Diet Supplementation During Early Lactation with Non-alcoholic Beer Increases the Antioxidant Properties of Breastmilk and Decreases the Oxidative Damage in Breastfeeding Mothers

Abstract

Background:

After delivery and birth, mothers and neonates are exposed to oxidative stress. We tested whether supplementing the diet of breastfeeding mothers with non-alcoholic beer, a product rich in antioxidants, could improve their oxidative status and the antioxidant content of their milk. A prospective trial begun on Day 2 postpartum was conducted in mother–infant dyads.

Subjects and Methods:

Sixty breastfeeding mothers and their infants were allocated to either a control group (n=30) on a free diet or a study group (n=30) on a free diet supplemented with 660 mL of non-alcoholic beer/day. The oxidative status of the mothers' breastmilk, plasma, and urine and the infant's urine was analyzed on Days 2 and 30 postpartum. The before–after difference was compared within and between the groups.

Results:

The increase in antioxidant capacity and coenzyme Q10 content in the breastmilk of the study group at Day 30 was higher than in that of the control group (p<0.001). There was also a change in the oxidative status of the mothers' plasma in the supplemented group regarding the control group; higher values of total antioxidant capacity (p<0.05) and lower levels of 8-hydroxydeoxyguanosine (p<0.05), indicative of DNA oxidative damage, were found. These results indicate a positive effect of non-alcoholic beer supplementation on oxidative stress in mothers. However, no difference in oxidant markers was found in the infant's urine.

Conclusions:

The consumption of non-alcoholic beer appears to enhance the antioxidant capacity of breastmilk and decrease oxidative damage in breastfeeding mothers.

Introduction

Human milk is recognized as the ideal food for the human infant. It contains numerous components that protect the infant from infection and, presumably, diseases in adulthood.¹ In addition to the numerous clinical benefits of breastfeeding, human milk has bioactive components that protect newborns from the relative hyperoxic challenge resulting from their transition to an environment far richer in oxygen than intrauterine life. How the full-term newborn can cope with this excess oxidation is not yet understood,² as the antioxidant defenses are immature during the neonatal period.³ This oxidative stress begins to diminish at 72 hours after birth⁴ but probably remains until the sixth month.²

This is likely why there is a great amount of antioxidant substances in breastmilk; these antioxidants include enzymatic substances (catalase, superoxide dismutase, and glutathione peroxidase) and nonenzymatic substances, such as vitamins (α-tocopherol, carotenoids, ascorbic acid), proteins (lactoferrin), minerals (selenium), and many others that are yet not known. Recently, specific lipophilic antioxidants such as coenzyme Q10 (CoQ₁₀) have received a great deal of interest because they directly correlate with the antioxidant capacity of the milk.⁵ Diverse studies have shown that human milk can suppress oxidative stress and oxidative damage in newborn infants more effectively than formula.^6,7 Some of the benefits of breastfeeding, like specific defense mechanisms needed by the infant, have been recently attributed to this antioxidant content.⁸

Furthermore, the antioxidant status of breastmilk may be influenced by the mother's antioxidant status, which in turn can depend on her diet. Previous studies have reported the effects of maternal diet and vitamin intake on human milk, showing that a higher intake of certain antioxidant nutrients results in a higher concentration of antioxidants in milk.^9,10

Beer is a popular drink that can stimulate prolactin secretion, thus enhancing lactogenesis,¹¹ and traditionally it has been recommended for breastfeeding mothers with the goal of increasing the milk production. Although beer has been demonstrated to be a rich source of antioxidants, mainly polyphenols and B vitamins,¹² its alcohol content, although low, precludes its inclusion in the diet of lactating women. It has recently been shown that non-alcoholic beer retains much of the antioxidant content attributed to polyphenols without the adverse effects of an alcoholic drink.^13,14 However, little is known about the influence of non-alcoholic beer consumption in lactating women and in their breastmilk.

