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Determination of Macronutrients, by Chemical
Analysis, of Home-Prepared Milk Feeding Bottles
and their Contribution to the Energy and Protein
Requirements of Infants from High and Low
Socioeconomic Classes
Tania Beninga Morais PhDa & Dirce Maria Sigulem MD, PhDa
a Department of Pediatrics, Federal University of São Paulo Medical School, São Paulo,
BRAZIL
Published online: 24 Jun 2013.
To cite this article: Tania Beninga Morais PhD & Dirce Maria Sigulem MD, PhD (2002) Determination of Macronutrients, by
Chemical Analysis, of Home-Prepared Milk Feeding Bottles and their Contribution to the Energy and Protein Requirements
of Infants from High and Low Socioeconomic Classes, Journal of the American College of Nutrition, 21:3, 284-288, DOI:
10.1080/07315724.2002.10719222
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Original Research
Determination of Macronutrients, by Chemical Analysis,
of Home-Prepared Milk Feeding Bottles and their
Contribution to the Energy and Protein Requirements of
Infants from High and Low Socioeconomic Classes
Tania Beninga Morais, PhD, and Dirce Maria Sigulem, MD, PhD
Department of Pediatrics, Federal University of Sa˜o Paulo Medical School, Sa˜o Paulo, BRAZIL
Key words: infant food, milk, food composition, nutritional requirements
Objectives: To determine the macronutrients composition of home-prepared milk feeding bottles, by
chemical analysis, and assess their contribution to the energy and protein requirements of children under two
years of age from high (HSE) and low (LSE) socioeconomic classes.
Methods: 72 samples were analyzed for energy density and protein, fat and carbohydrate content: 41 from
the LSE group and 31 from the HSE group. The assessment of the percentages of the energy and protein
requirements met by the consumption of the milk bottles was calculated as follows: the energy and protein per
100 mL obtained through chemical analysis were multiplied by the volume consumed at each feeding, then by
the number of feedings per day, the results divided by the energy and protein requirements and multiplied by 100.
Energy and protein requirements were those recommended by the FAO/WHO/UNU Committee and the Food
and Nutrition Board. The children’s weight-for-age index was assessed.
Results: Unmodified cow’s milk was largely consumed by both groups. The addition of sugar and other
ingredients to the milk was significantly higher in the LSE group. Moisture, protein and fat content were lower
in the LSE group, whereas carbohydrate and energy content were higher. The percentages of energy and protein
requirements provided by feeding bottles were higher in the LSE group. Children in the LSE group had lower
z-scores for weight-for-age.
Conclusions: Differences in the preparation practices led to differences in the chemical results. The feeding
bottles in the LSE group were high in energy, due to the addition of sugar and cereals to the milk in the bottle.
The milk feeding bottles were an important weaning food providing more than 50% and 100% of the children’s
energy and protein requirements, respectively. The children’s weight-for-age index was within the normal limits.
Few studies have been made on their actual nutritional com-
position as defined by chemical analysis [6,7], even though
incorrect dilution in home-prepared formula and powdered
INTRODUCTION
The weaning process is a period of particular concern in
cow’s milk has been found both in developed and in developing
developing countries because it is often accompanied by mal-
countries. Mothers in affluent societies are more likely to
nutrition, most commonly during the second semester of life
over-concentrate [8], whereas mothers in less developed coun-
when foods other than breast milk are added to the diet [1].
tries usually over-dilute formula and powdered cow’s milk
Malnutrition can be attributed to high rates of infection, insuf-
ficient energy and nutrient intake and poor dietary quality [2].
In developing countries, complementary foods can have energy
densities as low as 25–30 Kcal/100 g [2,3]. Bottle-fed porridges
prepared with cow’s milk and various kinds of cereals are a
common complementary food in developing countries [4–7].
[6,7]. The socioeconomic class has been shown to influence
feeding-bottles preparation practices [9–11]. Milk and formula
play a major role as a source of energy in the infants diet
[12–14]. However, in these studies, that role was assessed by
food composition tables, not by chemical analysis. Home-
prepared foods do not always have an adequate nutritional
Address reprint requests to: Tania B. Morais, Ph.D., R. Araioses, 75, 05442-010 Sa˜o Paulo, SP, BRAZIL. E-mail: tania.pnut@epm.br
Journal of the American College of Nutrition, Vol. 21, No. 3, 284–288 (2002)
Published by the American College of Nutrition
284

Chemical Analysis of Home-Prepared Feeding Bottles
profile, even in developed countries [15,16]. Knowing their
chemical composition would allow health professionals to bet-
ter advise the mothers on the preparation practices of the foods.
