration on biofilm formation by pathogenic bacteria isolated from human milk on model hydrophobic polystyrene surfaces. Model studies confirm that pathogenic bacteria appearing in human milk as a result of cross-contamination tend to form a biofilm. The majority of isolates formed biofilm at both 25 and 37 °C after 12 h at 1 × 103 CFU/mL inoculum count. Multivariate principal component analysis (PCA) showed that at lower temperatures, biofilm formation by bacterial isolates was the main determinant of biofilm formation, other factors were less important; however, at 37 °C, time was a factor in biofilm formation. The model research performed underlines the importance of maintaining the proper hygiene of rooms, surfaces, and devices for expressing, storing, and preparing mothers' milk and powdered infant formula (PIF) in facilities responsible for feeding newborns and premature babies.

Evaluation of pH and total soluble solid (ºbrix) on the cariogenic and erosive potential of milk powder infantile formula

The objective of this study is to analyze the influence of pH and the amount of Total Soluble Solids on the ºBrix scale on the cariogenic and erosive potential of powdered infant formula. This is a laboratory study with 5 brands of powdered infant formula for children aged 6 to 36 months. Brands with intact packaging, stored in cool places and within the expiry date for consumption were included. Obtaining the Brixº was performed by refractometer and pH with a pHmeter with a cycle of three repetitions. Of the five brands tested, the sugar levels on the ºBrix scale were between 10.67 and 12.0, with all maras having total carbohydrate values ​​between 9.0 and 14.0 g, which are considered healthy. Regarding the pH, four had a pH lower than 7, being considered acidic and all above the critical pH (5.5) for dissolution of dental structures. It was concluded that by measuring pH and sugars on the Brixº scale, the brands analyzed were within acceptable standards for maintaining oral health, making these two factors positive determinants so as not to influence the cariogenic and/or erosive potential of milk powder infant formula.

Spray-Dried Infant Formula Emulsion Stability as Affected by Pre-Heat Treatment and Total Solids Content of the Feed.

Pre-spray-drying processing may affect stability after reconstitution of emulsion-based powders, such as infant formulas. This study aimed to evaluate the effects of pasteurization temperature and total solids (TS) of the feed on the stability of the emulsions obtained from the reconstituted powders. Four infant formula powders (50%-75 °C, 50%-100 °C, 60%-75 °C, and 60%-100 °C) were produced at pilot scale, from emulsions with 50 or 60% TS pasteurized at 75 or 100 °C for 18 s. Both the emulsion feeds and the emulsions from the reconstituted powders (12.5% TS) were analyzed. The results showed that feeds with 60% TS were flocculated, as indicated by the large particle size and viscosity and the pseudoplastic behavior. Light microscopy revealed that, during spray drying, the flocs were disrupted in 60%-100 °C, while the 60%-75 °C emulsion remained flocculated, reducing its stability post-reconstitution. Although all four emulsions were mainly stabilized by caseins, the presence of β-lactoglobulin was also detected at the oil-water interface, in native state in the formulas preheated at 75 °C and aggregated in the formulas preheated at 100 °C. In conclusion, both the degree of whey protein denaturation (resulting from pasteurization) and the TS of the concentrates during infant formula production affected the emulsion stability of the reconstituted powders.

Cronobacter sakazakii Cue for the Attraction and Its Impact on the Immunity of Caenorhabditis elegans.

Cronobacter sakazakii, an opportunistic foodborne pathogen prevalently detected in contaminated powdered infant formula, is associated with different diseases, including meningitis. It can cross the blood-brain barrier and affects the CNS. The impact of C. sakazakii on host neuronal cells and behavior is largely unknown. Hence, detailed molecular data are required to understand its severity. Caenorhabditis elegans is a unique model for studying chemical communication, as it relies on chemosensation for searching nutritional supplements. Although, C. sakazakii is pathogenic to C. elegans, our analysis indicated that C. elegans was highly attracted toward C. sakazakii compared to its food source, E. coli OP50. To study the cue for the attraction, bioactive components (RNA/Protein/Lipopolysaccharides/Metabolites) of C. sakazakii were isolated and used for observing the chemotaxis behavior of C. elegans. The results signified that C. elegans was more attracted toward acid extracted metabolites than those of the other extraction methods. The combined action of acid extracted metabolites of C. sakazakii and a candidate pathogen drastically reduced the survival of C. elegans. In addition, qPCR analysis suggested that the exposure of isolated metabolites through acid extraction to C. elegans for 24 h modified the candidate immune regulatory genes involved in pathogen recognition and kinase activity such as clec-60, clec-87, lys-7, akt-2, pkc-1, and jnk-1.

Validation of the Assurance® GDS for Cronobacter Tq II in Infant Formulas, Infant Cereals, Ingredients, and Environmental Samples Collaborative Study: First Action 2021.08.