We hypothesized that regular consumption of non-alcoholic beer can increase the antioxidant content of human milk. The aim of the present study was to test whether diet supplementation with non-alcoholic beer could benefit the antioxidant status of lactating mothers and their infants. We measured before and after diet supplementation the antioxidant capacity and two types of antioxidants (one hydrophilic [polyphenols] and one lipophilic [CoQ₁₀]) in breastmilk and the biomarkers of oxidative stress in lipids, proteins, and DNA in mother–infant dyads. The results were compared with those of a group without non-alcoholic beer supplementation.

Subjects and Methods

Participants

A prospective clinical study was performed during a 6-month period enrolling women consecutively after delivery in the Maternity Clinic of our hospital. The inclusion criteria for participants were as follows: (1) postpartum mothers without any acute or chronic illness, who were not receiving medications or vitamin supplements, nonsmokers, and who were willing to breastfeed and accept 1 month of follow-up; (2) vaginal delivery at term without complications; (3) singleton-birth newborns with no acute perinatal or chronic postnatal disease, who did not need oxygen at birth or after; and (4) exclusively breastfeeding. The exclusion criteria were as follows: (1) mothers on restricted diets due to allergies, intolerances, or personal beliefs; (2) mothers who ceased exclusively breastfeeding during the study period; and (3) failing to attend the follow-up visits. All of the participating mothers were required to avoid consuming polyphenol-rich foods such as coffee, tea, chocolate, or vitamin supplements during the study period.

The 60 women included were divided into two groups according to the supplementation of non-alcoholic beer (0.0% alcohol): a control group of lactating women on a free diet and a study group of lactating women who supplemented their diet with 660 mL of non-alcoholic beer daily (330 twice a day) during the study period that started on Day 2 postpartum and lasted up to 30 days thereafter. The allocation to the two groups was consecutive while respecting individual preferences for beer. If a woman did not like beer, she was allocated in the control group, and the next consecutive woman was assigned to the beer group. The beer of the same type and brand was freely provided and delivered to the mother's domicile from Day 2 to Day 30, covering all study period.

Follow-up, breastfeeding support, baby health supervision, and collection of biological samples all took place in the Maternity Clinic (first visit or baseline at Day 2) and at the associated Primary Care Pediatric and Breastfeeding Clinic. Mothers and newborns were scheduled for at least two visits in the follow-up (15 and 30 days). Biological samples were obtained at baseline and at the end of study period (Day 30) to assess the effect of regular supplementation of beer. Other visits were based upon individual needs until breastfeeding was well established. Sociodemographic, clinical, and anthropometric data were collected from the mother and newborns. The prepregnancy weight and height of the participating women were obtained from their clinical record. Because diet may affect the body's antioxidant content, the women were asked to complete a 3-day dietary record of all the food and beverages consumed (including one Sunday), and a 24-hour recall with composition and portion sizes was recorded at each visit. Energy and nutrient intakes, both at baseline and during the intervention, were calculated using the validated software Food and Health 0698.046 (BiatASDE General Médica Farmacéutica, Valencia, Spain).

The study protocol was approved by the Ethics Committee of the Health Department, Dr. Peset University Hospital, Valencia. All of the participants signed a written informed consent.

Biological samples

Breastmilk, mothers' blood and urine, and infants' urine were obtained between 8 and 10 a.m. after overnight fasting on Day 2 postpartum (in the hospital) and on Day 30 postpartum (in the outpatient Breastfeeding Clinic). The plasma was separated by centrifugation at 1,500 g for 10 minutes. The infants' urine samples were collected using a single-use collecting device, a Hollister U-Bag^® (Hollister Inc., Kirksville, MO). Breastmilk was extracted using a Medela (Baar, Switzerland) Symphony^® double pump, transported on ice to the laboratory, and centrifuged at low speed (500 g for 15 minutes) to separate the cellular components. It was then centrifuged at high speed (5,000 g for 25 minutes) to separate the defatted milk, which was used for the measurements. All of the samples (plasma, milk, and urine) were frozen at −80°C in the first hour after collection and stored in Eppendorf tubes until they were processed. The samples were analyzed directly after thawing. The researchers were blinded to the group allocation of the samples.