The objectives of this study were 1) to determine by chem-
ical analysis the macronutrients composition of home-prepared
milk feeding bottles and 2) to assess their contribution to the
energy and protein requirements (according to the FAO/WHO/
UNU Committee and the Food and Nutrition Board) of children
under two years of age from high (HSE) and low (LSE)
socioeconomic classes. The contribution of other dietary foods
was not taken into account.
weight-for-age below the cut-off value, length or height mea-
sures were not done. This project was approved by the Medical
Ethics Committee of the Federal University of Sa˜o Paulo.
Parent/guardian written consent was also obtained.
Energy and Protein Provided by Feeding Bottles as
a Percentage of Requirements
In order to calculate the total intake of milk consumed per
day, the mothers were also asked how many milliliters of milk
the child consumed at each feeding (measured on the scale of
the bottles) and the number of feedings per day. The percent-
ages of energy and protein provided by the contents of the
feeding bottles was calculated as follows: The energy and
protein per 100 mL were obtained through chemical analysis
and were multiplied by the volume consumed at each feeding.
The results were multiplied by the number of feedings per day,
divided by the energy and protein requirements, then multiplied
by 100. Energy and protein requirements were those recom-
mended by the FAO/WHO/UNU Committee [18] and the Food
and Nutrition Board [19].
MATERIALS AND METHODS
Subjects and Study Design
The study was carried out in Sa˜o Paulo (a city of approxi-
mately 10,300,000 inhabitants in Southern Brazil). The subjects
of LSE group were selected from the records of the families
living in three slum areas, where the graduate course of nutri-
tion of the Federal University of Sa˜o Paulo has a field project
that provides free primary health care. Mothers attending a
private pediatric clinic and selected from the medical appoint-
ments of the day were allocated to the HSE group. The final
study sample contained 72 subjects, 41 in the LSE group and 31
in the HSE group. For both groups, the children could not
exceed two years of age and had to be fully weaned and
bottle-fed. The median age of the children in both groups was
12 months. The number of children, according to age was Ͻ6
months ϭ 7 (HSE) and 9 (LSE), 6–12 months ϭ 9 (HSE) and
11 (LSE), 12–24 mo ϭ 15 (HSE) and 21 (LSE). The mothers
were interviewed and requested to give specific details of how
they prepared the feeding bottles, specifically, the type of milk
used (formula or unmodified cow’s milk, either pasteurized or
powdered), other ingredients added and the amount of each one
used. All interviews were performed by the first author
(T.B.M.). Samples of the contents of the feeding bottles were
collected from all 72 subjects in order to perform the chemical
analyses. In the LSE group, the samples were collected from
bottles at home, whereas in the HSE group they were collected
from bottles brought to the pediatric clinic by the mothers.
None of the mothers had previously been informed that sam-
ples would be collected, thus ensuring that the bottles had been
prepared in the normal way. The children’s weight-for-age
index was assessed in relation to the current international
growth reference (NCHS/WHO) and expressed as z-scores.
Field constraints in the slum areas prevented the study from
obtaining accurate height or length measures of the children. In
these areas a two-step evaluation has been used [17]. The index
weight-for-age is used for growth monitoring. The children
found to be faltering in growth or below a selected cut-off value
had their length or height measured to assess their weight-for-
height and height-for-age. As none of the children showed
Chemical Analyses
The samples were transported to the laboratory at Ͻ10°C
within two hours of collection and analyzed for moisture,
protein, fat and ash [20]. The moisture content was obtained by
heating the samples to 102°C until a constant weight was
attained. The protein level was obtained by determination of
Kjeldahl nitrogen and multiplied by 6.38 [21]. After acid hy-
drolysis, the fat was extracted by ether using the Soxhlet
apparatus. Ash was obtained by incineration at 500–550°C
until the ash was carbon-free. Carbohydrate content was deter-
mined by difference. The energy density was calculated by
multiplying the protein and total carbohydrate content by 4
Kcal and adding the result to the fat content multiplied by 9
Kcal. The results for energy, protein, carbohydrate and fat were
compared with the guidelines of the European Society of Pe-
diatric Gastroenterology and Nutrition (ESPGAN) for fol-
low-up formulas [22]: energy: 60–80 Kcal ⅐ dLϪ1, protein:
2.1–3.1 g ⅐ dLϪ1, carbohydrate: 5.7–8.6 g ⅐ dLϪ1, fat: 2.7–4.0
g ⅐ dLϪ1
.
