Perspective: Assuring the Quality of Protein in Infant Formula.

Current regulations require that the assessment of protein quality in infant formula be determined using the protein efficiency ratio (PER) rat bioassay where the growth of rats fed a test protein is compared with the growth of rats fed casein. This review cites authoritative body opinions that the PER is not a preferred method for scoring protein quality, particularly as applied to the infant formula. Methodological recommendations specified by FDA and recent guidance propose to control nonprotein dietary variables in the PER. In contrast, the essential amino acid pattern of human milk has been adopted internationally as the standard for protein quality in infant formula. Because casein, the control protein in the PER fails to meet the standard of human milk essential amino acids, the PER based on casein can generate a false assurance of the quality of protein in an infant formula. FDA should revise the method of demonstrating the quality factor for the biological quality of protein to the essential amino acid pattern of human milk, which would be simpler, conform to international standards, and should be considered by FDA under a new statute. Alternate methods of determination of protein quality can be used selectively when there are questions about the digestibility of new protein sources or the effects of manufacturing processes.

Effects of addition of 2-fucosyllactose to infant formula on growth and specific pathways of utilization by Bifidobacterium in healthy term infants.

Oligosaccharides in human milk support health via intestinal microbiome. We studied effects of addition of 2-fucosyllactose (2'FL) to the infant formula on infant growth, occurrence of adverse events (AE), and infant microbiome, including expression of microbial genes that metabolize 2'FL. Our hypothesis was that while 2'FL would not affect growth, it would cause changes in microbiome metabolism. In a double-blinded randomized controlled study fashion, the infant formula ± 2'FL or human milk was fed to healthy term infants for 16 weeks. Fecal samples obtained at baseline and week 16 were analyzed for microbial populations, metagenomic species concept (MGS), and genetics of gut metabolic modules (GMMs). There were no effects of addition of 2'FL on growth or AEs. There were no significant differences by feeding group in MGS richness or Shannon diversity at baseline, but formula groups each had significantly greater richness (p < 0.05) and diversity (p < 0.05) after 16 weeks of feeding than the breastfed group. While two glycosyl hydrolase (GH) families (GH42 and GH112) were significantly increased, two other GH families (GH20 and GH2) were significantly decreased in the test formula group compared to the control formula group; although modest, addition of 2'FL resulted in changes in microbiome in the direction of breastfed infants, consistent with internal metabolism of HMOs by Bifidobacterium.

Assessing the safety of bioactive ingredients in infant formula that affect the immune system: recommendations from an expert panel.

Bioactive ingredients for infant formula have been sought to reduce disparities in health outcomes between breastfed and formula-fed infants. Traditional food safety methodologies have limited ability to assess some bioactive ingredients. It is difficult to assess the effects of nutrition on the infant immune system because of coincident developmental adaptations to birth, establishment of the microbiome and introduction to solid foods, and perinatal environmental factors. An expert panel was convened to review information on immune system development published since the 2004 Institute of Medicine report on evaluating the safety of new infant formula ingredients and to recommend measurements that demonstrate the safety of bioactive ingredients intended for that use. Panel members participated in a 2-d virtual symposium in November 2020 and in follow-up discussions throughout early 2021. Key topics included identification of immune system endpoints from nutritional intervention studies, effects of human milk feeding and human milk substances on infant health outcomes, ontologic development of the infant immune system, and microbial influences on tolerance. The panel explored how "nonnormal" conditions such as preterm birth, allergy, and genetic disorders could help define developmental immune markers for healthy term infants. With consideration of breastfed infants as a reference, ensuring proper control groups, and attention to numerous potential confounders, the panel recommended a set of standard clinical endpoints including growth, response to vaccination, infection and other adverse effects related to inflammation, and allergy and atopic diseases. It compiled a set of candidate markers to characterize stereotypical patterns of immune system development during infancy, but absence of reference ranges, variability in methods and populations, and unreliability of individual markers to predict disease prevented the panel from including many markers as safety endpoints. The panel's findings and recommendations are applicable for industry, regulatory, and academic settings, and will inform safety assessments for immunomodulatory ingredients in foods besides infant formula.

Perspective: Structure-Function Claims on Infant Formula.

In the context of a food product label, the term "claim" refers to information that attributes value to the product. The term extends to many different types of information, from product identity, descriptors of intended use, and identification of characteristic properties to the physiologic effects in the body of substances in the food, including the reduction of risk of disease. Food labeling, which includes claims, provides information that consumers want and use to improve their diets. Consumers prefer short statements on the front label claims to longer, more detailed information, including ingredients statements and a nutrition panel. Three types of claims are permitted in the United States. Nutrient content claims describe the level of the nutrient in the food relative to an established daily value, e.g., "Excellent source of choline," and are subject to composition limits for other nutrients, such as total fat, saturated fat, and cholesterol. Health claims describe the relation between a food substance and the risk of disease, e.g., "Adequate calcium and vitamin D throughout life, as part of a well-balanced diet, may reduce the risk of osteoporosis." They must undergo a premarket evaluation by the FDA to ensure that there is significant scientific agreement about the relation in question. The third type of claim, structure-function (SF) claims, has recently come under scrutiny, particularly regarding their use on infant formula. Such claims represent a food's effect on the structure or function of the body for maintenance of good health and nutrition. These claims must be truthful and not misleading, but are not subject to premarket approval before use. The purpose of this perspective is to describe the origins and unique niche of SF claims, and to comment on recent proposals to further regulate such claims on infant formula.

World Health Organization (WHO) infant and young child feeding indicators: associations with growth measures in 14 low-income countries.

