Essay: OLIGOSACCHARIDES AS AN ADDITION TO INFANT FORMULA RESULTS IN GROWTH OF THE NUMBER OF BIFIDOBACTERIA IN THE INTESTINAL FLORA OF INFANTS

ABSTRACT
Introduction: NEC is a common disease in premature infants with very low birthweight. It is known that human milk oligosaccharides(HMO) and bifidobacteria have a protective effect against the development of NEC. Studies have shown that the growth of the number of bifidobacteria is stimulated by the oligosaccharides in human milk. The aim of this review is to observe if addition of oligosaccharides to infant formula also results in a growth of bifidobacteria in the intestinal flora of infants.
Method: To answer the research question we have used the Mesh-terms ‘Infant formula’ AND ‘oligosaccharides’ AND ‘bifidobacteria/bifidobacterium’, and have used MedLine and Web of Science as databases. During the search we found 10 randomized controlled trials, published between 2005 and 2014. Which meet our inclusion criteria.
Results: Supplementation of infant formula with oligosaccharides results in an increase of the number of bifidobacteria in the intestinal flora of infants. Six of the ten studies have shown a statistically significant difference among the intervention group versus the control(formula feeding)group. One out of ten studies have shown a significant difference in the number of bifidobacteria between the intervention group and the reference(breast fed)group. Three of ten studies have shown a higher number of bifidobacteria in the intervention group compared to the reference group(breast fed), but this differed not significantly.
Conclusion: Addition of oligosaccharides to infant formula results in an increase of the number of bifidobacteria in the intestinal flora of infants. The number of bifidobacteria seems to be comparable to infants who have been breast fed. ‘
INTRODUCTION
Prematurity is associated with high morbidity and mortality rates. A common disease in premature newborns is necrotizing enterocolitis(NEC). NEC is a severe and devastating disease, and it is associated with a high mortality. Premature infants are most susceptible for this disease. Of all the NEC cases, 90% of the patients are premature infants(Schnabl et al. 2008). According to The Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network(NICHD), the mean incidence of NEC in premature newborns with very low birthweight is between 5% to 15% observed over the years of 2003 to 2007(Yee et al. 2012). The pathogenesis of NEC is not fully understood and is thought to be multifactorial consisting of an imbalance of the inflammatory responses, alterations in intestinal flora and an impairment of the immunity of the mucosa(Yang et al. 2014). Also, the intestines of premature newborns are immature, it is thought that abnormal colonization of the immature gut plays a role(Yang et al. 2014).
It is widely acknowledged that breastfeeding has a lot of benefits for the health and growth of infants. One of these benefits is that breastfeeding has a protective effect on the development of necrotizing enterocolitis (Herrmann & Carroll, 2014). Human milk contains several components that stimulate the maturation of the intestines in newborns, like human milk oligosaccharides(HMO) (Smilowitz et al. 2014). Human milk contains 12-15g oligosaccharides per liter(Ben et al. 2008). Human milk oligosaccharides are complex structures and are indigestible, so these structures can reach the large intestines and can be found in the feces of breast fed infants(Smilowitz et al. 2014). These oligosaccharides contribute to the host defense of the intestines, by playing a role in the development of the function of the intestinal barrier(Smilowitz et al. 2014). Also probiotics, more specifically bifidobacteria, are thought to have a protective effect against the development of NEC(Yang et al. 2014 & Lin et al. 2005). It is known that probiotics have the potential to contribute to the maturation of the intestinal barrier, to contribute to the production of anti-inflammatory cytokines, to control apoptosis and to diminish the growth of potential pathogens(Yang et al. 2014). It is important that probiotics, when added to food products, can survive the gastrointestinal tract environment, to maintain their activity. Another challenge is to grow faster than the velocity of peristaltic movements, in order to have their effect before they reach the diaper. Probiotics have a strong affinity for enterocytes, subsequently they can prevent pathogenic organisms to bind to the mucosa of the intestines. Furthermore, the production of antimicrobial peptides and lactic acids by the probiotics also have anti-bacterial effects.(Pandey et al. 2015).
