BREASTFEEDING MEDICINE Volume 8, Number 2, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/bfm.2012.0078
Effects of Synbiotic Supplementation on Total Antioxidant Capacity of Human Breastmilk Leila Nikniaz,1 Reza Mahdavi,2 Alireza Ostadrahimi,2 Mohammad Amin Hejazi,3 and Amir Mansour Vatankhah 4
Abstract
Background and Objectives: The aims of this study were to determine the effects of synbiotic (probiotic plus prebiotic) supplementation on total antioxidant capacity (TAC) and malondialdehyde (MDA) levels of human breastmilk. Subjects and Methods: In this randomized, double-blind, placebo-controlled trial, 80 lactating mothers were randomly divided into two groups to receive a daily supplement of synbiotic (n = 40) or a placebo (n = 40) for 30 days. Information on dietary intake was collected from lactating women using the 24-hour recall method for 3 days before and after supplementation. The TAC was measured by using a Randox (Crumlin, County Antrim, United Kingdom) assay, and the MDA level of breastmilk as thiobarbitaric acid complexes was measured by the fluorometry method. Data analysis was carried out using Nutritionist IV (Axxya Systems, Stafford, TX) and SPSS (SPSS, Inc., Chicago, IL). Results: The TAC of breastmilk increased significantly from 0.312 – 0.16 to 0.481 – 0.2 mmol/L in the supplemented group ( p < 0.039), whereas it decreased from 0.317 – 0.18 to 0.255 – 0.13 mmol/L in the placebo group ( p > 0.13). Although the MDA level decreased slightly from 1.62 – 0.69 to 1.6 – 0.95 lmol/L in the supplemented group, it increased significantly in the placebo group from 1.71 – 0.86 to 2.16 – 0.277 lmol/L after the experimental period ( p < 0.001). Also, maternal vitamin A, E, and C, zinc, and selenium intake did not change significantly in both groups during the study period. Moreover, no significant correlation was found between weight for age Z-score of infants and TAC and MDA levels in breastmilk. Conclusions: Based on these results, synbiotic supplementation may have positive effects on the TAC and MDA levels in breastmilk; however, these findings require confirmation from future trials.
Introduction
T
he first food naturally ingested by newborn babies is breastmilk. Breastmilk from healthy women contains all the nutrients necessary for newborn infants and also contains a variety of growth and immune factors.1 In addition to numerous clinically significant aspects of breastfeeding, it seems breastmilk contains antioxidant molecules that can help prevent oxidative stress situations, offering a vital antioxidant protection.2 Breastmilk’s enzymatic and nonenzymatic antioxidant constituents, like superoxide dismutase, glutathione peroxidase, catalase, vitamin E, vitamin C, and b-carotene, may protect newborns against reactive oxygen species at early stages of life.3,4 The total antioxidant
capacity (TAC) is a parameter characterizing the sum of the activities of all antioxidants present in human breastmilk.5 The TAC of breastmilk seems to be affected by the maternal antioxidant status, which in turn could influence the antioxidant status of breastfed infants.6 Milk with a higher TAC value will reflect greater oxidative stability and a potentially greater protection for the breastfed infant from exposure to oxidative agents.7 Several studies have shown that TAC of breastmilk decreases with the passage of days after birth.8,9 It has been suggested that using proper nutritional interventions and different supplements may be beneficial to prevent the TAC in breastmilk from decreasing with time.8 In addition, the malondialdehyde (MDA) level of breastmilk reflects the level of lipid peroxidation in human milk.10 In particular,
1 Student Research Committee and 2Nutrition Research Center, School of Public Health & Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran. 3 Branch for the Northwest and West Region, Agricultural Biotechnology Research Institute of Iran, Tabriz, Iran. 4 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. This study is registered with the registration center for clinical trials in Iran with the registration code number IRCT201110181197N12.