Eight World Health Organization (WHO) feeding indicators (FIs) and Demographic and Health Survey data for children <24 months were used to assess the relationship of child feeding with stunting and underweight in 14 poor countries. Also assessed were the correlations of FI with country gross national income (GNI). Prevalence of underweight and stunting increased with age and ≥ 50% of 12-23-month children were stunted. About 66% of babies received solids by sixth to eighth months; 91% were still breastfeeding through months 12-15. Approximately half of the children were fed with complementary foods at the recommended daily frequency, but <25% met food diversity recommendations. GNI was negatively correlated with a breastfeeding index (P < 0.01) but not with other age-appropriate FI. Regression modelling indicated a significant association between early initiation of breastfeeding and a reduction in risk of underweight (P < 0.05), but a higher risk of underweight for continued breastfeeding at 12-15 months (P < 0.001). For infants 6-8 months, consumption of solid foods was associated with significantly lower risk of both stunting and underweight (P < 0.001), as was meeting WHO guidance for minimum acceptable diet, iron-rich foods (IRF) and dietary diversity (P < 0.001); desired feeding frequency was only associated with lower risk of underweight (P < 0.05). Timely solid food introduction, dietary diversity and IRF were associated with reduced probability of underweight and stunting that was further associated with maternal education (P < 0.001). These results identify FI associated with growth and reinforce maternal education as a variable to reduce risk of underweight and stunting in poor countries.

How well are infant and young child World Health Organization (WHO) feeding indicators associated with growth outcomes? An example from Cambodia.

We assessed eight World Health Organization (WHO) core child feeding indicators for their association with stunting and underweight in Cambodia in 2000 and 2005. We compared the feeding data from the Cambodian Demographic and Health Surveys for 2000 with 2005 for 0-24 months children using the WHO feeding indicators, with stunting and underweight as outcomes. Prevalence of stunting and underweight was significantly less in 2005 than in 2000 among children aged 0-5 and 6-11 months, but stunting among children 18-23 months remained >50%. Prevalence of compliance with seven of the eight core healthy feeding indicators was higher in 2005. Exclusive breastfeeding among 0-5 months infants increased more than fivefold; among 6-11 and 12-17 months children, prevalence of feeding diversity and meeting a minimally acceptable diet, while improved, remained ≈25%. Modelling showed compliance with breastfeeding indicators was associated with reduced risk of underweight in 0-5 months infants, no association between compliance with feeding indicators and growth outcomes in other ages, and a significant association of higher relative wealth with growth outcomes overall. Between 2000 and 2005, Cambodia stabilized and focused resources on infant feeding. Prevalence of meeting the WHO feeding indicators improved, but modelling indicated that, in general, relative wealth, not feeding practices, was associated with improved growth outcomes. Yet, over 50% of children 18-23 months were stunted in 2005. Similar to the success with breastfeeding, focus on complementary feeding of 6-23 months children may reduce the risk of stunting in Cambodia.

Fatty acids in Chinese edible oils: value of direct analysis as a basis for labeling.

Edible oil is an important element in the diet of most transitional countries; nevertheless, little is known about the fatty acid composition of these oils. We examined the consumption of edible oils and the fatty acid composition of these oils obtained from a market survey conducted in seven Chinese provinces and in Beijing. Three days of measured household food intake from the 1997 China Health and Nutrition Survey households provided data on the consumption of edible oils. Edible oils sold in the capital cities of eight provinces were purchased. One hundred twenty-six samples, representing 14 different oils according to their labels, were assayed for their fatty acid content in 2001. Fatty acids were analyzed by standard gas chromatographic methods. More than 76% of households in China consume edible oil, providing an average of 29.6 g of edible oil per day to persons aged two years or older. Rapeseed was consumed by one-quarter of individuals. Rapeseed is rich in C22:1n9 cis (erucic acid). About 33% of edible oils differed from their labeled identification. Rapeseed oil, identified by the presence of C22:1n9 (erucic acid), was most frequently not labeled as such. In another 28% of the samples, trans isomers of linolenic acid were detected. Deviations from the label identification were more common in southern than in northern provinces. Regulations requiring complete labeling of mixed edible oils in China might help prevent unintentional consumption of fatty acids associated with adverse health outcomes. In particular, consumption of erucic acid and trans fatty acids might be reduced. The results suggest the need for closer control of food oil labeling in China, especially in the South.

Simultaneous determination of (45)calcium and (65)zinc uptake by caco-2 cells.

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.

Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry.

We identified several low-abundance proteins of bovine colostrum and mature milk using the immunoabsorption technique and two-dimensional electrophoresis (2-DE) followed by microsequencing and mass spectrometry. Two major milk proteins, beta-casein and immunoglobulin G (IgG), were effectively removed from the milk using immunoabsorbents. Milk samples before and after immunoabsorption were separated by 2-DE. Protein identification of the spots on 2-DE was performed by either gel comparison, microsequencing, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF-MS), peptide mass fingerprinting or peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight-MS (Q-TOF). Significant differences in protein patterns were observed between the low-abundance proteins of colostrum and mature milk. In addition, several low-abundance proteins including fibrinogen beta-chain, chitinase 3-like 1, alpha-antitrypsin, complement C3 alpha-chain, gelsolin and apolipoprotein H were observed only in colostrum. However, the level of beta-casein fragments increased significantly during this lactation period. alpha-Lactalbumin and beta-lactoglobulin as well as some low-abundance proteins including bovine serum albumin, serotransferrin and lactoferrin were identified in both colostral and mature milk. Low-abundance proteins in bovine colostrum may have special physiologic relevance to the health and development of calves early in lactation.