1

In vitro comparison of the prebiotic effects of two inulin-type fructans 1

Anna Pompei1, Lisa Cordisco1, Stefano Raimondi2, Alberto Amaretti2, Ugo Maria Pagnoni2, 2

Diego Matteuzzi1 and Maddalena Rossi2* 3

4

1 Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40100, 5

Bologna, Italy. 6

2 Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100, 7

Modena, Italy. 8

9

Running headline: prebiotic effects of inulin-type fructans 10

* Corresponding author: Tel. +39-(0)-59-2055567; Fax: +39-(0)-59-373543 E-mail:

rossi.maddalena@unimore.it

* ManuscriptClick here to view linked References

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ABSTRACT 1

Fecal cultures were used to compare the prebiotic effects of a new fructan containing high 2

solubility inulin (HSI) and of a well established prebiotic containing oligofructose (OF) with a 3

negative control (CT). Changes in the intestinal microbiota, pH, ammonia, volatile organic 4

acids and lactic acid were monitored during incubation. Molecular techniques for microbial 5

enumeration indicated that both HSI and OF led to a significant increase in bifidobacteria (P ≤ 6

0.05) and lactobacilli (P ≤ 0.05) compared to the control. Significant changes in the pH and 7

levels of ammonia with both inulin-type fructans were observed, as well as higher levels of 8

acetic, lactic and formic acids (P ≤ 0.05). The fermentative metabolism appeared to be faster 9

on OF than on HSI. Both OF and HSI showed clear prebiotic effects, but had differences in 10

fermentation kinetics because of to the different degree of polymerization (DP). This study 11

provides proof for the prebiotic effectiveness of high solubility inulin, and shows that inulin-12

type fructans with higher degree of polymerisation might have a prolonged bifidogenic effect, 13

thus could extend the saccharolytic metabolism and low pH to the distal parts of the colon. 14

15

Keywords: prebiotic, oligofructose, inulin, fecal culture. 16

Abbreviations: HSI, high solubility inulin; OF, oligofructose; CT, negative control; FEM, 17

fecal extract medium 18

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1. INTRODUCTION 1

The gastrointestinal tract is a complex ecosystem containing up to 1011 CFU of 2

bacteria per g of intestinal content. This large population of bacteria plays a key role in 3

normal gut function, human health and well-being [1]. In particular, bifidobacteria and 4

lactobacilli exert beneficial effects on their host and are generally regarded as probiotics [2-5]. 5

Lactic acid bacteria and bifidobacteria effectively compete with other species of the intestinal 6

microbiota and transient organisms for nutrients and attachment sites in the gut [6], provide 7

lactic and acetic acids which acidify the large intestine, and restrict putrefactive and 8

potentially pathogenic bacteria [7]. They also exert beneficial effects such as immuno-9

stimulation [8], anti-carcinogenic activity [9], pathogen growth inhibition [10], vitamin and 10

amino acid production [11-13] and the reduction of the conversion of primary bile acids to 11

secondary ones. Hence, there is great interest in the manipulation of the intestinal microbiota, 12

aiming to increase the number of bifidobacteria and lactobacilli and to stimulate the 13

production of short chain fatty acids (SCFA) and lactate in the colon. Oligofructose (OF) and 14

inulin are effective prebiotics, defined as non-digestible food ingredients which beneficially 15

affect the host by selectively stimulating the growth and/or activity of the microbial groups 16

that improve the host health [14, 15]. Inulin and OF are extensively used in different foods, 17

either because of their technological applications as fat and sugar replacers and/or for 18

prebiotic properties and associated health benefits[16-18]. They occur naturally in many foods 19

of vegetable origin, such as onions, jerusalem artichokes, asparagus, leeks, garlic, and chicory 20

[19]. Inulin and OF are chains of fructose moieties linked by β-(2→1)-glycosidic bonds with 21

or without a terminal glucose unit. OF has a degree of polymerization (DP) of 2-9 and is 22

produced from inulin by controlled hydrolysis. Inulin is extracted from chicory roots and 23

contains fructose chains ranging in DP from 3 to 60. Inulin and OF pass the stomach and 24

small intestine unaltered, since they cannot be degraded or absorbed, and reach the colon 25