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218 lipid peroxides constitute a potent potential source of reactive oxygen species, which may greatly perturb intestinal homeostasis in the newborn infant.11 So, decreasing levels of lipid peroxides and MDA of breastmilk may be helpful in newborns. Among various supplements, synbiotics or probiotics may be beneficial to reach these purposes. Strains of probiotics (defined as ‘‘live microorganisms which, when administered in adequate amounts, confer a health benefit on the host’’12) have been shown to produce substances with antioxidant and free radical scavenging activities13 and improve gastrointestinal health.14 There are few data relating to the systemic impact of probiotics in defense against oxidative stress.15 To the authors’ knowledge, there is no study investigating the effect of probiotic or synbiotic supplementation on TAC and MDA levels of human milk. So, the aims of this study were to evaluate the effects of synbiotic supplementation on TAC and MDA levels of human breastmilk. Subjects and Methods In this randomized, double-blind, placebo-controlled trial, conducted between October 2011 and March 2012, 80 volunteer lactating women from urban areas of Tabriz City, Iran, who exclusively breastfed their infants for 90 days after birth were recruited from health centers. The inclusion criterion for mothers was having a second child (parity of 2) who was full term with a birth weight between 2,500 and 4,000 g. Mothers and infants were excluded if they had clinical evidence of chronic illness or gastrointestinal disorders or if they had received antioxidant, probiotic, or synbiotic supplements and antibiotics in the month preceding recruitment. The study protocol was approved by the Ethics Committee of Tabriz University of Medical Sciences. All subjects were made aware of the content of the study, and a written informed consent document was obtained. Lactating mothers were assigned randomly to receive the synbiotic supplement or the placebo. The synbiotic supplement (Protexin; Probiotics International Ltd., Lopen Head, Somerset, United Kingdom) consisted of freeze-dried Lactobacillus casei PXN 37, Lactobacillus rhamnosus PXN 54, Streptococcus thermophilus PXN 66, Bifidobacterium breve PXN 25, Lactobacillus acidophilus PXN 35, Bifidobacterium longum PXN 30, Lactobacillus bulgaricus PXN 39, and fructooligosaccharide (as documented by the manufacturer). During the intervention period, the synbiotic group consumed a daily dose of a mixture of these strains (2.0 · 108 colony-forming units) and fructooligosaccharide (394 mg), 30 minutes after meal for 30 days. As a measure of compliance, unused capsules were counted by an independent investigator. A computer-generated random sequence was kept in a remote secure location and administered by an independent third party who was not involved with the clinical conduct of study until all study data were collected and verified. Lactating mothers and those involved in enrolling participants, administering interventions, assessing outcomes, and analyzing data were blind to group assignments. Three mothers in the supplemented group and two in the placebo group did not complete the study because of having gastrointestinal disturbances or antibiotic treatment of mothers or infants.
NIKNIAZ ET AL. Data collection from mothers and infants Information on food intake was collected by using a 24hour recall method for 3 days (one weekend day included) a week before and at the end of supplementation. Dietary intake of subjects was analyzed with the Nutritionist IV software program (Axxya Systems, Stafford, TX). Demographic data (place of residence and age) and clinical data (health status) were obtained through an interview. Body weight of each subject was measured with the woman barefoot and wearing light clothing to the nearest 0.1 kg with a Seca (Dubai, United Arab Emirates) scale. Height of each subject was measured with the woman barefoot and using a measuring tape with the subject’s arm hanging freely at her sides and recorded to the nearest 0.5 cm. The body mass index (BMI) was calculated as weight (kg) divided by height2 (m2). Infants’ body weight, height, and head circumference were measured by accurately calibrated instruments (body weight measurement by Soehnle [Leifheit AG, Nassau, Germany] electronic scales, maximum weight of 20 kg, accurate to 10 g; height measurement by a stadiometer from Schorr Industries [Glen Burney, MD]; and head circumference measurement by a plasticized