Statistical Analyses
The statistical analysis were performed using the softwares
EPI INFO 6 ANTHRO (CDC/WHO, 1994) and Sigma Stat for
Windows 2.0௡ (SPSS, 1997). The chi-square test was used to
compare the proportions of the addition of sugar and cereals
to the milk in the bottle. The Mann-Whitney test was used to
compare the two groups’ data. The level of significance was
p Ͻ 0.05.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
285

Chemical Analysis of Home-Prepared Feeding Bottles
Table 1. Types of Milk Used in the Preparation of Feeding
Bottles in the HSE (High Socioeconomic) and LSE (Low
Socioeconomic) Groups
Table 3. Macronutrient Composition of Feeding Bottles in
the HSE (High Socioeconomic) and LSE (Low
Socioeconomic) Groups
HSE
n ϭ 31
LSE
n ϭ 41
Mann-
Whitney
Test
Total
HSE
n ϭ 31
LSE
n ϭ 41
Unmodified Cow’s
Milk
Powdered
Pasteurized
Formula
24 (77.4%)
12 (38.7%)
12 (38.7%)
7 (22.6%)
40 (97.6%)
25 (61.0%)
15 (36.6%)
1 (2.4%)
64 (88.9%)
37 (51.4%)
27 (37.5%)
8 (11.1%)
Per dLϪ1
Moisture (g)
Protein (g)
Fat (g)
Median (P25–P75)
87.5 (83.8–87.9) 82.9 (79.7–85.9) pϽ0.001*
3.4 (2.9–3.9)
3.0 (2.6–3.7)
0.6 (0.4–0.6)
3.2 (2.2–3.7)
2.4 (1.8–3.3)
0.5 (0.4–0.7)
11.2 (8.1–13.2)
pϭ0.063
pϭ0.019*
pϭ0.539
pϽ0.001*
Ash (g)
Carbohydrates (g) 5.7 (4.8–8.2)
Energy (Kcal)
64.3 (58.6–81.6) 78.1 (64.5–92.7) pϭ0.017*
RESULTS
As reported by the mothers and shown in Table 1, unmod-
ified cow’s milk was largely consumed by both groups. The
HSE group consumed both powdered and pasteurized cow’s
milk, while the LSE group tended to use powdered cow’s milk.
The consumption of formula was low in the HSE group and
practically non-existent in the LSE group. The addition of sugar
and other ingredients to the milk was significantly higher in the
LSE group (Table 2).
Macronutrient composition of the feeding bottle contents is
given in Table 3. Moisture and fat content were significantly
lower in the LSE group, whereas carbohydrate and energy
content were significantly higher. Protein content was lower in
the LSE group, although the difference did not reach statistical
significance.
Table 4 shows the percentages of feeding bottles that fell in
the various intervals for energy, protein, carbohydrate and fat
proposed in the ESPGAN guidelines. Only 35.5% and 46.3% of
the feedings bottles, in the HSE and LSE groups, respectively,
had an energy content in the interval of 60–80 Kcal. Further-
more, the HSE group presented a higher percentage of feeding
bottles with less than 60 Kcal than the LSE group, while the
LSE group presented with a higher percentage of feeding
bottles Ͼ80 Kcal. Because unmodified cow’s milk was used in
the majority of the feeding bottles, most of them presented a
protein content above 3.1 g ⅐ dLϪ1, although the LSE group
presented a higher percentage of feeding bottles with values
below 2.1 g ⅐ dLϪ1. Approximately 29% of the feeding bottles
for both groups fell within the normal ESPGAN guidelines for
carbohydrate (5.7–8.6 g ⅐ dLϪ1). The HSE group had a higher
percentage of feeding bottles with a content below 5.7 g ⅐ dLϪ1
,
while the LSE group presented a higher percentage of feeding
bottles with a content above 8.6 g ⅐ dLϪ1. The results for fat
were similar in both groups.