4

where they are fermented by the intestinal microbiota [20, 21]. Human in vivo trials have 1

demonstrated that the addition of OF and/or inulin to the diet leads to a significant increase in 2

bifidobacteria [22-26] and several studies have described in vitro fermentation of OF in pure 3

cultures of Bifidobacterium [21, 27-35]. Although the increase in bifidobacteria has been 4

clearly demonstrated in human studies with both OF and long chain inulin, it is noteworthy to 5

mention that most Bifidobacterium strains when studied in vitro are not able to ferment the 6

long inulin chains. In fact, the fermentation of oligo- and polysaccharides in the colon is the 7

result of a complex microbial metabolic activity and long inulin chains are hydrolysed by 8

extracellular enzymes, supplying OF to bifidobacteria for selective growth [22, 32, 33]. 9

During this process, SCFA and lactic acid are formed and an increase in the bacterial biomass 10

is observed [36]. SCFA are absorbed by the colon in a concentration-dependent manner and 11

are the major respiratory fuels for colonocytes, supplying up to 60-70% of their energy needs 12

[37-41]. SCFA also stimulate the growth of colorectal mucosal cells, retard mucosal atrophy 13

and decrease the risk of malignant transformation in the colon. Butyrate has been shown to be 14

particularly effective in decreasing the risk of malignant transformation of the colon [42]. 15

This study investigated the prebiotic effects of a new inulin-type fructan containing a 16

high soluble inulin by means of in vitro faecal fermentation experiments and molecular 17

techniques for microbial enumeration. OF was chosen as the positive control because of its 18

well established prebiotic properties. The impact of the two prebiotics on ammonia 19

concentration, pH, and SCFA and lactic acid production was studied in faecal cultures to 20

monitor the predominance of a saccharolytic or proteolytic metabolism. 21

22

2. MATERIALS AND METHODS 23

2.1. Chemicals and media 24

5

Inulin-type fructans investigated were oligofructose (OF, BeneoTM P95, Orafti, 1

Tienen, Belgium) and high solubility inulin (HSI, BeneoTM HSI, Orafti, Tienen, Belgium). 2

The former is composed of 95% oligosaccharides (DP 2-9) and 5% glucose, fructose and 3

sucrose; the latter is composed of 60% oligosaccharides with DP 2-9, 28% oligosaccharides 4

with DP ≥ 10 and 12% sugars. All other chemicals were obtained from Sigma-Aldrich 5

(Steinheim, Germany) unless otherwise stated. 6

To compare the selectivity of fermentation of HSI and OF, faecal cultures were carried 7

out in Faecal Extract Medium (FEM) [43]. FEM medium was obtained as follows: frozen 8

faecal samples from 8 healthy volunteers were mixed and resuspended 1:5 (w/v) with water. 9

The suspension was centrifuged (10000 x g, 15 min, 4°C) and insoluble matter was washed 10

twice with water. Supernatants from each centrifugation step were collected; the volume was 11

adjusted to obtain the final dilution factor 1:10 (w/v). The suspension was filtered through 12

filter paper and 1.2-µm pore size membrane, then autoclaved at 110°C for 30 minutes. The 13

sterile medium was stored at -20°C until the use. All the experiments were performed with the 14

same lot of FEM medium. Faecal cultures were carried out in N2-flushed anaerobic serum 15

bottles with pierceable cap containing 80 ml of FEM medium, 0.5 g l-1 L-cysteine · HCl and 16

pH adjusted to 6.0. Carbohydrates were autoclaved separately and added to the sterile basal 17

medium to obtain the concentration of 10 g L-1. Control bottles were prepared without 18

addition of any carbohydrate. 19

Inocula for faecal cultures were prepared from the fresh faeces of seven healthy 20

volunteers (four men and three women) who had not been treated with prebiotics and/or 21

probiotics for one month and antibiotics for at least three months. Faecal samples were diluted 22

100-fold in half-strength Wilkins-Chalgren anaerobe broth (Oxoid) in an anaerobic cabinet 23

