Prebiotic Effects of a Novel Combination of Galactooligosaccharides and Maltodextrins

Abstract

Prebiotics are used for stimulating the growth of beneficial microorganisms in the gut. However, it is very difficult to find a suitable prebiotic mixture that exclusively supports the growth of beneficial microbes such as bifidobacteria and lactobacilli. We tested the effects of a prebiotic mixture in vitro by incubating it with fecal samples and in vivo by administration of the prebiotic supplement to healthy adult volunteers, followed by analysis of their fecal microbiota. The effect of the oligosaccharides on bacterial metabolism was studied by analyzing short-chain fatty acid (SCFA) production in vitro and the SCFA pattern for the stool samples of volunteers. In the in vitro test, a higher proportion of bifidobacteria (25.77%) was seen in the total bacterial population after cultivation on a prebiotic mixture than on the control medium (7.94%). The gram-negative anaerobe count significantly decreased from 8.70 to 6.40 log CFU/g (from 35.21% to 0.60%) and the Escherichia coli count decreased from 7.41 to 6.27 log CFU/g (from 1.78% to 0.44%). Administration of a prebiotic mixture in vivo (9 g of galactooligosaccharides [GOS]+1 g of maltodextrins; daily for 5 days) significantly increased the fecal bifidobacterial count from 9.45 to 9.83 log CFU/g (from 40.80% to 53.85% of total bacteria) and reduced the E. coli count from 7.23 to 6.28 log CFU/g (from 55.35% to 45.06% of total bacteria). The mixture comprising GOS and maltodextrins thus exhibited bifidogenic properties, promoting the performance of bifidobacteria by boosting their growth and inhibiting the growth of undesirable bacteria.

Introduction

The composition of intestinal microbiota is significantly influenced by diet. Recently, the food industry has begun focusing on functional foods, such as probiotics and prebiotics, and their impact on human health. Prebiotics are one of the most popular supplements used to influence the composition of the gut microbiota by stimulating the growth of beneficial microorganisms. Supplementation of the diet with prebiotics has considerable therapeutic and preventive potential and may delay and/or reverse the progression of immune and metabolic diseases.¹

Little is known of the role played by many of the dominant bacterial species in the gut. It is well accepted that bifidobacteria and lactobacilli are the bacterial species that directly contribute to human health.² Some oligosaccharides can exhibit a prebiotic effect, such as stimulation of the growth of bifidobacteria and lactobacilli, in the intestinal tract.³

Bifidobacteria constitute a predominant part of the population in the guts of neonates and infants,⁴ and the bacterial numbers decrease with age.⁵ However, bifidobacteria still represent a significant proportion of the adult gut microbiota.⁶ The composition of gut microbiota correlates with diet and health. Dairy products fermented with lactic acid bacteria such as lactobacilli and fortified with bifidobacteria—along with prebiotics such as fructooligosaccharides (FOS) or inulin—have gained popularity in the food market over the past decade. The control of intestinal microbial composition by the use of prebiotics is likely to affect the development of metabolic diseases through modulation of the immune system. Supplementation with prebiotics may delay and/or reverse the progression of metabolic diseases. The first food and the first prebiotic for humans is human milk, which contains high quantities of prebiotic human milk oligosaccharides (HMOs). There have been many attempts to replicate HMOs and their unique effects, and it has been difficult to find a suitable prebiotic mixture that would support the growth of lactobacilli and bifidobacteria in the intestine. The bifidogenic effect of a prebiotic mixture containing 90% galactooligosaccharides (GOS) and 10% FOS mimics the molecular size distribution of HMOs, as demonstrated by Boehm et al.⁷ and Roberfroid.⁸ FOS and GOS are the most frequently used prebiotics in human food and infant formulas.^8,9 However, commercially available prebiotics are not fully selective, and they inadvertently support the growth of potentially pathogenic bacteria such as gram-negative anaerobes and Escherichia coli. ¹⁰ Therefore, it is very important to find a suitable prebiotic mixture that supports the growth of beneficial microbes, including lactobacilli and bifidobacteria.

In the current study, we investigated the effects of a prebiotic mixture of GOS and maltodextrins on the composition of fecal bacteria, both in vitro and in vivo. The prebiotic effect of the oligosaccharide mixture was tested in vitro by incubating fecal samples and in vivo by administration of prebiotic supplements to healthy adult volunteers, followed by analysis of their fecal microbiota. Many microbiota-associated activities have a direct impact on the host's health, partly because of the production of short-chain fatty acids (SCFAs), which comprise the major bacterial fermentation products in the large intestine. Up to 95% of the SCFAs (acetate, propionate, and butyrate) produced during carbohydrate fermentation may be taken up and utilized by the host.¹¹ The effect of the oligosaccharides on bacterial metabolism was studied by analyzing the SCFA production in vitro and the SCFA in the stool samples of the volunteers.