Analytical methods

Biochemical standard determinations were performed at the Clinical Laboratory of Dr. Peset University Hospital using automated methods (Aeroset System^® and Architect c8000^® [Abbott Clinical Chemistry, Wiesbaden, Germany]).

The total antioxidant capacity of the mothers' milk and plasma was measured using the 2,2′-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging activity analysis method according to Brand-Williams et al.¹⁵ The total amount of polyphenols of the breastmilk and mothers' plasma was determined using the Folin–Ciocalteau method,¹⁶ based on the oxidation of polyphenols with a mixture of phosphomolybdate and phosphotungstate used in the colorimetric assay. The CoQ₁₀ levels in the breastmilk were determined by the method of high-pressure liquid chromatography described by Tang et al.¹⁷

Oxidative damage was determined in samples of the mothers' plasma and the mothers' and infants' urine. The level of malondialdehyde, a lipid oxidation marker, was measured in the plasma and urine at 532 nm following a reaction with thiobarbituric acid and reverse-phase isocratic high-performance liquid chromatography separation according to the protocol from Mateos et al.¹⁸ To evaluate protein oxidation in the mothers' plasma, we measured the carbonyl groups released during the incubation of the plasma with 2,4-dinitrophenylhydrazine using the spectrophotometric method reported by Levine et al.¹⁹ The concentration of 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, was used to assess DNA damage because 8-OHdG is produced upon DNA repair. We have determined the 8-OHdG concentration by means of a commercially available competitive enzyme-linked immunosorbent assay kit (8-OHdG EIA; Cayman Chemical Co., Ann Arbor, MI) following the manufacturer's instructions.²⁰

Statistical analysis

The normality of each variable was determined by the Kolmogorov test. Non-normal variables were analyzed after logarithmic transformation. A paired Student's t test was used to compare the human milk at baseline (Day 2, colostrum) with the human milk at the end of the study (Day 30, mature milk) and to compare the oxidative stress parameters in the mothers' plasma and urine and in the infants' urine at the initiation (Day 2) and at the end (Day 30) of the study. The before–after difference in the intervention group was compared with that in the control group. The mean changes and SDs are presented. Next, an independent two-tailed t test was used to compare the mean change in oxidative status between the intervention and control groups. Differences were considered statistically significant at a value of p<0.05. The statistical analysis was conducted with the SPSS^® for Windows program (version 17; SPSS Inc., Chicago, IL).

Results

Characteristics of the participants

The general characteristics of the study participants at recruitment are given in Table 1. No significant differences were noted at baseline between the groups of mothers based on their anthropometric or biochemical characteristics, the gestational age and birth weight of their newborns, or the macro- or micronutrient content of their diet.

Table 1.

Participants' Clinical and Biochemical Data

	Control group (n=30)	Study group (n=30)	p value
Age (years)	31±3	30±5	0.229
Prepregnancy weight (kg)	63±7	62±7	0.323
Predelivery weight (kg)	73±6	74±9	0.669
Mother's height (cm)	162±4	163±6	0.736
Newborn birth weight (g)	3,320±266	3,220±443	0.239
Serum total cholesterol (mmol/L)	6.06±1.11	5.77±0.98	0.261
Serum HDL-cholesterol (mmol/L)	1.55±0.28	1.47±0.26	0.164
Serum triglycerides (mmol/L)	2.01±0.71	1.80±0.48	0.163
Serum uric acid (μmol/L)	256±59	250±42	0.871
Serum total proteins (g/L)	59±3	58±4	0.137
Serum iron (μmol/L)	9.7±3.9	8.8±3.7	0.373
Serum glucose (mmol/L)	4.1±0.5	4.2±0.5	0.388
Serum creatinine (μmol/L)	48±6	49±7	0.861
Energy intake (kJ/day)	8,828±1673	8,978±1410	0.475
Protein intake (g/day)	96±21	102±17	0.172
Lipid intake (g/day)	82±22	85±20	0.467
Vitamin C intake (mg/day)	107±42	98±34	0.391
Vitamin E intake (mg/day)	11±5	9±6	0.260
Iron intake (mg/day)	17±4	16±5	0.362

Data are mean±SD values.