(Anaerobic System, Mod. 2028, Forma Scientific Co., Marietta, OH) under a 85% N2, 10% 24

CO2, 5% H2 atmosphere. Anaerobic serum bottles were then inoculated 1% (v/v) with the 25

6

faecal suspension to obtain approximately 106 bacteria ml-1. The cultures were incubated at 1

37°C; samples were anaerobically collected by means of a syringe. 2

3

2.2. Experimental design 4

Samples from faecal cultures containing OF or HSI, and from control (CT, in absence 5

of inulin-type fructans) were collected at 0, 6, 12, 24 and 48 hours. pH, ammonia, SCFA, 6

lactic acid concentrations and microbiota composition were determined. Faecal cultures were 7

carried out in triplicate and mean values from measurements were compared using Student’s 8

t-test. For the comparison between cultures containing fructans vs. control cultures, 9

differences were considered statistically significant when P ≤ 0.05. 10

11

2.3. Bacterial enumeration by FISH technique 12

Intestinal bacterial groups were enumerated using specific Fluorescent In-Situ 13

Hybridisation (FISH) technique commercial kits (Microscreen B.V., Microbial Diagnostics, 14

Groningen, The Netherlands) for the following bacterial groups: Bifidobacterium genus (10-15

ME-H001), Lactobacillus group (10-ME-H006), Escherichia coli group (10-ME-H004), 16

Bacteroides genus (10-ME-H008), cluster I clostridia (Clostridium sensu strictu species or 17

Clostridium butyricum group) (10-ME-H009), and cluster XIV clostridia (Clostridium 18

coccoides - Eubacterium rectale group) (10-ME-H011). The slides were evaluated with a 19

Nikon Eclipse E-600 epifluorescence microscope equipped with a mercury arc lamp (Nikon, 20

HBO, 100W) and the FITC specific filter Nikon BA 520. Each sample was enumerated in 21

triplicate. Depending on the number of fluorescent cells, 30 to 100 microscopic fields were 22

counted and averaged in each slide. 23

24

2.4.Chemical analyses 25

7

The samples collected from faecal cultures were immediately chilled at 0°C, 1

centrifuged and filtered (cellulose acetate syringe filter 0.22 µm, Albet Filalbet, Barcelona, 2

Spain) to remove cells and to avoid changes in analytes concentration resulting from active 3

metabolism. 4

Ammonia was measured using a commercial kit (Urea/BUN – Color, BioSystems 5

S.A., Barcelona, Spain). 6

Formic, acetic, propionic, butyric, valeric, isovaleric, and lactic acids were measured 7

using a gas chromatographic method for direct analysis of short-chain (C2-C12) volatile 8

organic acids [44]. The standard solutions were made in water with each volatile organic acid 9

at the final concentration of 0.5% (w/v) and were used to calculate the relative response factor 10

to internal standard 1,3-propanediol. 800 µl of each sample were mixed with 100 µl 1,3-11

propanediol (1%) and 100 µl of 0.4 M HCl. 1 µl of each treated sample was directly injected 12

into Hewlett Packard 5890 SERIES II gas chromatograph equipped with a flame ionization 13

detector (H2 flow rate 30 ml min-1, air flow rate 300 ml min-1) and a CP-Wax 52 CB fused 14

silica column (20 m x 0.53 mm, 1.0 mm film thickness, Chrompack, Netherlands). Glass wool 15

was inserted in the glass liner of the injection port. Detector temperature and injector port 16

temperature were 280 and 240°C, respectively. Helium was used as the carrier gas with 3 ml 17

min-1 flow rate. The oven temperature was programmed at 90°C for 1 min, raised to 180°C at 18

6°C min-1, then increased to 230°C at 20°C min-1, and finally held at 230°C for 5 min. 19

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3. RESULTS 21

3.1. Kinetics of microbial groups and total bacteria in faecal cultures 22

The numbers of bifidobacteria, lactobacilli, E. coli, Bacteroides, cluster I clostridia, 23

and cluster XIV clostridia after 0, 6, 12, 24 and 48 hours of fermentation are given in figure 1. 24

