What Is Known
* Oligosaccharides provide prebiotic effects when added to infant's formula.
* Microbiota in infants’ gut is associated with long-term health consequences.
What Is New
* Medically graded honey has prebiotic effect when added to infants’ formula.
* The prebiotic effect of honey is dose dependent.
* Higher doses of honey are associated with enhanced head growth of premature infants.
Preterm infants develop a portfolio of intestinal microbiota that differs largely from full terms regardless of being fed breast milk or milk formula. Enterobacteriaceae, enterococci, clostridia, staphylococci, and yeasts are the predominant intestinal organisms in preterm infants, whereas bifidobacteria are significantly less common. These differences could be attributed to decreased exposure to the maternal microbiota, increased exposure to hospital organisms, use of antibiotics, and delayed enteral feeding (1).
The absence of protective microbiota in the intestine of premature infants is yet another challenge to their growth; the gastrointestinal barrier function, gut motility, mucosal immunity, and the digestive and absorptive capacity are all underdeveloped in this population, thereby increasing their risk of nosocomial infections and necrotizing enterocolitis (NEC) (1). Therefore, modifying the intestinal microbiota to more closely resemble that of term breast-fed infants has been a focus of research (2). Interest in bifidogenic diets has increased after the findings that demonstrated the addition of bifidobacteria to infant formula could reduce the risk and severity of NEC (3) and the incidence of allergic diseases in preterm infants (4).
Honey contains fructose, glucose, and to fructo-oligosaccharides (FOS) that can serve as prebiotics. The consumption of honey is not encouraged for infants younger than 12 months of age because of the risk to develop infantile botulism in the rare event that it is contaminated with the spores of Clostridium botulinum(5). Such risk can be avoided when using medical-grade, spore-free honey that has been shown in multiple reports to efficaciously and safely treat neonatal wounds and infantile pressure ulcers (6). It is, however, unknown whether honey can function as a prebiotic agent and is able to change the intestinal microbiota in premature infants.
In the present study, we hypothesized that the introduction of medical-grade, spore-free honey to infants’ milk formula would produce a bifidogenic effect and stimulate the immune response of premature infants. We aimed in this prospective double-masked randomized trial to assess the effect of enteral honey on the intestinal microbiota and somatic growth of preterm infants, determine its effect on the immune system of preterm infants, and determine the optimal dose to achieve these effects, if any.
PATIENTS AND METHODS
This pilot prospective randomized trial was approved by the institutional review board at Cairo University Children's Hospital. Parental consent was obtained before enrollment of subjects. Subjects included in this trial fulfilled the following inclusion criteria born prematurely with gestational age ≤34 weeks, their postnatal age was >3 days, parental wish to use milk formula with no intention to use maternal breast milk or to breast-feed, and enteral feeds were started and well tolerated but the feeding volume did not reach half of the goal feed. It is important to note that donor's breast milk banks are not available in the country of Egypt. Thus, the only feeding option for nonlactating mothers is the use of cow-based milk formula. In the neonatal intensive care unit, all premature formula-fed infants receive the same type of formula that has no prebiotic ingredient in it (Danone, Barcelona, Spain). Infants were excluded from the study if they had maternal conditions suggestive of chorioamnionitis or peripartum infections; major chromosomal abnormalities or major congenital anomalies of the cardiovascular, pulmonary, or central nervous system, including neuromuscular disorders and neural tube defects; intestinal atresia, tracheoesophageal fistulas, omphalocele, gastroschisis, and other major congenital GI anomalies; and sepsis, either before or during enrollment. None of the recruited subjects should receive antimicrobials at the time of enrollment.
Maternal and perinatal history was collected for each infant. Gestational age was estimated using the last menstrual date, ultrasonography, and the Ballard scoring system performed in the first day of life (7). Anthropometric measurements were recorded for all subjects at recruitment and weekly throughout the study. Birth weight was measured just after delivery on a calibrated scale after daily zero adjustment (8). The length of the newborn was measured with the subject in the supine position and the head in contact with a fixed board while the angles were gently held to extend the legs (9). Head circumference was determined by applying a measurement tape around the head over the glabella and supraorbital ridges anteriorly and the occiput so that the maximal circumference can be recorded (10).