Materials and Methods

Composition of the experimental prebiotic mixture

A dose of the prebiotic mixture (10 g) contained 9 g GOS syrup (Vivinal^®; FrieslandCampina Domo, Amersfoort, The Netherlands) and 1 g maltodextrins (Nutriose^® FM 06, soluble fiber; Herford, Germany). Both components were obtained from Humana GmbH.

Fermentation of the substrate in vitro

Fresh fecal samples from nine healthy volunteers (four women and five men; mean age, 34.44±9.99 years) were incubated in roll tubes containing a cultivation medium with the prebiotic mixture. The cultivation medium (10 g tryptone, 10 g peptone, 5 g yeast extract, 1 g sodium pyruvate, 1 mL Tween 80, and 0.5 g cysteine per liter) contained 9% GOS and 1% maltodextrins as the sole carbon source. The samples were incubated at 37°C for 24 h under anaerobic incubations and were then analyzed by the plate technique using selective media according to Vlková et al. ¹² Appropriate dilutions were transferred to sterile Petri dishes, which were immediately filled with the media for the following: total anaerobes (Wilkin-Chalgren; Oxoid, ThermoFisher Scientific, Carlsbad, CA, USA), bifidobacteria (TPY agar; Scharlau Schemie, Spain; modified by the addition of 100 mg/L mupirocin and 1 mL/L acetic acid according to Rada and Petr¹³ lactobacilli (Rogosa agar; Oxoid), gram-negative anaerobes (Wilkins-Chalgren agar, Oxoid; supplemented with G-N Anaerobe Selective Supplement, Oxoid), and E. coli (TBX; Oxoid). Total anaerobes, gram-negative anaerobes, and bifidobacteria were incubated in anaerobic jars (Anaerobic Plus System; Oxoid) at 37°C for 48 h. Lactobacilli were cultivated under microaerophilic conditions at 37°C for 3 days, and E. coli were cultivated aerobically at 37°C for 24 h. Cultivation medium with glucose as a sole carbon source was also used as a control.

Fermentation of the substrate in vivo

The prebiotic mixture was administered to 11 healthy adult volunteers (6 women and 5 men; mean age, 35.18±10.91 years) each morning for 5 days; the volunteers were not prescribed a specific diet. During the experiment, none of the volunteers consumed any other prebiotic or probiotic products and did not consume fermented products with probiotic bacteria. Fresh fecal samples were obtained and analyzed (estimation of total anaerobic bacteria, gram-negative anaerobes, bifidobacteria, lactobacilli, and E. coli) by cultivation on selective agar media before and after the experiment.¹²

Chemical analyses of SCFA

The total SCFA concentration was estimated by titration after steam distillation.¹⁴ The molar profile of volatile fatty acids was estimated by gas chromatography at 140°C using a Chromosorb WAW glass column (2 m×3 mm i.d.) with 15% SP 1220 and 1% H₃PO₄ (Supelco, Bellefonte, PA, USA), as described by Ottenstein and Bartley.¹⁵ Nitrogen at an inlet pressure of 80 kPa was employed as the carrier gas.

Lactic acid and pH estimation

All the mixtures and fecal samples (for the in vitro and in vivo testing) were homogenized for enzymatic determination of lactic acid using the Reflectoquant apparatus (Merck, Darmstadt, Germany). The pH of all samples was measured directly at room temperature using a Handylab pH meter (Schott, Mainz, Germany) equipped with a pH THETA 90 HC 163 electrode (Czech Republic).

Statistical analysis

Statistical analysis was performed using Student's t-test.