Both OF and HSI produced a significant increase in bifidobacteria (fig. 1a) and lactobacilli 25

8

(fig. 1b) compared to the control (CT) cultures (P ≤ 0.05). Different kinetics of growth of 1

Bifidobacterium and Lactobacillus were observed on OF and HSI. The major increase 2

occurred after 6 h on OF and 12 h on HSI for bifidobacteria (fig. 1a), and after 24 h on OF 3

and 48 h on HSI for lactobacilli (fig. 1b). Moreover, the final concentration of both these 4

microbial groups was higher on HSI than on OF (fig. 1a, 1b). No significant differences in E. 5

coli counts were observed in the first 12 h of incubation. After 24 h, E. coli were significantly 6

lower in the cultures supplemented with fructans compared to the CT (P ≤ 0.05). After 48 h 7

the lowest concentration was obtained in the cultures containing HSI (fig. 1c). No statistically 8

significant differences in numbers of cluster XIV clostridia were observed between groups 9

(fig. 1f). Cluster I clostridia were significantly lower in the cultures with inulin-type fructans 10

compared to the CT (P ≤ 0.05) at 6, 12, and 48 h. Such difference was remarkable during the 11

first 12 h of fermentation (fig. 1e). The levels of Bacteroides increased in the control cultures 12

from the first 6 h. On the fructans, levels were significantly lower (P ≤ 0.05) at 6 h and 24 h 13

for HSI and OF respectively compared to the CT (fig. 1d). 14

15

3. 2. pH and ammonia concentration 16

pH and ammonia concentration in faecal cultures were monitored during fermentation. 17

In the absence of prebiotic fructans, pH slightly decreased in the first 12 h, then increased 18

again reaching pH 6.0 at 48 h (fig. 2). In faecal cultures that contained OF or HSI the pH 19

progressively decreased down to about 3.7 and was significantly lower (P ≤ 0.05) than in 20

control cultures from 12 h. Differences between pH values on OF and HSI were not 21

statistically significant. 22

Ammonia concentration constantly increased in control cultures, mostly during the last 23

24 hours (fig. 3). Ammonia increase was minor on both fructans (P ≤ 0.05). The levels were 24

9

significantly lower on OF than CT from the first 12h. On HSI, ammonia concentration 1

decreased in the last 24 h, leading to a null net balance at 48 h. 2

3

3. 3. Production of volatile organic acids 4

Formic, acetic, propionic, butyric, valeric, isovaleric, and lactic acids were monitored 5

during fermentation (fig. 4). Acids production was much higher in cultures containing 6

fructans than in control cultures, coherently with the pH reduction. In particular, acetic and 7

lactic acids were the major products of fructan fermentation. 8

No production of formic acid was observed during cultivation without carbohydrates, 9

whereas both fructans led to a slight accumulation of this acid (P ≤ 0.05). Differences 10

between formic acid concentration on OF and HSI were not statistically significant (fig. 4a). 11

Little or no acetic acid was accumulated in control cultures, while a significantly higher 12

concentration (P ≤ 0.05) was produced by both fructan-containing cultures (0.4 and 0.3 g l-1 13

on OF and HIS at 48 h, respectively). Production rate and yield for acetic acid were both 14

higher on OF than on HSI (fig. 4b). Amounts of propionic acid produced by all faecal cultures 15

and the final yields were not statistically different (fig. 4c, 4d). Butyric acid was significantly 16

higher on HSI at 6 and 12 h and on OF at 12 h compared to the CT (P ≤ 0.05). A small 17

amount of valeric acid was produced by all the cultures, without any significant difference 18

(fig. 4e). Little or no net accumulation of isovaleric acid was found in all the faecal cultures 19

(fig. 4f). Lactic acid was not accumulated in control cultures, while a significantly higher 20

concentration (0.2 g l-1) was produced on both fructans (P ≤ 0.05) (fig. 4g). 21

22

4. DISCUSSION 23

The aim of this study was to assess the prebiotic properties of a high solubile inulin 24