nonelastic measuring tape accurate to 1 mm). Weight for age Z-score (WAZ) and height for age Z-score were calculated according to the median value of the international reference population recommended by the National Center for Health Statistics/World Health Organization.16 Breastmilk analysis Breast milk samples (15 mL) were collected into sterile glass bottles by self-expression before the baby was nursed in the morning and stored at - 70C until analysis. Before taking measurements, we prepared defatted milk through a double centrifugal process. The breastmilk was centrifuged at low speed (500 g for 25 minutes) to settle out the suspended cellular components. Next, the cellular components were removed, and the decelled milk was centrifuged at high speed (5,000 g for 30 minutes) to separate it into defatted milk and fat content. The defatted milk was used for taking the measurements. Breastmilk TAC was measured using a Randox (Crumlin, County Antrim, United Kingdom) total antioxidant status kit in which 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfanate) (ABTS) is incubated with a peroxidase and H2O2 to produce the radical cation ABTS + . This has a stable blue-green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree that is proportional to their concentration. MDA levels were determined by the thiobarbitoric acid reaction with acid, which was extracted with n-butanol and measured spectrophotometrically at a wavelength of 523 nm and compared with a standard curve. Statistical analysis Statistical analysis was performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL) and included means and SDs or SEMs. Normal distribution of data was verified with the Kolmogorov–Smirnov test. Between-group comparisons were made by independent t test. The differences between variables before and after intervention were compared by
SYNBIOTIC SUPPLEMENTATION AND TAC IN BREASTMILK
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Table 1. Comparison of Anthropometric Measures and Daily Intake of Energy, Macronutrients, and Antioxidant Micronutrients Between the Two Study Groups Supplemented group (n = 37)
Mothers’ weight (kg) BMI (kg/m2) WAZ HAZ Head circumference (cm) Energy intake (kcal) Lipid (kcal) Carbohydrate (kcal) Protein (kcal) Vitamin A (lg) Vitamin E (mg) Vitamin C (mg) Zinc (mg) Selenium (mg)
Placebo group (n = 38)
Before
After
p value
Before
After
p value
70.6 – 12.3 28.7 – 4.2 1.2 – 0.85 0.37 – 0.81 40 – 1.2 2,260.6 – 82 553.5 – 19.3 1,460 – 17 247.1 – 16.4 549.6 – 99 8.9 – 1 100.2 – 15.6 9.1 – 2.2 0.08 – 0.007
70.8 – 12.4 28.8 – 4.3 1.1 – 0.92 0.34 – 0.83 41.2 – 1.3 2,296 – 88 537 – 21.7 1,492 – 15.1 267 – 17.8 600.9 – 107.2 9.2 – 1 110.5 – 18 9.6 – 2.5 0.087 – 0.007
0.26 0.2 0.34 0.37 0.01 0.4 0.57 0.35 0.22 0.4 0.33 0.45 0.2 0.51
70.3 – 8.9 28.6 – 2.9 1.2 – 0.8 0.39 – 0.67 40.7 – 1.2 2,312.3 – 92 600.5 – 28 1,451.2 – 20.4 260.8 – 12.4 469.9 – 80 7.9 – 1.2 113.2 – 26.5 9 – 2.3 0.079 – 0.009
68.7 – 9 28.2 – 2.8 0.97 – 0.73 0.38 – 0.98 41.8 – 1.2 1,985 – 100 458.6 – 22.6 1,322.8 – 15.4 203.6 – 10.4 533.3 – 108.5 7.5 – 1.2 116.4 – 25 7.8 – 2 0.072 – 0.007
0.01 0.01 0.03 0.52 0.01 0.023 0.008 0.03 0.042 0.57 0.38 0.92 0.14 0.42
Anthropometric measures are given as mean – SD values, and energy and nutrient measures are given as mean – SEM values. BMI, body mass index; HAZ, height for age Z-score; WAZ, weight for age Z-score.
paired t test. Multiple linear regressions using the backward technique were used to analyze the association of each potential factor with WAZ of infants. Potential factors like infants’ birth weight, maternal BMI, maternal energy intake, and breastmilk TAC and MDA levels were selected as independent variables, while the WAZ of infants was chosen as the dependent variable. All of the variables were entered in the model as continuous variables. All of the tests were done two-sided, and a p value of < 0.05 was considered statistically significant. Results The two groups were similar with respect to maternal age, type of delivery, and education and infants’ gender and birth weight at baseline ( p > 0.05). Anthropometric measures (mean – SD) and daily intake of energy, macronutrients, and antioxidant micronutrients (mean – SEM) between the two groups are compared in Table 1. There were no significant differences in the baseline measures between the supple-
FIG. 1. Total antioxidant capacity (TAC) levels in breastmilk before and after intervention in the two study groups. *p < 0.039 in comparison with before intervention, by paired t test.