Results

In vitro test

The bifidobacterial counts were almost identical after cultivation on the control medium (8.06±0.26 log CFU/g) and the prebiotic mixture (8.03±0.45 log CFU/g) in vitro (Table 1). However, because the total bacterial counts differed between the control (total count, 9.16±0.20 log CFU/g) and prebiotic mixture-treated (total count, 8.62±0.25 log CFU/g) groups, it was necessary to consider the proportion of bifidobacteria from the analyzed bacteria. The proportion of bifidobacteria after cultivation on the prebiotic mixture increased (from 7.94% to 25.77%) than on the control medium. The gram-negative anaerobe count significantly decreased (P<.05) from 8.70±0.45 to 6.40±1.22 log CFU/g (from 35.21% to 0.60%) and the E. coli count from 7.41±0.78 to 6.27±0.71 log CFU/g (from 1.78% to 0.44%; P<.01; Table 1). All changes in the percentage distribution of microorganisms correlated with changes in primary metabolites, including SCFA (Table 2). There were significant changes in the proportions of acetate, propionate, and butyrate. The predominant SCFA was acetate (95.55±3.63 mol%) in the experimental samples; the acetate level was only 63.15±17.16 mol% in control samples. The changes in the ratio of SCFA and the higher representation of acetate were correlated with the drop in pH (Table 2).

Table 1.

Counts of Bacteria After In Vitro Testing of Prebiotic Mixture

	log CFU/g
In vitro	Total counts	Bifidobacterium	Lactobacillus	G-anaerobes	E. coli
Control medium	9.16±0.20	8.06±0.26	6.16±1.12	8.70±0.45^*	7.41±0.78^*
Prebiotic mixture	8.62±0.25	8.03±0.45	5.82±1.09	6.40±1.22^*	6.27±0.71^*

Counts of bacteria after cultivation of donor feces (average from nine samples) on control medium with glucose and on prebiotic mixture in vitro in log CFU/g. Data are mean±standard deviation (SD). Values in columns differ (^* P<.01). The differences among bacterial counts were evaluated by t-test.

Table 2.

The Concentration of Short-Chain Fatty Acids After Cultivation of Donor Feces on Control Medium and on Prebiotic Mixture in In Vitro

	In vitro
SCFA (mol%)	Control medium	Prebiotic mixture
Acetate	63.15±17.16^*	95.55±3.63^*
Propionate	10.94±5.23^*	3.43±2.73^*
Isobutyrate	0.79±1.25	0.09±0.23
Butyrate	21.39±11.65^*	1.45±1.32^*
Isovalerate	2.24±3.73	0.01±0.02
Valerate	1.53±2.56	0.03±0.06
Lactic acid, mg/L	1425.00±985.74^*	5155.89±1083.36^*
pH	6.66±0.57^*	3.83±0.18^*

Data are mean±SD. Values in rows differ (^* P<.05). The differences among values were evaluated by t-test.

SCFA, short-chain fatty acids.

It is possible to evaluate the effect of the prebiotic mixture in vitro by using a prebiotic index (PI).¹⁶ A positive value indicates that the tested prebiotic was able to specifically stimulate the growth of probiotic bacteria under the experimental conditions, while a negative value indicates that the addition of prebiotics had no effect on probiotic growth. Our results, using the plate technique, confirmed the selective stimulation of probiotic bacteria (bifidobacteria and lactobacilli), which was calculated using a modified PI according to the following equation: PI=(bifidobacteria/total bacteria)+(lactobacilli/total bacteria) – (gram-negative anaerobes/total bacteria) − (E. coli/total bacteria). All the bacterial counts have been given as log CFU. The prebiotic mixture was found to have prebiotic properties with a modified PI of +0.138, as opposed to the control (with glucose) with a modified PI of −0.207.

In vivo test

Administration of the prebiotic mixture (9 g of GOS+1 g of maltodextrins daily for 5 days) to volunteers significantly increased (P<.01) the bifidobacterial count in the gut from 9.45±0.47 to 9.83±0.39 log CFU/g and reduced the E. coli count from 7.23±0.68 to 6.28±0.88 log CFU/g (Table 3). Additionally, it was observed that the pH of feces decreased after administration of the prebiotic mixture in 8 of 11 volunteers (Table 4). However, according to the Student's t-test, the changes in pH were not significant.

Table 3.

Counts of Bacteria After In Vivo Testing of Prebiotic Mixture

	log CFU/g
In vivo	Total counts	Bifidobacterium	Lactobacillus	G-anaerobes	E. coli
Before administration	9.97±0.39	9.45±0.47^*	6.06±1.86	9.74±0.40	7.23±0.68^*
After administration	10.25±0.36	9.83±0.39^*	6.98±1.37	9.75±0.46	6.28±0.88^*

Counts of bacteria before and after administrations of prebiotic mixture to volunteers (average from 11 samples) in log CFU/g. Data are mean±SD. Values in columns differ (^* P<.05). The differences among bacterial counts were evaluated by t-test.

Table 4.