(HSI) and to compare it against oligofructose (OF), a well established prebiotic, using in vitro 25

10

faecal batch cultures. FEM medium [43], entirely based on faecal matter, was supplemented 1

with the appropriate fructan mixtures, while negative control did not contain any additional 2

carbon source. Hence, no alterations in microbiota could occur as a consequence of 3

exogenous nutrients in the cultural medium. 4

In the CT cultures, both microbiological and chemical data demonstrate that 5

proteolytic and putrefactive processes prevailed over saccharolytic and fermentative 6

metabolism. The increase in Bacteroides, cluster I clostridia, cluster XIV clostridia, and E. 7

coli prevailed over the raise in beneficial bifidobacteria and lactobacilli. Despite no additional 8

carbon source was added to the control cultures, a limited growth of saccharolytic bacteria 9

was observed. It is likely that bifidobacteria and lactobacilli grew in control cultures because 10

of fermentable residual carbohydrates, such as residual non-digestible sugars, dietary fibres 11

and mucins, present in the faecal medium [45]. Coherently, the pH decreased in the early 12

phase of incubation and started to increase when fermentable carbohydrates were depleted 13

and proteolysis overcame. The growth of proteolytic and putrefactive groups was consistent 14

with the constant increase of ammonia concentration during the whole incubation period. 15

Moreover, scarce low molecular weight organic acids were produced: the main products were 16

propionic and butyric acids, coming from Bacteroides, clostridia and eubacteria metabolism 17

[22, 46-50], whereas formic, acetic and lactic acids were almost absent. 18

The addition of the inulin-type fructans to the cultures induced a shift from proteolytic 19

into a predominant saccharolytic metabolism. A marked increase in Bifidobacterium and 20

Lactobacillus groups was seen in the presence of inulin-type fructans at the expense of 21

potentially pathogenic microbes. Preliminary results revealed that the monocultures of 22

Bifidobacterium strains belonging to the species B. adolescentis, B. infantis, B. breve, and B. 23

longum grew abundantly on both the OF or HSI. On the other side, the monocultures of 24

Lactobacillus strains belonging to the species L. plantarum, L. rhamnosus, L. reuteri, and L. 25

11

delbrueckii poorly grew on OF and grew less on HIS (unpublished data). It’s conceivable that 1

lactobacilli grew in faecal cultures by crossfeeding on mono- and oligosaccharides produced 2

by primary inulin intestinal degraders. Consistent with these microbiological changes, also the 3

microbial metabolism was favourably changed with increased production of organic acids and 4

significantly lower pH values. High levels of acetic, lactic and formic acids accumulated in 5

the presence of both OF and HSI, which is in agreement with the high numbers of 6

bifidobacteria and lactobacilli in these cultures [47, 49, 51, 52]. Moreover, propionic and 7

butyric acids were produced at a higher rate on fructans than in control cultures. Previous 8

studies demonstrated that propionic and butyric acids result from cross-feeding between 9

lactate/acetate producers and propionigenic and butyrogenic bacteria (e.g. eubacteria and 10

clostridia) [22, 48-50]. Butyrate production is distributed across many clostridial clusters, but 11

cluster XIV is a particularly abundant group of butyrate-producers in human faeces, 12

accounting for approximately 7% of total faecal bacteria [48, 53]. It comprises relatives of 13

Roseburia and Eubacterium rectale, that show high levels of butyrate production together 14

with acetate utilization [48, 54] and relatives of Eubacterium hallii and Anaerostipes caccae, 15

that convert acetate and D or L lactate into butyrate [55]. In the present study, no statistically 16

significant differences in numbers of cluster XIV clostridia were observed between groups. 17