Using opaque sealed envelopes, subjects were randomized into 4 intervention groups: group A received 5 g of bee honey suspension daily for 2 weeks, group B received 10 g of bee honey suspension daily for 2 weeks, group C received 15 g of bee honey suspension daily for 2 weeks, and group D included control subjects who received regular feeding without any modifications. Honey was added to a bottle of milk formula by one of the investigators. None of the managing team was aware of the group assignment of the infant. Pathology and microbiology personnel were not aware of the group assignment at the time of assays.
Infants were fed cow's protein–based premature formula (80 cal/100 mL) as requested by parents. Feeds were started according to unit protocol every 2 hours and advanced until full feed goal calories of 120 cal/kg reached around 2 to 3 weeks of life. While advancing on enteral feeds, infants received parenteral nutrition.
Honey was added to milk formula according to the randomization arm the infant belonged to. Honey was added only when infants’ enteral feeding was advanced to half of the full feeding goal.
Unprocessed clover honey was used after being sterilized by the Egyptian Atomic Energy Authority via 25 kGy Cobalt-60 gamma radiation (11). This dose should not change the physical, chemical, or mineral contents of the honey. It may alter moisture content, vitamins C and E, and hydroxymethylfurfural compositions (12). After sterilization, the honey was further examined by the Egyptian Ministry of Health to confirm absence of C botulinum spores.
Methodologic details for blood counts and cytokines and for stool cultures and quantitative real-time polymerase chain reaction (PCR) are described in the Supplemental Digital Content, Methods, http://links.lww.com/MPG/A973.
Comparison of numerical variables between the study groups was done using Student t test for independent samples. Paired t test was used to compare repeated measures within the same group. Analysis of variance test was used for multiple comparisons. For comparing categorical data, Chi square (χ2) test was performed. Exact test was used instead when the expected frequency is <5. P values <0.05 were considered statistically significant.
Forty preterm newborns were enrolled in the study with the intervention started during the second week of life in 22 infants and during the third week of life in 18 infants. The characteristics of the study population are presented in Table 1. As per unit policy, all enrolled infants received empiric antibiotics for 72 hours. None of the infants received any subsequent antibiotics beyond 72 hours throughout the study period. None of them were diagnosed with sepsis or NEC during or after enrollment. At enrollment, the 4 groups did not differ in weight and the administration of honey was associated with significant weight increase. Compared to control group, groups B and C had significant increase in weight after 1 week (P < 0.0001), and groups A, B, and C had significant weight increase by 2 weeks (P < 0.0001). No significant changes in length during the study except for group A at 2 weeks had greater increase compared to controls (P = 0.009). Head circumference increased significantly in groups B and C when compared to group D after 2 weeks (P = 0.0056) (Fig. 1).
CD4 and CD8 concentrations (ng/dL) did not differ among groups at enrollment or after 2 weeks of randomization (Table 2).
Enterobacteriaceae colony count by culture was significantly greater in group A before intervention. After 1 week of intervention, a significant decrease in colony count was recognized in the 3 intervention groups A, B, and C (P < 0.0001). After 2 weeks, only groups A and B continued to have less growth in colonies compared to group D (P = 0.002) (Fig. 2).
Bifidobacterium bifidum colony count by culture did not differ among the 4 groups at baseline. After 1 week, colony counts in groups A, B, and C did not differ from group D. After 2 weeks, all 3 groups (A, B, and C) had significantly greater colony growth compared to group D (P = 0.002). Lactobacilli mean colony number by culture was less in groups B and C compared to groups A and D (P = 0.015). After 1 week, the number of colonies significantly increased in group B only (P = 0.0007). After 2 weeks, the colony count increased in group B only (P < 0.0001) (Fig. 2).
Applying real-time PCR, for B bifidum, the copy numbers were similar in the 4 groups (A, B, C, and D) at enrollment (8.6 ± 11.1 vs 33.7 ± 46.6 vs 8.4 ± 2.6 vs 14 ± 8.3, respectively; P = 0.08). The increase in copy number was significant in groups B and C when compared to group D after 1 week (59.6 ± 67.6 and 65.9 ± 7.3 vs 7.3 ± 11.4, respectively; P = 0.0001). After 2 weeks, the increase in copy number was significant only in group C when compared to group D (1359 ± 983.8 vs 0.8 ± 2.9; P < 0.0001). For lactobacilli, there was no difference among groups at enrollment with increased growth in groups B and C when compared to group D (112.6 ± 142.9 and 53.3 ± 26.1 vs −4.1 ± 8.5, respectively; P = 0.013) after 1 week and in group C compared to group D after 2 weeks (1718.4 ± 1434 vs −6.4 ± 7.6, P < 0.0001).