The intestinal flora of breast fed infants predominantly consist of bifidobacteria(Smilowitz et al. 2014 & Chichlowski et al. 2012). A mature colonized intestinal flora helps to protect against infections. Several studies have showed a correlation between human milk and the growth of bifidobacteria in the intestines of premature infants. These studies showed that growth of bifidobacteria is stimulated by HMO in the intestines(Smilowitz et al. 2014 & Chichlowski et al. 2012 & Zivkovic et al. 2011 & Musilova et al. 2014).
Unfortunately, some infants can not be breast fed, due to maternal diseases or medication use, or diseases of the newborn. These infants are fed with infant formula. Infant formula contains several components, but interesting for this review is the addition of oligosaccharides like galacto-oligosaccharides and/or fructo-oligosaccharides(Oozeer et al. 2013). In this review we would like to search in literature what the effect is on the number of bifidobacteria in the intestinal flora of infant formula with addition of these oligosaccharides compared to other forms of feeding, like breastfeeding or standard infant formula. This information can help us to assess the potency of this supplementation for infants who can not be breastfed.
Our hypothesis is that the addition of oligosaccharides to infant formula results in an increase in the number of bifidobacteria in the intestinal flora of infants, similar to breast milk. ‘
METHOD
Search terms:
To answer the research question ‘What is the effect of addition of oligosaccharides to infant formula on the growth of the number of bifidobacteria in the intestine of infants who can not be breast fed’?, we have performed two searches, using the databases MedLine and Web of Science.
For the search in MedLine we have used the following Mesh terms: ‘Infant formula’, ‘Oligosaccharides’ and ‘Bifidobacterium/Bifidobacteria’. Additionally, we specified the term ‘Infant formula’ by adding [title], to eliminate studies who did not use infant formula as an intervention. This resulted in10 articles from which 5 were useful for this review. In a snowball search, with use of the articles from ‘Bakker-Zierikzee’, and ‘Salvini’ we have found 2 extra articles.
For the search in Web of Science we have used the same search terms, resulting in 3 extra eligible articles in addition to the articles we already found in MedLine. In order to retrieve up to date evidence, we have made the decision to exclude articles published before 2005.
In total we have found 10 suitable studies which address our research question. In the flowchart below, we present an overview of the search.
Figure 1: flowchart using the search terms: ‘Infant formula’, ‘Oligosaccharides’ and ‘Bifidobacterium/bifidobacteria’.
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Inclusion and exclusion criteria:
To include studies for this review we have used the following inclusion and exclusion criteria:
Figure 2. inclusion and exclusion criteria.
We have chosen to include randomized controlled trials, because these studies provide the strongest level of incidence. Also we had made the decision not to base the results of this review on other reviews, therefore this was chosen as an exclusion criteria.
In addition, we selected studies in which the intervention consists of infant formula with supplements of galacto-oligosaccharides(GOS) and/or fructo-oligosaccharides(FOS).
The comparison had to be an infant formula without supplements, this is interesting because in this way we can compare the total number of bifidobacteria in infants received standard formula to the ones who received study formula.
Data collection:
All the included randomized controlled trails also examined other outcomes than the number of bifidobacteria, out of the focus of our study. Relevant other outcomes will be discussed in the section ‘Discussion’.
We have put the characteristics of the studies and the results, defined as the number of bifidobacteria in a table to be able to compare the characteristics and the results of the different studies.
We will discuss the results of the studies below.
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RESULTS
Author, year Participants Method Groups Results
(in absolute number or percentage of total bacterial content)
Euler et al. 2005 Healthy
Age = 2-6 weeks
GA = 37-42 weeks
N= 72
Follow-up = 5 weeks Prospective
Randomized
Crossover
Outpatient
Single-site study
Used CASBA 4 system for measurement of bifidobacteria. Intervention group:
1. Infant formula + FOS 1,5g/L(n=28)
2. Infants formula + FOS 3,0g/L(n=30)
Reference group(n=14)
Breast feeding Intervention group:
1. ‘9,1 log10 CFU/g
2. ‘ 8,6 log10 CFU/g
Control group ‘ 8,0 log10 CFU/g
P=0,0450
Bakker-Zierikzee et al. 2005 Healthy
Age = 3 days
GA = unknown
N=120
Follow-up = 16 weeks RCT
Used FISH analysis. Intervention group:
1. Standard infant formula + mixture of GOS and lcFOS 6g/L(n=19).
2. Standard infant formula + bifidobacterium animalis(n=19).
Control group(n=19):
Standard formula(Nutrilon).