The Concentration of Short-Chain Fatty Acids After In Vivo Testing of Prebiotic Mixture

	In vivo
SCFA (mol%)	Before administration	After administration
Acetate	67.12±7.89	64.62±7.68
Propionate	11.47±3.53	12.44±2.96
Isobutyrate	1.64±1.02	1.73±1.83
Butyrate	15.20±4.59	16.66±4.67
Isovalerate	2.27±1.59	2.13±1.97
Valerate	2.31±1.18	2.42±1.52
Lactic acid, mg/L	NT	NT
pH	6.47±0.35	6.46±0.50

Data are mean±SD.

NT, not tested.

The differences in the proportion of bifidobacteria were significant both in vitro and in vivo. The bifidobacterial count increased from 9.45±0.47 to 9.83±0.39 log CFU/g (from 40.80% to 53.85%) in vivo.

Discussion

A prebiotic is “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health.”^17,18 Prebiotics play an important role in human nutrition. In recent years, numerous studies have characterized several prebiotics and verified potential health benefits associated with their consumption. Considerable evidence exists on prebiotics and their positive effects on human intestinal microbiota, but the studies are usually performed in vitro. The health effects of prebiotics (prevention of diarrhea or constipation, modulation of the metabolism of intestinal microorganisms, cancer prevention, improved lipid metabolism, and enhanced mineral adsorption and immunomodulatory properties) are indirect, i.e., mediated by the intestinal microbiota, and therefore not fully understood. All the criteria for prebiotic classification have been verified many times in vitro. However, their fermentation profiles and the dosages required for beneficial health effects to manifest in vivo are poorly understood.¹⁹

A majority of the health effects of prebiotics are associated with the metabolism of nutrients, changes in the concentrations and composition of SCFA, the related reduction in colon pH, the increased expression of binding proteins, and immune system modulation.^20
–22

In this study, we observed changes in the proportion of SCFA. The predominant SCFA in the in vitro test was acetic acid. This finding is likely associated with the higher proportion of bifidobacteria, whose main primary metabolites are acetate and lactate (in a 3:2 proportion; Tables 1 –4). These changes correlate with the pH values noted in our in vitro test, as shown in Table 2. The second predominant SCFA in the in vivo conditions was butyrate, which can be associated with miscellaneous bacteria (e.g., Faecalibacterium spp., clostridia, eubacteria, and heterofermentative lactobacilli). Butyric acid is used by the epithelial cells of the colon mucosa as a source of energy and as a growth factor²³ and it also exerts preventive effects on carcinogenesis,²⁴ which particularly protects against colonic cancer.²⁵

In summary, we verified the prebiotic properties of the examined prebiotic mixture, which comprised GOS and resistant maltodextrins, by using the PI as an indicator. Prebiotic and bifidogenic effects were expected, but they were not significant.

In vitro test was a kind of batch cultivation. Under such conditions, growth of bacteria that can utilize simple saccharides such as monosaccharides is seen and not regulated by factors such as enzymes, digestive juices, and excretory and resorption activities that are seen in the intestine.

In contrast, our in vivo experiment was a kind of continuous cultivation. This was also the reason why the decrease in pH was not as radical as that in the in vitro test. However, some features were similar in both in vitro and in vivo experiments. For example, the increase in the percentage of bifidobacteria caused by the prebiotic mixture was accompanied by decrease in the E. coli and gram-negative anaerobe counts. The fact that the SCFA concentration before administration of the prebiotic mixture did not significantly differ from that after administration (Table 4) may be attributable to the distribution of standard deviation over a large range.

The prebiotic mixture should exhibit statistically significant prebiotic effects when administered to more volunteers than those involved in the current study, or at higher doses. The mixture also showed a bifidogenic effect and its fermentation led to the production of SCFA, which may confer positive effects on the colonic mucosa. Increase in the butyrate and propionate content of feces indicates the influence of prebiotic effects. We observed changes in the proportion of the monitored bacteria during the in vitro and in vivo tests. The differences in the absolute numbers of bifidobacteria were significant for the in vitro and in vivo testing. The percentage of bifidobacteria increased by 13.05%, and the ratio of gram-negative anaerobes to E. coli decreased by more than 10%. On the basis of the results of the in vitro and in vivo experiments, it can be concluded that the novel prebiotic mixture developed can modulate the intestinal microbiota to benefit the host.

Footnotes

Acknowledgments

The authors thank the volunteers for their participation, the company Humana GmbH, and the NAZV QJ1210093 project for the financial support.

Author Disclosure Statement

No competing financial interests exist for any of the authors.

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