mented and placebo groups. Although maternal weight and BMI increased slightly in the supplemented group, these two parameters decreased significantly in the placebo group at the end of the study ( p < 0.01). The WAZ of infants decreased in both treatment groups but was only significantly different in the placebo group. Moreover, the comparison of changes in infants’ WAZ showed a significant difference ( p < 0.044) between the two groups during the study. In contrast to the synbiotic group, a significant reduction was observed in daily mean energy intake and energy intake from lipid, carbohydrate, and protein after intervention in the placebo group. However, maternal vitamin A, E, and C, zinc, and selenium intake did not change significantly in the two groups. Figure 1 depicts the TAC levels in breastmilk before and after intervention in the two study groups. The differences in baseline levels were not statistically significant between the supplemented and placebo groups ( p > 0.05). The TAC of breastmilk increased significantly from 0.312 – 0.16 to 0.481 – 0.2 mmol/L in the supplemented group ( p < 0.039), whereas it decreased from 0.317 – 0.18 to 0.255 – 0.13 mmol/L
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FIG. 2. Malondialdehyde (MDA) levels in breastmilk before and after intervention in the two study groups. *p < 0.001 in comparison with before intervention, by paired t test.
in the placebo group ( p > 0.13). Also, the comparison of changes in the TAC of breastmilk showed a significant difference ( p < 0.02) between the two groups during the study. Figure 2 shows breastmilk MDA levels before and after intervention in both groups. Although the MDA level decreased slightly in the supplemented group from 1.62 – 0.69 to 1.6 – 0.95 lmol/L, it increased significantly in the placebo group from 1.71 – 0.86 to 2.16 – 0.277 lmol/L after the experimental period ( p < 0.001). Also, the comparison of changes in the MDA of breastmilk showed a significant difference ( p < 0.04) between the two groups during the study. In addition, after adjusting for maternal BMI, energy, macronutrients, and vitamin A, E, and C, zinc, and selenium intake, no significant association were observed between these factors and TAC and MDA levels of breastmilk (data not shown). Table 2 shows the association of different maternal factors with infants’ WAZ. After adjusting for infants’ birth weight, maternal BMI, maternal daily energy intake, and TAC and MDA levels in breastmilk, association of infants’ WAZ with birth weight and maternal BMI was significant in both groups before and after intervention ( p < 0.05).
Table 2. Association of Factors with Infants’ Weight for Age Z-Score b for association with infants’ WAZ Supplemented group (n = 37)
Placebo group (n = 38)
Factor
Before
After
Before
After
Infant birth weight Maternal BMI Daily energy intake Breastmilk level TAC MDA
0.9a
0.99a
0.45a
0.41a
0.4a 0.03
0.5a 0.01
0.19a 0.11
0.27a 0.06
0.3 0.1
0.45 0.16
0.4 0.22
0.28 0.13
a p < 0.05. BMI, body mass index; MDA, malondialdehyde; TAC, total antioxidant capacity; WAZ, weight for age Z-score.