As is shown in Table 5, the intake of milk per bottle, the
energy per bottle and the total energy per day from feeding
bottles were significantly higher in the LSE group. The per-
centage of energy requirement met by the contents of the
feeding bottles was also higher in the LSE group, although it
did not reach statistical difference. The percentage of protein
Table 4. Distribution of the Number and Percentage of
Feeding Bottles in the HSE (High Socioeconomic) and LSE
(Low Socioeconomic) Groups in the Intervals Proposed by
ESPGAN
Table 2. Addition of Sugar and Other Ingredients in the
Preparation of Feeding Bottles in the HSE (High
Socioeconomic) and LSE (Low Socioeconomic) Groups
HSE
LSE
HSE
n ϭ 31
LSE
n ϭ 41
Per dLϪ1
Energy (Kcal)
60–80
Ͻ60
Ͼ80
Protein (g)
2.1–3.1
Ͻ2.1
n (%)
n (%)
2
␹
Test
Sugar
Yes
No
Other Ingredients
Yes
11 (35.5%)
12 (38.7%)
8 (25.8%)
19 (46.3%)
7 (17.1%)
15 (36.6%)
4 (12.9%)
27 (87.1%)
37 (90.2%)
4 (9.8%)
p Ͻ 0.001*
p ϭ 0.046*
13 (42.0%)
28 (68.3%)
10 (32.3%)
1 (3.2%)
20 (64.5%)
13 (31.7%)
7 (17.1%)
21 (51.2%)
Cornstarch
Corn flour
Infant cereals
Rice flour
Chocolate
Oat
2
0
17
2
Ͼ3.1
1
0
3
1
4
2
1
2
Carbohydrates (g)
5.7–8.6
Ͻ5.7
9 (29.0%)
15 (48.4%)
7 (22.6%)
12 (29.3%)
1 (2.4%)
28 (68.3%)
Ͼ8.6
Sweeteners
Sustagen®
Honey
2
2
2
0
0
0
Fat (g)
2.7–4.0
Ͻ2.7
13 (42.0%)
13 (42.0%)
5 (16.0%)
17 (41.4%)
20 (48.8%)
4 (9.8%)
No
18 (58.0%)
13 (31.7%)
Ͼ4.0
286
VOL. 21, NO. 3

Chemical Analysis of Home-Prepared Feeding Bottles
Table 5. Intake Volume and Energy per Bottle, Number of Feedings per Day, Total Energy from Feeding Bottles per Day,
Percentages of Energy and Protein Requirements from Feeding Bottles per Day and z-Score (Weight-for-Age) in the HSE (High
Socioeconomic) and LSE (Low Socioeconomic) Groups
HSE
LSE
Mann-
n ϭ 31
n ϭ 41
Whitney Test
Median (P25–P75)
Intake (mL)/bottle
Energy (Kcal)/bottle
Feedings/day
200.0 (162.5–232.5)
123.6 (103.2–157.5)
3.0 (3.0–5.0)
220.0 (200.0–240.0)
162.8 (149.3–197.5)
3.0 (3.0–5.0)
pϭ0.021*
pϭ0.002*
pϭ0.950
Total energy (Kcal) from feeding bottles/day
% of energy requirements from feeding
bottles (RDA: 600–1300 Kcal/day)
% of protein requirements from feeding
bottles (RDA: 13–16 g/day)
z-Score (weight-for-age)
450.7 (362.5–562.9)
599.0 (453.2–773.8)
pϭ0.010*
46.7 (32.6–74.4)
61.1 (42.7–96.2)
pϭ0.096
160.0 (120.0–220.0)
ϩ0.535 (Ϫ0.010/ϩ0.930)
160.0 (130.0–200.0)
ϩ0.100 (Ϫ0.850/ϩ0.595)
pϭ0.914
pϭ0.049*
requirement provided by the contents of the feeding bottles
were similar in both groups. Children in the LSE group had
significantly lower z-scores for weight-for-age, though in both
groups they were within the normal limits.
consumption of sugar and cereals in the HSE group might be
linked with a concern, on the part of the mothers, of the risk of
obesity. Nowadays, obesity in children is a cause of concern in
Brazil, affecting about 11.0% of children under four years of
age in the high socioeconomic class [27].
The different preparation practices gave rise to differences
in the chemical composition of the feeding bottle contents, with
the addition of sugar and other ingredients leading to a signif-
icantly higher carbohydrate and energy content in the LSE
group. The protein content (3.2 g/dLϪ1) was higher than that
found by Hibbert et al. [6] (2.8 g/dLϪ1) in Jamaica and by
Dorea et al. [7] (2.0 g/dLϪ1) in the Brazilian capital, Bras´ılia.
The energy content (78.1 Kcal/dLϪ1) was similar to that found
in Jamaica (74.0 Kcal/dLϪ1), lower than in Bras´ılia (96.0
Kcal/dLϪ1), but higher than other complementary foods con-
sumed in developing countries (25–30 Kcal/100 g) [2,3]. The
lower protein and fat contents in the LSE group’s feeding
bottles could be due to over dilution of the powdered cow’s
milk and to the relative dilution resulting from the addition of
sugar and cereals.