Reference group(n=63):
Breast feeding Intervention group:
1. ‘ 64%
2. ‘ 53%
Control group ‘ 53%
Reference group ‘ 49%
P>0,05
Brunser et al. 2006 Healthy
Age = 3,5 months
GA = full-term
N= 116A
Follow-up = 13 weeks.
Prospective RCT
Blinded
Used FISH analysis. Intervention group:
1. Infant formula + FOS 2g/L(n=26).
2. Infant formula + lactobacillus johnsonii 108 per gram powder(n=33)
Control group(n=25):
Standard infant formula (Nestl??)
Reference group:
Breast feeding (n=32) After 7 weeks:
Intervention group:
1. ‘9,41 log10 CFU/g
2. ‘ 9,99 log10 CFU/g
Control group ‘ 9,55 CFU/g
Reference group ‘ 9,49 CFU/g
P-value is not mentioned
Costalos et al. 2007 Healthy
Age =
GA = 37-42 weeks
N=140
Follow-up = 6 weeks RCT
Double blind
Used FISH analysis. Intervention group(n=70):
Infant formula + GOS/lcFOS unknown concentration
Control group(n=70):
Infant formula Intervention group ‘ 39,69%
Control group ‘ 14,87%
P=0,262
Ben et al.
2008 Healthy
Age = direct after delivery
GA = 37-42 weeks
N=82
Follow-up = 3 months
RCT
Used culture technique.
Intervention group(n=37):
Infant formula + GOS 2,4g/100mL
Control group(n=45):
Infant formula (Frisolac)
Reference group(n=24):
Breast feeding
Combination group(n=58):
Breast milk + supplemented infant formula Intervention group ‘ 9,01 log10 CFU/g
Control group ‘ 8,16 log10 CFU/g
Reference group ‘ 9,25 log10 CFU/g
Combination group ‘ 8,97 log10 CFU/g
P=0,01
Salvini et al. 2011 Healthy
Age = direct after delivery
GA = 37-42 weeks
N=20
Follow-up = 6 months RCT
Placebo-controlled
Double blinded
Used culture technique. Intervention group(n=10):
Bovine milk formula + scGOS/lcFOS 8g/L.
Control group(n=10):
Bovine milk + Maltodextrin. Intervention group ‘ 9,4 log10 CFU/g
Control group ‘ 7,3 log10 CFU/g
P=0,0014
Xia et al.
2012 Healthy
Age = 0-6 months
GA = unknown
N = 65
Follow-up = 4 weeks
RCT
Multi-center
Double blind
Used real-time PCR. Intervention group:
1. Cow milk based infant formula + 2,0g FOS/L(n=14)
2. Cow milk based infant formula + 3,0g FOS/L(n=20)
Control group(n=14):
Cow milk based infant formula.
Reference group(n=17):
Breast feeding. Intervention group:
1. ‘ 8,75 log10 CFU/g
2. ‘ 9,38 log10 CFU/g
Control group ‘ 9,29 log10 CFU/g
Reference group ‘ 8,49 log10 CFU/g
P-value is not mentioned
Holscher et al. 2012 Healthy
Age = 2-8 weeks
GA = 37-42 weeks
N = 102
Follow-up = 6 weeks.
Prospective RCT
Double blind
Used FISH analysis. Intervention group(n=36):
Infant formula + GOS/scFOS 4g/L.
Control group(n=33)
Infant formula
Reference group(n=33):
Breast feeding Absolute number:
Intervention group ‘ 3,72×109 CFU/g
Control group ‘ 2,05×109
Reference group ‘ 3,02×109
P=0,0083
Relative number:
Intervention group ‘ 44,10%
Control group ‘ 32,62%
Reference group ‘ 43,48%
P=0,0219
Sierra et al.
