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Growth of selected probiotic strains with fructans from different sources relating to degree of polymerization and structure
Journal of Functional Foods 24 (2016) 264–275
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Growth of selected probiotic strains with fructans from different sources relating to degree of polymerization and structure Monika Mueller *, Jacqueline Reiner, Lisa Fleischhacker, Helmut Viernstein, Renate Loeppert, Werner Praznik Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
A R T I C L E
I N F O
A B S T R A C T
Article history:
Fructans as prebiotics are an important factor in the functional food industry due to their
Received 17 December 2015
beneficial effect on health. However, the influence of structure and degree of polymeriza-
Received in revised form 7 April
tion (dp) on their prebiotic effect is not fully elucidated so far. Unbranched inulin-type (β-
2016
2,1 linked) fructans from chicory and branched mixed-type (β-2,1 and β-2,6 linked) fructans
Accepted 10 April 2016
from agaves were separated into fractions with different dps using preparative size exclu-
Available online
sion chromatography, and the growth curves of selected probiotic strains were determined. All fructans exerted a significant growth enhancement, being higher with lower dp and with
Keywords:
branching. Lactobacillus acidophilus and L. paracasei CRL431 quickly used fractions indepen-
Fructans
dent of the dp, whereas other strains (i.e. L. reuteri) did not use or only slowly used high dp
Prebiotics
fractions. Most strains cleaved fructans into smaller units before uptake into the cells. Our
Probiotics
findings may contribute significantly to the development of improved prebiotic formulations. © 2016 Published by Elsevier Ltd.
Agave Chicory Degree of polymerization
1.
Introduction
Due to their beneficial effects on health, fructans as prebiotics are an important factor in the functional food industry. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health (Gibson & Roberfroid, 1995). Fructans are unbranched or branched fructose oligo- or polymers, either β-2,1-linked inulin-type, β-2,6linked levan-type or β-2,1 and β-2,6-linked mixed-type, and
indigestible for the human gut (Roberfroid & Delzenne, 1998). Fructose polymers are accumulated by a great variety of plants, including composites (e.g. chicory, Jerusalem artichoke), liliales (e.g. garlic, onion), asparagales (asparagus) or agavaceae (div. agave species) (Praznik & Loeppert, 2016; Praznik, Löppert, & Huber, 2007; van Loo, Coussement, de Leenheer, Hoebregs, & Smits, 1995). Only a few fructan-containing plants are currently used in the functional food industry, including chicory (Cichorium intybus), Jerusalem artichoke (Helianthus tuberosus) and agave (Agave tequilana). Chicory and Jerusalem artichoke – as composites – contain inulin-type fructan being predominantly
* Corresponding author. Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. Tel.: +43 1 4277 55414. E-mail address: [email protected] (M. Mueller). http://dx.doi.org/10.1016/j.jff.2016.04.010 1756-4646/© 2016 Published by Elsevier Ltd.
Journal of Functional Foods 24 (2016) 264–275
unbranched. Fructan from agaves possesses a mixed-type structure containing β-2,1 and β-2,6 linkages with branching characteristics (Praznik & Loeppert, 2016; Roberfroid & Delzenne, 1998). Until now the scientific focus was more concentrated on unbranched inulin-type fructan isolated from chicory and Jerusalem artichoke and not so much on the effect of mixedtype branched fructan from agaves. Fructan can exert its beneficial effect via direct or indirect mechanisms (Vogt et al., 2015). Indirect mechanisms involve a stimulation of the growth of probiotic bacteria and can be caused by their fermentation products such as short chain fatty acids. A direct effect was suggested for inulin-type fructans on lipid profile (dos Reis, da Conceição, Rosa, dos Santos Dias, & do Carmo Gouveia Peluzio, 2015) or on immunomodulation via activation of Toll-like receptors, nucleotide oligomerization domain containing proteins, C-type lectin receptors, or galectins, eventually inducing pro- and anti-inflammatory cytokines (Capitán-Cañadas et al., 2014; Vogt et al., 2015). A direct effect for agave fructooligosaccharide (FOS) on metabolic parameters (Márquez-Aguirre et al., 2013) and as immunomodulator (Moreno-Vilet et al., 2014) was suggested as well. The dp of fructans has a major impact on the kinetics of fermentation by probiotic bacteria and thus on the beneficial effect. For most probiotic strains, inulin-type fructans with lower dp lead to an earlier growth of lactobacilli and bifidobacteria than those with higher dp. Longer chain inulins, however, showed a more pronounced prebiotic effect affecting not only probiotics in the proximal colon, but also in the distal colon (Ito et al., 2011; Pompei et al., 2008; Stewart, Timm, & Slavin, 2008; van de Wiele, Boon, Possemiers, Jacobs, & Verstraete, 2007). Furthermore, the range of dp from inulin-type fructan has an impact on the short chain fatty acid profile, in such a way that increase of butyrate can only be found in faecal samples from rats fed with inulin of high dp (Kleessen, Hartmann, & Blaut, 2001). For mixed-type branched fructans from agave, a similar dependency was shown. Probiotic strains prefer fructan sources with different dps and grow only or faster with fructans of low dp (Velázquez-Martínez et al., 2014; Mueller et al, 2016). The effect of fructans from agave and chicory on food intake and weight gain was shown to be dependent on the dp and structure of fructan as well. In fact, agave fructan had a significant effect on weight reduction and on the increased secretion of peptides involved in appetite regulation, whereas inulin from chicory did not significantly show such an effect (Santiago-García & López, 2014). Recently we showed the dependency of dp and structure of fructan on the formation of short chain fatty acids playing a major role in the gut’s health. Lactate and butyrate production was higher from fructans with lower dp. Branched fructan with high dp led to a higher butyrate formation than unbranched fructans with high dp (Koenen, Cruz Rubio, Mueller, & Venema, 2016). An elucidation of the degradation pattern of fructans may help to explain the ability of probiotics to use fructans with different dp. Inulin-type fructans may be degraded either extracellularly or taken up by the bacteria and metabolized intracellularly (Tsujikawa, Nomoto, & Osawa, 2013). The presence of fructanase in the cell wall of several probiotic strains was found previously. The expression of the enzyme was en-
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hanced using inulin as sole carbon source for Lactobacillus paracasei, even more than for FOS or fructose (Goh, Lee, & Hutkins, 2007). However, the influence of mixed-type branched fructans on the induction of fructanase expression was not shown so far. In general the influence of structure and dp on the prebiotic effectivity of fructan for developing of probiotic strains is not fully elucidated yet. Thus, we compared the growth enhancement of selected probiotic strains on different samples of dps from unbranched chicory inulin and agave fructan with mixed-type and branching characteristics. In this manner we obtained information about degradation of low and high molecular fructans during bacteria development and allowed an evaluation of their metabolism.
2.
Materials and methods
2.1.
Materials
Yeast extract was obtained from Oxoid (Hampshire, UK). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MA, USA). The source for inulin-type chicory fructan was Raftiline® or Raftilose® (Orafti, Oreye, Belgium) and for mixedtype agave fructans Metlin® or Metlos® (Nekutli, Guadalajara, Mexico). The prebiotic effect of fructan was tested on seven selected probiotic strains, namely Lactobacillus paracasei ssp. paracasei CRL 431 (ATCC 55544), Lactobacillus paracasei ssp. paracasei DN114001, Lactobacillus paracasei ssp. paracasei (DSM20315), Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus reuteri (ATCC 55730), Lactobacillus acidophilus LA-5 (DSM 13241) and Bifidobacterium animalis ssp. lactis BB12 (DSM 15954). Man-Rogosa-Sharpe (MRS) liquid medium was prepared by dissolving 10 g of peptone from casein, 8 g of meat extract, 4 g of yeast extract, 1 g di-potassium-hydrogen phosphate, 2 g Tween 80, 2 g of di-ammonium-hydrogencitrate, 5 g of sodium acetate, 0.2 g of magnesium sulphate, 0.5 g cysteine hydrochloride, and 0.04 g manganese sulphate per litre. The medium was autoclaved and stored at 4 °C until further usage. MRS plates were prepared with the same components as MRS broth and additionally agar (Sigma Aldrich).
2.2.