The Assurance® GDS for Cronobacter Tq II assay is a nucleic acid amplification system for the qualitative detection of Cronobacter. The method uses an upfront concentration of the target organism from the enrichment by immunomagnetic separation (IMS) using the PickPen®device. The Assurance GDS for Cronobacter Tq II method was evaluated for Official Methods of AnalysisSM certification. The matrix was compared to the ISO 22964:2017: Microbiology of the Food Chain-Horizontal Method for the Detection of Cronobacter spp. standard and using an alternative confirmation procedure. The alternative method was evaluated using 10 g test portions in an unpaired study design for powdered infant formula (milk based with iron and DHA) containing probiotics. Eleven technicians from eight laboratories located within the United States and Europe participated in the collaborative study. Statistical analysis was conducted according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval of 0.03 (-0.18, 0.15). The dLPOD results indicate equivalence between the candidate method and reference method for the matrix evaluated. The method also demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. There were no false negative results; the false positive rate was determined and produced a value of <2%. Based on the data generated, the method demonstrated Assurance GDS for Cronobacter Tq II assay produced acceptable interlaboratory reproducibility data and statistical analysis. The Assurance GDS for Cronobacter Tq II method is suitable for the rapid qualitative detection of Cronobacter in infant formulas, infant cereals, ingredients, and environmental samples.

Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis.

Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, blaACT-7, blaACT-14,qacJ, oqxAB,aac(6')-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics-together with resistance and virulence genes-pose a health risk for infants consuming these food products.

Perspective: Challenges with product testing in powdered infant formula.

Right now, many concerned parents are probably thinking, how could Cronobacter sakazakii get through the powdered infant formula (PIF) production system? Shouldn't a food safety monitoring system find such a serious problem? Aren't those products tested before arriving on shelves? And the very challenging answer is yes, these products were likely tested with a standardized sampling and testing method consistent with regulatory guidance. In fact, in an official statement, the company said all finished products are tested for Salmonella spp.

Enhancement of Thermal Inactivation of Cronobacter sakazakii in Apple Juice at 58°C by Inclusion of Butyl Para-Hydroxybenzoate and Malic Acid

After studies with powdered infant formula indicated that the enhancement of thermal inactivation of Cronobacter sakazakii by butyl para-hydroxybenzoate (BPB) was blocked by high protein concentrations, we hypothesized that BPB would retain its synergistic activity in foods with limited protein and lipid concentrations. This hypothesis was tested by examining the ability of BPB to enhance the thermal inactivation of C. sakazakii 607 at 58°C in commercial apple juice, including examining the effects of pH and possible synergistic effects with malic acid. Apple juice was adjusted to designated pH values of 3.2 to 9.0, supplemented with selected concentrations of BPB (≤125 ppm), inoculated with early-stationary-phase C. sakazakii 607, and thermally treated (58°C) for 15 min with a submerged coil apparatus. The same methods were used to study the enhancement of thermal inactivation by malic acid. Samples were plated on tryptic soy agar for recovery and enumeration. Survival curves were plotted, and D-values were calculated by linear regression and compared using the Tukey honestly significant difference test. BPB significantly enhanced thermal inactivation in a concentration dependent manner, with D-values of a few seconds at the original pH (3.8). The enhancement of thermal inactivation was pH dependent over the pH range of 3.4 to 9.0. Malic acid enhanced thermal inactivation; the pH was decreased from 3.8 to 3.2. These results support the hypothesis that BPB can enhance the thermal inactivation of C. sakazakii in low-protein and low-lipid foods.

Bacterial community analysis of infant foods obtained from Chinese markets by combining culture-dependent and high-throughput sequence methods

In this study, twenty-two baby foods including cereal-based products and powdered infant formula (PIF) obtained from local markets were comprehensively investigated for their bacterial contamination using culture-dependent and high-throughput sequence (HTS) methods. In addition, the genetic diversity and biofilm-forming capacity of the most abundant species were analyzed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Results showed that 170 mesophilic isolates collected from 22 samples were clustered into 15 genera and 41 species. Bacillus (77.65%) was the most prevalent genus, followed by Paenibacillus (7.06%), Alkalibacillus (3.53%), and Lysinibacillus (2.35%). Bacillus licheniformis (49.41%) proved to be the most dominant species in infant foods, and a high genetic diversity with six different RAPD profiles was observed. A total of 87.5% of B. licheniformis isolates were identified as strong biofilm formers, and heterogeneous biofilm-forming ability was observed among the isolates sharing the same RAPD pattern. HTS analysis revealed an 18-fold higher biodiversity at the genus level, and a significantly different bacterial community of infant foods was dominated by Lactococcus, Streptococcus, and Bifidobacterium. Foodborne pathogens including Bacillus cereus, and potentially pathogenic microorganisms such as Acinetobacter baumannii, were identified in infant foods by HTS. The current results could expand the crucial information about bacterial contamination of baby foods.

Comparison of Origanum Essential Oil Chemical Compounds and Their Antibacterial Activity against Cronobacter sakazakii.