2014 Healthy
Age = <2 months
GA = 37-42 weeks
N = 81
Follow-up = 4 months RCT
Multi-centered
Double-blind
Placebo-controlled
Used real-time PCR. Intervention group(n=44):
Infant formula + GOS 0,44g/dl.
Control group(n=37):
Infant formula.
Follow-up group:
Infant formula + GOS 0,50g/dl. Intervention group ‘ 8,65 log10 CFU/g
Control group ‘ 8,02 log10 CFU/g
P=0,010
Paineau et al. 2014 Healthy
Age = 0-7 days
GA = 37-42 weeks
N=61
Follow-up = 3 months RCT
Double blind
Used real-time PCR. Intervention group(n=43):
Infant formula + scFOS 4g/L
Control group(n=39):
Infant formula + Maltodextrin. Intervention group ‘ 3,01×107 CFU/g
Control group ‘ 0,04×107 CFU/g
P=0,03
Table 1. Results of the articles included for this review.
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In this section we describe the results of the included studies. At last we will review if the number of bifidobacteria in the intervention groups is similar with the number of bifidobacteria in the breast fed groups.
The overall result of the included articles is that supplementation of infant formula with oligosaccharides results in an increase of the number of bifidobacteria in the intestinal flora of infants.
All studies have quantified bifidobacteria in CFU/g, except for Costalos et al and Bakker-Zierikzee et al. They have described the number of bifidobacteria as a percentage of total microorganisms in the infant gut. Holscher et al have described the number of bifidobacteria in both CFU/g and as a percentage of the total microorganisms.
Intervention group:
The studies that added FOS supplement to infant formula as their intervention(Euler et al., 2005 & Brunser et al. 2006 & Xia et al. 2012 & Paineau et al. 2014), all showed an increase in the number of bifidobacteria, compared to the control group. Euler et al (2005), and Paineau et al (2014) have shown a statistically significant difference among the feeding groups, the differences showed by the other studies were not statistically significant. Xia et al (2012) and Brunser et al (2006) have shown no statistically significant difference among the feeding groups, but do have showed an increase in the number of bifidobacteria in the intervention group. In the study of Euler et al (2005), they have compared two different concentration of FOS supplements. At the end the concentration of 1,5g FOS/L had the highest count of bifidobacteria compared to the concentration of 3,0g FOS/L, this difference is statistically significant(P=0,045). Also Xia et al (2012) have compared two different concentrations of FOS supplements. At the end the concentration of 3,0g FOS/L had the highest count of bifidobacteria compared to the concentration of 2,0g/L. The differences between the feeding groups were not statistically significant. Brunser et al (2006) have compared FOS supplements with lactobacillus supplements, resulted in a higher number of bifidobacteria in the group of lactobacillus supplements. The group with FOS supplements showed an increase in the number of bifidobacteria. The differences between the feeding groups was not statistically significant, the p-value is not mentioned in the article. Paineau et al (2014) have used scFOS supplements, resulted in an statistically significant increase of the number of bifidobacteria compared to the control group(P=0,03).
Other studies have used a mixture of 10% lcFOS and 90% GOS(9:1) added to infant formula as their intervention(Bakker-Zierikzee et al. 2005 & Costalos et al. 2007 & Salvini et al. 2011 & Holscher et al. 2012). The ratio of this mixture resembles most the molecular masses of the oligosaccharides found in human milk(Bakker-Zierikzee et al. 2005 & Costalos et al. 2007). The studies of Holscher et al (2012) and Salvini et al (2011) have shown a statistically significant difference between the intervention group and the control group. The studies of Bakker-Zierikzee et al (2005) and Costalos et al (2007) did not have shown a significant difference between the feeding groups. But all studies have showed an increase in the number of bifidobacteria in the intervention group. The increase in the number of bifidobacteria was significant in the study of Salvini et al (2011) (P<0,005). The other studies did not calculated a p-value for the increase in number of bifidobacteria.
Two studies have used only GOS supplement as an intervention(Ben et al. 2008 & Sierra et al. 2014), in different concentrations. These two studies both shown a significant difference between the feeding groups, and both shown an increase in the number of bifidobacteria in the intervention group. In the study of Ben et al (2008), the increase in the number of bifidobacteria was significant in the intervention group and reference group.