HDL, high-density lipoprotein.

Breastmilk

The results of the tests measuring the antioxidant capacity, total polyphenolic content, and CoQ₁₀ levels in colostrum and mature milk are shown in Table 2. In the control group, colostrum showed a greater capacity to reduce the stable radical DPPH content in comparison with mature milk. This decrease in the antioxidant capacity from colostrum to mature milk was not observed in the study group; the antioxidant capacity of milk from mothers who supplemented their diet with non-alcoholic beer did not differ significantly from Day 2 to Day 30 and was significantly higher than that of mothers in the control group. The content of polyphenols was significantly higher in colostrum and decreased as lactation progressed, with no differences between the two groups. Furthermore, the CoQ₁₀ levels were significantly higher in mature milk compared with colostrum only in the mothers in the study group. The difference between groups in change over time was significant (p<0.05) for both the DPPH test and the CoQ₁₀ levels. Supplemented mothers had a significant change in the values for these two variables relative to control mothers.

Table 2.

Changes in Antioxidant Capacity, Total Polyphenolic Content, and Coenzyme Q10 Content in Breastmilk During the Study Period

	Control group (n=30)			Study group (n=30)
	Colostrum	Mature milk	Change	Colostrum	Mature milk	Change
DPPH (μmol/100 mL)	30±8	26±7^a	−5±6	28±10	29±8^c	0.7±6^d
Total polyphenols (μg/mL)	1.2±0.3	0.7±0.3^b	−0.4±0.3	1.2±0.3	0.6±0.2^b	−0.5±0.3
Coenzyme Q10 (μmol/L)	0.7±0.2	0.6±0.2	−0.02±0.2	0.6±0.3	0.8±0.2^a,c	0.2±0.3^e

Data are mean±SD values.

DPPH, 2,2′-diphenyl-1-picrylhydrazyl free radical.

p<0.01, ^bp<0.0001 versus colostrum in the same group.

p<0.05 versus mature milk in the control group.

p<0.05, ^ep<0.001 versus change in the control group.

Mothers' plasma

Free radical scavenging activity in the mothers' plasma, when measured with a DPPH assay at Day 30, was higher relative to Day 2 in the mothers of the study group (Table 3). Furthermore, the total polyphenol content decreased significantly at Day 30 compared with Day 2 postpartum only in the control group. The difference in the mean changes between the groups was significant for the two variables; the mothers who consumed non-alcoholic beer maintained higher levels of polyphenols and antioxidant activity than the mothers in the control group during the time of study.

Table 3.

Changes in Oxidative Status Measured in the Mothers' Plasma and Urine and the Infants' Urine Between Day 2 (Baseline) and Day 30 (End Point)

	Control group (n=30)			Study group (n=30)
	Day 2	Day 30	Change	Day 2	Day 30	Change
Mother's plasma
DPPH (μmol/L)	238±59	223±54	−15±43	243±66	268±82^b,d	25±96^f
Total polyphenols (μg/mL)	105±27	92±30^b	−13±24	103±23	104±20^d	1.1±20^f
CG (nmol/mg of protein)	1.5±1.0	1.3±0.8	−0.2±0.4	1.9±1.5	1.6±1.2^a	−0.3±0.6
MDA (nmol/mL)	1.4±0.4	1.3±0.5	−0.2±0.4	1.4±0.5	1.3±0.4^a	−0.1±0.2
8-OHdG (ng/mL)	3.4±2.3	3.9±3.3	0.5±3.4	3.4±2.7	1.9±1.3^b,e	−1.5±2.4^f
Mother's urine
CG (nmol/mg of protein)	0.06±0.04	0.05±0.03	−0.01±0.05	0.05±0.03	0.04±0.02^a	−0.01±0.06
MDA (nmol/mL)	0.8±0.6	0.7±0.6	−0.1±0.6	0.8±0.6	0.7±0.4	−0.1±0.5
8-OHdG (ng/mL)	23±10	21±5	−1.8±14	22±7	20±6	−1.7±9
Infant's urine
CG (nmol/mg of protein)	0.9±0.5	0.8±0.6	−0.1±0.7	1.1±0.6	1.1±0.8	−0.01±0.9
MDA (nmol/mL)	0.6±0.2	0.2±0.1^c	−0.4±0.3	0.5±0.3	0.2±0.1^c	−0.3±0.3
8-OHdG (ng/mL)	22±6	19±5^c	−2.6±2.4	22±6	19±4^c	−3.1±3.5