Origanum vulgare L. (oregano) is an aromatic plant with wide applications in the food and pharmaceutical industries. Cronobacter sakazakii, which has a high detection rate in powdered infant formula, adversely impacts susceptible individuals. Oregano essential oil (OEO) is a natural antibacterial agent that can be used to fight bacterial contamination. Here, OEO chemical compounds from eight oregano varieties were analyzed by gas chromatography–mass spectrometry and their antibacterial properties were assessed. The eight OEOs were clustered into two groups and were more diverse in group 2 than in group 1. Six compounds, including p-cymene, 3-thujene, γ-terpinene, thymol, carvacrol, and caryophyllene, were shared by eight OEOs. Among the eight oregano varieties, OEOs from O. vulgare sc2 had the strongest antibacterial activity against C. sakazaki, with the inhibition zone of 18.22mm. OEOs from O. vulgare jx, O. ‘Nvying’, O. vulgare ‘Ehuang’, and O. vulgare ssp. virens were also potent. Moreover, the antibacterial activity of OEOs was positively correlated with the relative content of thymol. As the main OEO antibacterial compound, thymol affected the normal growth and metabolism of C. sakazakii cells by destroying the bacterial membrane and decreasing the intracellular ATP concentration. Thus, in light of the antibacterial activity detected in the OEOs from the eight oregano varieties, this study provides a theoretical foundation for oregano cultivar management and development.

Emerging of Multidrug-Resistant Cronobacter sakazakii Isolated from Infant Supplementary Food in China.

ABSTRACTCronobacter is a foodborne pathogen associated with severe infections in restricted populations and particularly with high mortality in neonates and infants. The prevalence and antimicrobial resistance (AMR) phenotype of Cronobacter cultured from powdered infant formula and supplementary food were studied. The virulence factors, AMR genes, and genomic environments of the multidrug-resistant isolates were further studied. A total of 1,055 Cronobacter isolates were recovered from 12,105 samples of powdered infant formula and supplementary food collected from 29 provinces between 2018 and 2019 in China. Among these, 1,048 isolates were from infant supplementary food and 7 were from powdered infant formula. Regarding antimicrobial resistance susceptibility, 11 (1.0%) isolates were resistant and two showed resistance to four antimicrobials (ampicillin [AMP], tetracycline [TET], sulfamethoxazole-trimethoprim [SXT], and chloramphenicol [CHL]), defined as MDR. These two MDR isolates were subsequently identified as Cronobacter sakazakii sequence type 4 (ST4) (C. sakazakii Crono-589) and ST40 (C. sakazakii Crono-684). Both MDR isolates contain 11 types of virulence genes and 7 AMR genes on their genomes. Meanwhile, the IncFIB plasmids of both MDR C. sakazakii isolates also harbored 2 types of virulence genes. Results of the genomic comparative analysis indicated that food-associated C. sakazakii could acquire antimicrobial resistance determinants through horizontal gene transfer (HGT).IMPORTANCE As a foodborne pathogen, Cronobacter can cause serious infections in restricted populations and lead to death or chronic sequelae. Although a number of investigations showed that Cronobacter isolates are susceptible to most antimicrobial agents, MDR Cronobacter isolates, isolated mainly from clinical cases but occasionally from foods, have been reported in recent years. In this study, we successfully identified two MDR Cronobacter sakazakii isolates from infant foods based on nationwide surveillance and genome sequencing in China. Genomic analysis revealed that these two MDR C. sakazakii strains acquired resistance genes from other species via different evolution and transmission routes. It is important to monitor MDR C. sakazakii isolates in infant foods, and appropriate control measures should be taken to reduce the contamination with and transmission of this MDR bacterium.

The Role of ptsH in Stress Adaptation and Virulence in Cronobacter sakazakii BAA-894.

Cronobacter sakazakii, an emerging foodborne pathogen that was isolated primarily from powdered infant formula, poses an important issue in food safety due to its high stress tolerance and pathogenicity. The Hpr (encoded by ptsH gene) has been shown to regulate carbon metabolism as well as stress response and virulence. However, the functional properties of ptsH in C. sakzakii have not been investigated. In this study, we clarified the role of ptsH in the C. sakzakii stress response and virulence, and explored its possible regulatory mechanism by RNA-seq. Compared with wild-type, the ΔptsH mutant showed a slower growth rate in the log phase but no difference in the stationary phase. Moreover, the resistance to heat stress (65 °C, 55 °C), simulated gastric fluid (pH = 2.5), biofilm formation and adhesion to HT-29 cells of ΔptsH mutant were significantly decreased, whereas the oxidative resistance (1, 5, 10 mM H2O2), osmotic resistance (10%, 15%, 20% NaCl), and superoxide dismutase activity were enhanced. Finally, RNA-seq analysis revealed the sulfur metabolism pathway is significantly upregulated in the ΔptsH mutant, but the bacterial secretion system pathway is dramatically downregulated. The qRT-PCR assay further demonstrated that the ΔptsH mutant has elevated levels of genes that are related to oxidative and osmotic stress (sodA, rpoS, cpxA/R, osmY). This study provides a great understanding of the role of ptsH in diverse stress responses and virulence in C. sakazakii, and it contributes to our understanding of the genetic determinant of stress resistance and pathogenicity of this important foodborne pathogen.