Both Paineau et al (2014), and Salvini et al (2011), have feed the control group with infant formula + Maltodextrin. In both studies there was a statistical significant difference between the intervention and the control group. Also Salvini et al (2011) have shown a significant increase in the number of bifidobacteria. Paineau et al (2014) did not have calculated a p-value for the increase in the amount of bifidobacteria.
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Techniques for assessment of the outcome:
When we compare the techniques used to quantify bifidobacteria we can distinguish studies which utilized culture technique, FISH analysis and real-time PCR.
Ben et al (2008) and Salvini et al (2011) have used culture technique for the measurement of bifidobacteria. Both studies have shown a statistically significant increase in the number of bifidobacteria. Ben et al (2008) did not describe a p-value for this increase, but Salvini et al (2011) have shown a p-value <0,005 for this increase.
Four studies have utilized FISH analysis for the measurement of bifidobacteria(Bakker-Zierikzee et al. 2005 & Brunser et al. 2006 & Costalos et al. 2008 & Holscher et al. 2012). All studies have used the probe Bif164 during the FISH analysis. All four studies found an increase in the number of bifidobacteria, while using different oligosaccharides in the intervention group. Holscher et al (2012) have shown a statistically significant difference in the number of bifidobacteria between the intervention group and the control group. Costalos et al (2007) and Bakker-Zierikzee et al (2005) have shown a higher number of bifidobacteria in the intervention group compared to the control group, but this difference was not statistically significant. Brunser et al (2006) have shown an increase in the number of bifidobacteria in the intervention group, but this amount was lower than the amount of bifidobacteria in the control group.
Sierra et al (2014) and Paineau et al (2014), have used real-time PCR for measurement of bifidobacteria. Both studies have shown a statistically significant difference in the number of bifidobacteria between the intervention group and the control group. Both studies have shown an increase in the number of bifidobacteria in the intervention group.
Comparison with number of bifidobacteria in human milk:
Six studies have also included a reference group with breast fed infants(Euler et al. 2005 & Bakker-Zierikzee et al. 2005 & Brunser et al. 2006 & Ben et al. 2008 & Xia et al. 2012 & Holscher et al. 2012). Euler and colleagues (2005) have shown a statistically significant difference in the number of bifidobacteria between the intervention group and the reference group. Bakker-Zierikzee et al (2005), Holscher et al (2012) and Xia et al (2012) have shown a higher number of bifidobacteria in the intervention group compared to the reference group, but this is not statistically significant. Brunser et al (2006) and Ben et al (2008) have shown a lower number of bifidobacteria in the intervention group compared to the reference group, but this is not statistically significant.
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DISCUSSION
The included studies in this review have all shown that addition of oligosaccharides to infant formula results in an increase of the number of bifidobacteria in the intestinal flora of infants. The studies have used different oligosaccharides supplements, FOS, GOS or a mixture of GOS/FOS, in different concentrations. The studies in this review have used different concentrations of oligosaccharides added to infants formula, ranging from 0,44g/L to 8,0g/L. The studies also have utilized different techniques to quantify the amount of bifidobacteria, like culture technique, FISH analysis and real-time PCR. All studies have shown a positive effect on the growth of bifidobacteria in the intestinal flora.
In this review only RCT’s are includes, because these studies provide the strongest level of evidence. For this review we have used specific inclusion criteria, because during the search we found studies which used other forms of supplements but we wanted to focus us on the specific supplements of galacto-oligosaccharides and/or fructo-oligosaccharides. We also found studies which did not examined the outcome worded as the number of bifidobacteria. By using these specific inclusion criteria we have found similar studies. The included articles in this review are published between 2005 and 2014, this indicate that this review is based on recent studies.
All studies have shown a statistically significant difference in the number of bifidobacteria among the feeding groups, except for Costalos et al (2007), Bakker-Zierikzee et al (2005), Xia et al (2012) and Brunser et al(2006). When comparing the different characteristics of the studies it is hard to conclude the reason for this phenomenon. Although, the technique used in the studies of Costalos et al (2007), Bakker-Zierikzee et al (2005) and Brunser et al (2006) is FISH analysis. According to Xia et al (2012), FISH analysis overestimate the outcome in abundance of bifidobacteria, this might have influenced the validity of the results.