The present study demonstrated the introduction of medically graded honey to cow's milk formula was associated with changes in microbiota of stool in premature infants. Infants who received honey had decreased colonization with Enterobacteriaceae and increased colonization with B bifidum and lactobacilli. Consumption of honey was associated with enhanced growth evidenced in increased weight gain and head circumference. There was no difference in CD4 and CD8 counts between the intervention and control groups.
Infants who received honey gained more weight than controls that can be explained by the increased caloric intake in association with honey consumption. Some trials demonstrated increased weight gain in infants receiving prebiotics (13), whereas others did not show any benefit of adding prebiotic combinations of galacto-oligosaccharides (GOS) and FOS or FOS alone on weight gain (14). The present study showed that supplementing preterm formula with honey had no significant effect of on the linear growth of infants. This finding was similar to a recent meta-analysis that showed no effect on the linear growth when GOS/FOS were added to preterm formula; however, an effect was present when FOS was used alone (14). Our study also showed that the intervention groups had a greater increase in head circumference than the control group. Previous studies, however, did not show any effect of prebiotics on head growth (14,15). We speculate that, in addition to the prebiotic properties, honey contains multiple trace elements that could have nootropic, synaptic plasticity, and antioxidants effects and may affect brain growth (16). Of note, the study was not powered to test this outcome. Therefore, future studies are needed to examine whether honey has an effect on brain growth of premature infants.
B bifidum and lactobacilli colonies in the stool of premature infants, as detected by culture and quantitative PCR were significantly increased with honey supplementation. This effect was even more pronounced in the groups of infants who received 10 and 15 g/day but was not significant in infants who received 5 g/day (group A). Thus, the increased dose of honey is more efficacious in establishing this prebiotic effect. These findings are comparable to previously on the use of a mixture of GOS/FOS that showed a dose-dependent prebiotic effect (17–20). In addition, a previous study reported the addition of honey to enhance the growth, the activity, and the viability of commercial strains of bifidobacteria; such effect was not generalized and was only strain specific (21). The synergic effect of honey on bifidobacteria could be partially reproduced when using the carbohydrate components of honey. Therefore, the prebiotic properties of honey are not restricted to its saccharide components. Further studies are needed to explore which component(s) of honey has the most efficacious prebiotic property.
Enterobacteriaceae colony count decreased in all of the 3 groups of infants who received honey within 1 week. After 2 weeks, this favorable effect, however, did not continue in infants who received 15 g/day. Therefore, it seems this favorable effect of honey to decrease Enterobacter was dose dependent and was best achieved with 5 and 10 g/day of honey. In fact, the dose of 15 g/day increased the growth of Enterobacter significantly after 2 weeks. It seems that the dose of 10 g/day was the optimal dose in the present study; it decreased the growth of Enterobacter and had a positive bifidogenic effect.
The present study is the first to examine the role of honey on the colonization of gut flora in human neonates. The study demonstrated the feasibility of using medical-graded sterilized honey to neonates. The present study is, however, not without limitations. Similar to other pilot studies, it has the inherit limitation of small sample size that makes it difficult to detect significant relationships. Although managing physicians were unaware of subjects’ group assignments, investigators could not ascertain the practicality of masking the bedside nurses who would notice the change in the color of honey-treated milk. Stool culture and quantitative PCR were used to characterize microbiota; however, more information could have been obtained with sequencing and metabolome analysis. The study was conducted on hospitalized preterm infants; therefore, the findings cannot be extrapolated to other populations with different gestational or postnatal age. The microbiota of subjects’ mothers were not mapped or considered during the analysis, although they may have been an independent variable affecting outcomes. Infants tolerated the honey-treated milk formula without noticeable complications. Nevertheless, it is important to emphasize the pilot nature of the study with its primary goal to test feasibility without any safety claims. Of note, honey contains trace amounts of manganese, copper, zinc, and alkaloids. The effect of regular consumption of these elements on preterm infants needs to be studied.