Data are mean±SD values.

p<0.05, ^bp<0.01, ^cp<0.0001 versus colostrum in the same group.

p<0.05, ^ep<0.01 versus end point (Day 30) in the control group.

p<0.05 versus change in the control group.

CG, carbonyl groups; DPPH, 2,2′-diphenyl-1-picrylhydrazyl free radical; MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

Significant decreases were observed in the levels of biomarkers measuring oxidative damage to lipids (malondialdehyde), proteins (carbonyl groups), and DNA (8-OHdG) in the plasma of mothers in the study group. However, these decreases were not observed in the mothers in the control group. When the mean changes were compared, the change in 8OHdG content remained significant; the mothers who supplemented their diet with non-alcoholic beer showed less damage to DNA reflected in their plasma on Day 30 relative to mothers in the control group.

Mothers' urine

A slight decrease in the biomarkers for oxidative damage was observed in both groups from Day 2 postpartum to Day 30 postpartum (Table 3). A significant decrease was only observed in the biomarker for protein damage (carbonyl groups) in the mothers in the study group. However, this change was not significant compared with the change in the control group.

Infants' urine

In the infants' urine, a significant decrease in the markers for lipid and DNA damage from birth to 30 days was found and was not different between the groups (Table 3).

Discussion

The present study was carried out to investigate the influence of supplementing the diet of lactating women with a product rich in antioxidants, such as non-alcoholic beer. In our research, the most significant result is that the intake of non-alcoholic beer can enhance the total antioxidant capacity of breastmilk and diminish the biomarkers for oxidative damage in the mother.

In agreement with several previous studies,^21,22 our study shows that the total antioxidant activity in human milk is highest immediately after birth in the colostrum and decreases in mature milk. Colostrum is the first secretion of the mammary gland, lasting 3–5 days; its composition considerably differs from milk in later stages.²³ Thus, colostrum should offer the most critical protection against peroxidation because oxidative damage can be much more severe during the first days after birth. Growing evidence indicates that oxidative stress occurs during delivery and the fetal-to-neonatal transition. In fact, a high degree of lipid peroxidation has been found in cord blood, which is indicative of oxidative stress during the birth process.²⁴ Susceptible newborns, especially preterm or diseased infants, are more vulnerable to oxidative stress because of the inefficiency of their antioxidant defense system or an increase in free radical production.²⁵ In these infants, breastfeeding is extremely important and useful in protecting against oxidative stress.⁶ However, even a full-term newborn has an increased oxidative status.² The high antioxidant content in human milk may aid the infants' capacity to deal with the oxidative damage.

It is noteworthy that there were several important findings in our study about the milk's antioxidant status. First, the total antioxidant capacity of breastmilk was maintained during the course of lactation without declining in the mothers who supplemented their diet with non-alcoholic beer. In this sense, we can prevent the loss of antioxidant capacity in breastmilk by including foods rich in exogenous antioxidants in the mother's diet.²⁶ Moreover, there is evidence that the antioxidant protection that breastmilk provides in the first years may be related to the diminished risk of several degenerative diseases.²⁷ Indeed, infants' and adults' health is closely related not only to the nutritional components and quality of the mother's milk but also to its antioxidant level.