Not all studies described whether they added long-chain FOS or short-chain FOS. Paineau and colleagues (2014) have specifically used short-chain FOS, while the other studies do not give information about the type of FOS. It would be interesting to investigate whether short-chain or long-chain FOS have an impact on the consistency of the infant flora of infants.
In the studies which used a mixture of GOS and FOS, two of them have shown a significant difference between the feeding groups. The other two studies did not show this phenomenon. Maybe this can be due to the fact that the studies of Bakker-Zierikzee et al (2005) and Costalos et al (2007) have quantified the number of bifidobacteria as a percentage of total microorganisms. The studies with significant difference have quantified the number of bifidobacteria in CFU/g.
Another remarkable detail is that all studies used a mixture of GOS and FOS, have all used long-chain FOS except for Holscher et al (2012) which have used short-chain FOS.
Interestingly, the number of bifidobacteria of the intervention and breast fed groups did not significantly differ from each other, except for Euler et al (2005). This would indicate that infants given an oligosaccharides supplemented infant formula have comparable intestinal flora to infants fed with human milk, focusing on the number of bifidobacteria. Xia et al (2012), Bakker-Zierikzee et al (2005) and Holscher et al (2012), have shown a higher number of bifidobacteria in the intervention group compared to the breast fed group, but these differences were not significant or that much.
The trials included for this review have also report other aspects next to the amount of bifidobacteria. Some of these results are interesting to shortly discuss. Some studies have determined a decrease in fecal pH in the intervention group compared to the control group(Bakker-Zierikzee et al. 2005 & Ben et al. 2008 & Costalos et al. 2008 & Salvini et al. 2011 & Holscher et al. 2012 & Sierra et al. 2014). This can indicate that there is a correlation between the growth of bifidobacteria and the decrease of fecal pH.
Some studies have focused on the amount of the harmful bacteria Clostridium Difficile. Costalos and colleagues (2007) have found a significant lower number of C. difficile in the intervention group compared to the control group. Holscher and colleagues (2012) had found a lower number of C. difficile in the intervention group compared to the control group. When comparing the breastfed group and the intervention group, the number of C. difficile was lower in the breast fed group. Xia and colleagues (2012) had found a higher number of C. difficile in the intervention group compared to the control group and the breast fed group. The reason for this phenomenon can be that Xia et al (2012) used only FOS supplements in their intervention group, and Costalos et al (2007) and Holscher et al (2012) used a mixture of GOS and FOS supplements as their intervention. But we can not state this with any consistency, based on two studies. So maybe it is interesting to find out if the addition of GOS is necessary for a reduction in growth of C.difficile.
Euler and colleagues (2005) have also focused on the presence of adverse events. In the group with lower concentrations of FOS supplements they found a lower incidence of adverse events, compared to the group with higher concentration of FOS supplements. These adverse events included decreased appetite, increased flatulence and increased spit-ups. When focused on more serious adverse events for which antibiotic therapy was necessary, there was no significant difference between the intervention group and the breast fed group. Also, Ben et al (2008) did not find a difference of the infant formula + GOS supplements on the incidence of crying, vomiting and regurgitation
This review showed that addition of oligosaccharides to infant formula results in an increase of bifidobacteria in the intestinal flora of infants. The intestines of premature infants is still immature compared to the intestines of full term born infants. It would be interesting to study, if attenuated growth of bifidobacteria in the intestinal flora of premature infants can stimulate maturation. And if that would be possible, can addition of oligosaccharides to infant formula result in a decrease in the incidence of necrotizing enterocolitis? These questions are interesting to be answered by further and larger randomized controlled trials.
CONCLUSION
In conclusion, addition of oligosaccharides supplements to infant formula results in an increase in the number of bifidobacteria in the intestinal flora of term born healthy infants. The amount of bifidobacteria in these intervention groups differ not significantly from the amount of bifidobacteria in the breast fed group, these amount seems to be comparable.
So the addition of oligosaccharides to infant formula might be a good alternative for infants who can not be breast fed.
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