In conclusion, honey supplementation was associated with changes in physical growth and colonic microbiota of preterm formula-fed infants. Further studies are needed to examine the sustainability of the prebiotic effect and the associated long-term outcomes. Studies are also needed to examine the reproducibility of these effects in breast-feeding infants.
1. Underwood MA, Salzman NH, Bennett SH, et al. A randomized placebo-controlled comparison of 2 prebiotic/probiotic combinations in preterm infants: impact on weight gain, intestinal microbiota, and fecal short-chain fatty acids. J Pediatr Gastroenterol Nutr
2. Mohan R, Koebnick C, Schildt J, et al. Effects of Bifidobacterium lactis
Bb12 supplementation on intestinal microbiota of preterm infants: a double-blind, placebo-controlled, randomized study. J Clin Microbiol
3. Lin H, Su B, Chen A, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics
4. Hanson LÅ, Korotkova M, Telemo E. Breast-feeding, infant formulas, and the immune system. Ann Allergy Asthma Immunol
5. Tanzi MG, Gabay MP. Association between honey consumption and infant botulism. Pharmacotherapy
6. Abdel-Rahman MA, Tayseer N. Not giving honey to infants, a recommendation that should be reevaluated. J Am Apither Soc
7. Ballard JL, Khoury JC, Wedig K, et al. New Ballard score, expanded to include extremely premature infants. J Pediatr
8. Pursely S, Cloherty J. Cloherty J, Stark A. Identifying the high-risk newborn and evaluating gestational age, prematurity, postmaturity, large-for-gestational age and small-for-gestational age infants. Manual of Neonatal Care 4
6th ed.Philadelphia, PA: Lippincott Williams & Wilkins; 1998. 37–49.
9. Mosby Year Book, Formon S, Nelson S. Forman S. Size and growth. Nutrition of Normal Infants
1993; St. Louis, MS, 529–534.
10. Dine M, Gartside P, Glueck C. Behrman R, Kliegman R, Nelson WE, et al. Relationship of head circumference to the length. Nelson Textbook of Pediatrics
14th ed.Philadelphia, PA: WB Suanders Company; 1992. 67–77.
11. Postmes T, Van Den Bogaard A, Hazen M. The sterilization of honey with cobalt 60 gamma radiation: a study of honey spiked with spores of Clostridium botulinum
and Bacillus subtilis
12. Hussein S, Yusoff K, Makpol S, et al. Does gamma irradiation affect physicochemical properties of honey. Clin Ter
13. Rao S, Srinivasjois R, Patole S. Prebiotic supplementation in full-term neonates: a systematic review of randomized controlled trials. Arch Pediatr Adolesc Med
14. Mugambi MN, Musekiwa A, Lombard M, et al. Probiotics, prebiotics infant formula use in preterm or low birth weight infants: a systematic review. Nutr J
15. Moro G, Arslanoglu S, Stahl B, et al. A mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age. Arch Dis Child
16. Mijanur Rahman M, Gan SH, Khalil MI. Neurological effects of honey: current and future prospects. Evid Based Complement Alternat Med
17. Boehm G, Lidestri M, Casetta P, et al. Supplementation of a bovine milk formula with an oligosaccharide mixture increases counts of faecal bifidobacteria in preterm infants. Arch Dis Child Fetal Neonatal Ed
18. Moro G, Minoli I, Mosca M, et al. Dosage-related bifidogenic effects of galacto-and fructooligosaccharides in formula-fed term infants. J Pediatr Gastroenterol Nutr
19. Moro G, Mosca F, Miniello V, et al. Effects of a new mixture of prebiotics on faecal flora and stools in term infants. Acta Paediatrica
2003; 92 (s441):77–79.
20. Fanaro S, Jelinek J, Stahl B, et al. Acidic oligosaccharides from pectin hydrolysate as new component for infant formulae: Effect on intestinal flora, stool characteristics, and pH. J Pediatr Gastroenterol Nutr
21. Ustunol Z. The effect of honey on the growth of bifidobacteria. Rep Natl Honey Board
Bifidobacterium bifidum; lactobacilli; microbiota; neonates; premature
Supplemental Digital Content
© 2017 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,