This is possibly due to the lack of a suitable detection method for the major butyrate and 18

propionate producers within this group. 19

As a result of increased fermenting population, limited ammonia increase by 20

proteolytic bacteria was observed in the cultures containing inulin and oligofructose, 21

compared to the control ones. The significantly lower levels of ammonia, indicate ammonium 22

consumption by bacteria fermenting carbohydrates, lower proteolyis and a more favourable 23

saccharolytic fermentation pattern [56]. 24

25

12

1

5. Conclusions 2

3

OF and HSI exerted similar effects in the in vitro faecal model. OF has a well 4

established prebiotic effectiveness, therefore HSI can be regarded as an effective prebiotic as 5

well. Nevertheless different kinetics in microbial population and metabolism (pH, ammonia 6

levels, SCFA and lactic acid production, etc.) were observed on OF and HSI. The increase in 7

lactobacilli and bifidobacteria concentrations occurred earlier on OF than on HSI. This 8

difference is attributable to the different DP distribution of the tested fructans. Previous 9

studies demonstrated that the short-chains of the OF are more easily metabolized by 10

bifidobacteria than the longer chains of inulin [33]. The different DP distribution affected the 11

kinetics of microbial changes and activities, and resulted in a faster production of butyric acid 12

on HSI than on OF. Compared to control cultures, a significant (P ≤ 0.05) accumulation of 13

lactate and acetate was observed in cultures containing inulin-type fructan after the first 24 h. 14

Nevertheless, differences in acetate yield and in accumulation rates of lactate and acetate were 15

observed on OF and HSI. These differences result from the diverse DP distribution and 16

confirm that colon microbial and metabolic interactions in the colon are particularly complex 17

and current knowledge is still incomplete. 18

One of the most highly sought-after targets for prebiotics is increased colonic 19

persistence, which can result in saccharolytic metabolism and in a lowered pH extended to the 20

distal colon. This study demonstrates the prebiotic potential of highly soluble inulin (HSI), 21

due to the beneficial SCFA produced in faecal cultures. Its higher DP, compared to OF, might 22

provide a long-lasting effect throughout the colon as well. Moreover, butyrate production 23

from HSI is noteworthy since butyrate is particularly effective in decreasing the risk of 24

malignant transformation of the colon [42]. It appears that inulin-type fructans, although 25

13

having a similar structure, affect colonic metabolism differently depending on their degree of 1

polymerization. 2

3

6. ACNOWLEDGEMENTS 4

This study was partially supported by Orafti, Tienen, Belgium. 5

6

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20

LEGENDS TO FIGURES 1

Fig. 1. 2

Enumeration of Bifidobacterium (a), Lactobacillus (b), E. coli (c), Bacteroides (d), cluster I 3

clostridia (e), and cluster XIV clostridia (f) in faecal cultures containing OF, HSI or no 4

prebiotic carbohydrates at 0, 6, 12, 24, and 48 h of anaerobic incubation at 37°C. Data are 5

reported as average values of three samples ± standard deviations. Significant differences (P ≤ 6

0.05) between cultures containing OF (*) or inulin (†) and the negative control at the same 7

time point are indicated. 8

9

Fig. 2. 10

pH in faecal cultures containing OF (), HSI () or no prebiotic carbohydrates () at 0, 6, 11

12, 24, and 48 h of anaerobic incubation at 37°C. Data are reported as average values of three 12

samples ± standard deviations. Significant differences (P ≤ 0.05) between cultures containing 13

OF (*) or inulin (†) and the negative control at the same time point are indicated. 14

15

Fig. 3. 16

Ammonia concentration in faecal cultures containing OF (), HSI () or no prebiotic 17

carbohydrates () at 0, 6, 12, 24, and 48 h of anaerobic incubation at 37°C. Data are reported 18

as average values of three samples ± standard deviations. Significant differences (P ≤ 0.05) 19

between cultures containing OF (*) or inulin (†) and the negative control at the same time 20

point are indicated. 21

22

Fig. 4. 23

Concentration of formic (a), acetic (b), propionic (c), butyric (d), valeric (e), isovaleric (f), 24

and lactic (g) acids in faecal cultures containing OF (), HSI () or no prebiotic 25

21

carbohydrates () at 0, 6, 12, 24, and 48 h of anaerobic incubation at 37°C. Data are reported 1

as average values of three samples ± standard deviations. Significant differences (P ≤ 0.05) 2

between cultures containing OF (*) or inulin (†) and the negative control at the same time 3

point are indicated. 4

5

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