Total antioxidant capacity includes the activity of all the antioxidant systems present in the milk sample such as vitamins, enzymatic radical scavenger systems, and yet unknown antioxidants. Our results indicate that powerful antioxidants present in beer such as phenolic compounds decrease in mature milk independently of non-alcoholic beer supplementation. Hence, other antioxidants can account for the high antioxidant capacity in human milk found in the present study.²⁸ Among them is CoQ_10, one of the most important lipohilic antioxidants,²⁹ the only one endogenously synthesized and exceeding other antioxidants both in quantity and in efficiency. In this way, an interesting and novel finding of our study is the stable or even slightly increasing CoQ₁₀ levels in the breastmilk of mothers who supplemented their diet with non-alcoholic beer. Moreover, levels of CoQ₁₀ in human milk directly correlate with the antioxidant capacity of the milk.⁵ The importance of this compound was recently highlighted in different experimental studies indicating a stimulation of the immune response with exogenous administration of CoQ₁₀.³⁰

A decrease in the total antioxidant capacity of the mothers' plasma from birth to Day 30 postpartum is observed, likely because less antioxidant activity is needed after the stress of delivery subsides. Additionally, the antioxidant reserves could be depleted after their consumption for counteracting oxidative stress. We have observed that the decrease is lower in the mothers in the study group. The significant reduction in markers for oxidative damage to proteins, lipids, and DNA in the plasma of these mothers reinforces the hypothesis that non-alcoholic beer intake increases the amount of antioxidants. Moreover, our hypothesis is further supported by the decrease in the polyphenol content in the plasma of the mothers who did not supplement their diet with non-alcoholic beer, which was not observed in the plasma of mothers who did. Beer is especially rich in polyphenols, which can account for the observed increase in antioxidant activity. However, this difference was not observed in milk; there was no increase in the polyphenolic content of breastmilk in the mothers who supplemented their diet with non-alcoholic beer. The reason why the increase in levels of plasma polyphenols is not also apparent in breastmilk will have to be studied further, leading to more in-depth research on the transfer from plasma to breastmilk or the secretion of different antioxidant substances by the mammary glands.

To avoid confounding effects in the characteristics of milk,³¹ we controlled for differences among the groups. The absence of significant differences after the comparison of somatometric, sociodemographic, dietary intake, and biochemical variables in both groups of mothers at baseline supports the thesis that the results should be attributed to the supplement given to the mothers in the study group.

We tested the possibility that antioxidant defenses were enhanced in the newborns of the mothers who supplemented their diets with non-alcoholic beer. To this goal we have used urine noninvasive tests. The results of our study do not demonstrate benefits of this supplementation to the infants. In both groups of infants, an improvement in oxidative damage was observed on Day 30, whether their mothers consumed non-alcoholic beer or not. The great antioxidant power naturally present in breastmilk might mask some differences. Likewise, there was no difference with supplementation in the biomarkers measured in mothers' urine. It is possible that longer periods are required to observe significant changes because kidney function differs from other life periods during the puerperium and newborn intervals.

We know the limitations of our study: the sample is limited in size and duration. We have studied colostrum and mature milk but not transitional milk. Moreover, oxidative status was determined in the baby only by biomarkers in the urine; we cannot inference their plasma status. However, its strength is the longitudinal design with simultaneous sampling of the mothers' breastmilk and blood and both the mothers' and infants' urine and the strict recording of the maternal diet. To our knowledge, there are no studies addressing these aspects. Our results deserve further research to investigate the effect of different foods on the characteristics of breastmilk and on the oxidative status of the mother and her infant.

Conclusions

Our results indicate that (1) the decrease in total antioxidant capacity observed from colostrum to mature milk was improved when the mother's diet was supplemented with non-alcoholic beer, (2) the concentration of CoQ₁₀ does not decrease with time in the milk of mothers who consumed non-alcoholic beer, (3) consumption of non-alcoholic beer produces a decrease in oxidative damage to biomolecules, mainly DNA, as reflected in the mother's plasma by a decrease in 8OH-dG levels, (4) the biomarkers of oxidative status in the urine of both the mother and infant were not affected by the supplementation, and (5) supplementation of the mother's diet with non-alcoholic beer may enhance the antioxidant properties of human milk and help mothers to decrease their oxidative status after delivery.

Footnotes

Acknowledgments

We thank the nurses Luisa Gallardo, Paz Rodriguez Castellano, and Purificación Rodos-Cordón for their invaluable support. This investigation was partially funded by Foundation “Cerveza y Salud.”

Disclosure Statement

No competing financial interests exist.

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