DISCUSSION
Home-prepared weaning foods in developing countries have
been regarded as nutritionally poor, with low caloric and pro-
tein densities [2,3]. In this study, however, such a pattern was
not found. According to the ESPGAN guidelines [22], which
were used as a framework to assess the nutritional quality of the
macronutrients in the feeding bottles (even though they were
intended for cow’s milk based follow-up formulas without the
addition of other ingredients), most of the feeding bottles, both
in the HSE and the LSE group, met or exceeded the recom-
mended values for energy, protein, carbohydrate and fat.
Addition of sugar and other ingredients to feeding bottles
was significantly higher in the LSE group. This is a common
practice in less developed countries [4–7,9] and in low-income
classes in the United States [10,11]. In Brazil, this practice is
also a cultural norm in low socioeconomic classes as was
reported by Tudisco et al. [4] in a study of weaning diet in four
Brazilian state capitals. Here, the main complementary food
was cow’s milk with sugar and cereals, consumed by 68% of
466 children under two years of age. Brazilian pediatric text-
books, however, recommend the addition of sugar and starch to
the milk just for malnourished children of low socioeconomic
class [23,24]. In this study, even though children in the LSE
group had significantly lower z-scores for weight-for-age, they
were within the normal limits.
Feeding bottles played a major role as a source of energy
and protein in the LSE group, with about 60% and 160% of
energy and protein requirements, respectively, being provided
by them. These results may explain the normal weight-to-age
index of the children of this group. However, as children seem
to regulate energy intake [28], the feeding bottles in the LSE
group could eventually displace a more varied diet. This leads
to a monotonous diet high in calories, poor in micronutrient
content and bioavailability, potentially resulting in another set
of nutritional problems, such as anemia and obesity. In the state
of S. Paulo, anemia has been found in 59% of the children
under two years of age attending health care units [29]. Ac-
cording to two large national surveys, underweight in children
less than four years old dropped from 20.6% in 1974 to 12.2%
in 1989 in the poorest economic stratum. In the intermediate
and richest strata, overweight percentages were higher than
underweight percentages [27].
In the HSE group, the percentages of mothers adding sugar
(12.9%) and other ingredients (42.0%) to feeding bottles were
higher than those observed by Fein et al. [25] (2.0% of the
mothers added sugar and 15.0% added cereal) and by Skinner
et al. [26] (33.1% of the mothers added cereal) in middle and
upper socioeconomic classes in the United States. The lower
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
287

Chemical Analysis of Home-Prepared Feeding Bottles
Mothers do not always recognize which practices are im-
portant for their infants’ health, because failure to follow rec-
ommendations does not generally have consequences that are
obviously linked to feeding practices [25]. The nutritional
issues are dynamic, and the professionals working in this area
should be prepared to address them.
12. Ernst JA, Brady MS, Rickard KA: Food and nutrient intake of
6-to-12-month-old infants fed formula or cow milk: A summary of
four national surveys. J Pediatr 117:S86–100, 1990.
13. Heinig MJ, Nommsen LA, Peerson JM, Lonnerdal B, Dewey KG:
Energy and protein intakes of breast-fed and formula-fed infants
during the first year of life and their association with growth
velocity: the DARLING Study. Am J Clin Nutr 58:152–161, 1993.
14. Capdevila F, Vizmanos B, Mart´ı-Henneberg C: Implications of the
weaning pattern on macronutrient intake, food volume and energy
density in non-breastfed infants during the first year of life. J Am
Coll Nutr 17:256–262, 1998.
CONCLUSIONS
Differences in the preparation practices led to differences in
the chemical results. The feeding bottles in the LSE group were
high in energy, due to the addition of sugar and cereals to the
milk in the bottle. The milk feeding bottles were an important
weaning food providing more than 50% and 100% of the
children’s energy and protein requirements, respectively. The
children’s weight for age index was within the normal limits.
15. Stordy BJ, Redfern AM, Morgan, JB: Healthy eating for infants—
mothers’ actions. Acta Paediatr 84:733–741, 1995.
16. Boom SAM, Kimber AC, Morgan JB: Nutritional composition of
home-prepared baby meals in Madrid. Comparison with commer-
cial products in Spain and home-made meals in England. Acta
Paediatr 86:57–62, 1997.
17. Gorstein J, Sullivan K, Yip R, On´ıs M, Trowbridge F, Fajans P,
Clugston G: Issues in the assessment of nutritional status using
anthropometry. Bull World Health Org 72:273–283, 1994.
18. Food and Agriculture Organization/World Health Organization/
United Nations University Expert Consultation: Energy and pro-
tein requirements. World Health Organization Technical Report
Series 724. Geneva: WHO, 1985.
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Received April 16, 2001; revision accepted January 16, 2002.
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