Discussion Human milk antioxidant capacity value represents a complex mixture of numerous compounds with antioxidant activities functioning by different chemical reactions that collectively culminate in a stable food source for the breastfed infant.17 The mean antioxidant concentration of the breastmilk in all subjects at baseline was 0.314 – 0.17 mmol/L, which was relatively in agreement with results (0.375 – 0.092 mmol/L) previously reported from Portugal by Matos et al.,18 and it was significantly lower than reported values from Japanese8 (3.8 mmol/L) and Nigerian19 (1.1 mmol/L) human milk samples. This discrepancy in TAC values between studies may be due to maternal diet and supplementation with vitamins during pregnancy and lactation, time of year during which the milk samples were collected, ethnic group and the geographical area to which the mother belongs, and analyzing methods.19 Obviously, milk with a higher TAC value will reflect greater oxidative stability and a potentially greater protection for the breastfed infant from exposure to oxidative agents.7 Derived from our results, TAC of breastmilk tends to level off in the placebo group. This result was similar to the results of previous studies conducted by Zarban et al.20 and Ezaki et al.,8 which showed that colostrum in comparison with transitional and mature milk has more total antioxidant activity and that this activity decreases during the course of lactation, which can be a natural result of decline in antioxidant storage of the mothers. Based on the results of this study, synbiotic supplementation resulted in a significant increase in TAC and a slight decrease in MDA levels in the supplemented group. Because maternal antioxidant micronutrients intake did not change significantly in the supplemented and placebo groups after the experimental period, it seems that synbiotic supplementation might be the most important factor affecting the TAC and MDA levels in this study. During the past decade, several studies have supported the potential health benefits of probiotics, such as the improvement of gastrointestinal microbiota ecosystems, stimulation of the immunological system, anticarcinogenic activities, and reduction of oxidative stress.21 To the best of our knowledge, there has been no human study investigating the effect of synbiotic or probiotic supplementation on TAC or MDA of breastmilk. However, the results of the only
SYNBIOTIC SUPPLEMENTATION AND TAC IN BREASTMILK animal model study, which was conducted by Maragkoudakis et al.,22 showed that feeding goats on probiotic diet has no effect on TAC of goat milk. This discrepancy may be due to differences between species and different strains of supplements. Some other human studies have shown the direct effect of probiotic or synbiotic on blood TAC levels. Songisepp et al.23 reported the suppressive effect of Lactobacillus fermentum ME3 on cardiovascular disease–related oxidative stress indices in healthy subjects. In addition, Mikelsaar et al.15 indicated that the consumption of the synbiotic can suppress oxidative stress indices in healthy volunteers. Probiotics exert antioxidant activity through several mechanisms. For example, the expression of high levels of antioxidant enzymes by probiotic strains can neutralize oxidants directly in the intestinal tract. Moreover, the stimulation of the immune system reduces inflammation and prevents cytokine-induced oxidative stress. Also, the inhibition of intestinal pathogens reduces inflammation and its associated oxidative damage. Furthermore, probiotics also seem to enhance the absorption of macro- and micronutrients, including antioxidants. In addition, probiotics reduce postprandial lipids that are related to significant oxidative damage, which is responsible for several food-related pathologies.24–28 The results of our study demonstrating that infants’ weight gain in the supplemented group was significantly higher than that of the placebo group at the end of intervention may be due to direct and indirect effects of probiotics. However, there was no significant association between WAZ of infants and TAC and MDA levels of breastmilk in this study. Oveisi et al.29 showed that infants’ height at birth was directly correlated with antioxidant capacity of breastmilk, and they suggested that this result was a consequence of nutritional and food intake status in the pregnancy period. However, in another study, Aycicek et al.30 reported no significant differences in growth or anthropometric measurements between term infants 3–6 months of age who were fed breastmilk (which provides better antioxidant power than formula) or a cow’s milk modified formula. Obviously, more studies with longer follow-up periods are needed to clarify this association and its underlying mechanisms. Conclusions In general, TAC of breastmilk decreases with the passage of time, and our results showed that administration of synbiotics may be beneficial in improving the TAC level of breastmilk and preventing its decreases with time. However, further studies using different species of probiotic bacteria and longer duration of supplementation are necessary to make concise conclusions. Acknowledgments The authors wish to thank the Nutrition Research Center at Tabriz University of Medical Sciences for financial support. This study would not have been possible without the help and participation of the health centers. The results of this article are from L.N.’s PhD thesis. Disclosure Statement No competing financial interests exist.
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Address correspondence to: Reza Mahdavi, PhD Nutrition Research Center School of Public Health & Nutrition Tabriz University of Medical Sciences Golgasht Street Tabriz, Iran E-mail:
[email protected]