Determination of growth curves of probiotic strains
Growth curves were derived using Bioscreen C (Oy Growth Curves Ab Ltd, Helsinki, Finland) based on a turbidity measurement as indicated by an increased OD600 value. A fresh overnight culture was prepared and cell density was determined. The cell amount for a final starting OD600 of 0.1 was calculated. The bacteria were washed three times in phosphate buffered saline (PBS) supplemented with 0.5 g/L cysteine hydrochloride and resuspended in MRS-medium without carbohydrate source. The negative control was incubated without sugar and the samples in the presence of 1% carbohydrates in honeycomb plates with a final volume of 200 µL. The plates were incubated at 37 °C for 48 hours and the OD600 was measured every hour after mixing by shaking for 15 sec. All samples were tested in triplicates at various concentrations, and the average was calculated and plotted in the graphs.
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The turning point of the growth curve corresponding to the time after which the medium of the maximal OD600 increase was reached was determined using TableCurve2D software (Systat Software Inc., San Jose, CA, USA). The growth effects obtained by turbidity measurements using Bioscreen were controlled using conventional cultivation on agar plates. A fresh overnight culture was prepared and the OD600 was diluted to 10−3, 10−4 and 10−5 and spread onto an agar plate containing 1% carbohydrates. Cells were incubated at 37 °C for 48 hours under anaerobic conditions and the colonies formed were counted.
2.3. Preparative size exclusion chromatography (SEC) of fructans Agave fructan and inulin were separated into three to five fractions with different dps by means of preparative SEC. Samples were prepared by dissolving 400 mg of fructan (Metlin® or Raftiline®) in 2 mL bi-distilled water and dissolved under heating. The column system consisted of Biogel P-2 (fine, 45–90 µm, MW range 100–1800; 90 × 2.5 cm) and Biogel P-4 (extra fine, <45 µm, MW range 800–4000; 89 × 2.5 cm) in series (gel: Bio Rad Laboratories, Inc.) as described previously (Praznik & Loeppert, 2016). As eluent sterile, bi-distilled water was used at a flow rate of 30–36 mL/h performed by peristaltic pump; the system was connected to an RI-detector (Knauer, Berlin, Germany), fraction collector and recorder (Healthcare, Co). The injected volume of sample was 2 mL, the elution volume was 800 mL, and the fraction volume was 8–9 mL (25 min). To obtain appropriate quantities, identical fractions from several runs were pooled together, freeze dried, and stored under dry condition.
2.4. Analysis of the oligo- and polysaccharides of fructan by means of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) The distribution of dps of fructan fractions was determined using an IC-3000 ion chromatography system (Dionex, Thermo Scientific) with a guard column (4 × 50 mm) and an analytical column (4 × 250 mm) of CarboPac Pa1 (applied method modified from Thermo Scientific application note 67 and CarboPac combined Product Manual; Praznik & Loeppert, 2016). The modified eluent system consisted of solvent A (0.2 M sodium hydroxide), solvent B (0.1 M sodium hydroxide) plus 0.7 M sodium acetate, solvent C (water), and solvent D (0.5 M sodium hydroxide). Samples (10 µL) were separated using a multi-stepgradient starting with equilibration using 0.1 M sodium hydroxide for 10 min, followed by an increase of sodium acetate to 21 mM for 15 min. From min 25 to 90, A was decreased from 48.5 to 15%, B was increased from 3 to 70%, and C was decreased from 48.5 to 15%. Finally the column was washed for 10 min using 0.2 M sodium hydroxide. During separation the column oven temperature was set to 30 °C and sugars were detected using an electrochemical detector (PAD-detector; Dionex). The number-average degree of polymerization (dpn) and the weight-average degree of polymerization (dpw) of fructan fractions were calculated from the distribution of dps using CPCwin software (a.h. group, uni-graz).
2.5.
Degradation analysis of fructans
The degradation patterns of the fructan samples by the probiotic strains were determined using thin layer chromatography (TLC). Bacteria were incubated at 37 °C, in the presence of 1% glucose or 1% of the respective fructan samples in MRS medium starting with an OD600 of 0.1. Every 2 hours samples were collected, centrifuged and stored at −20 °C until further analysis. Samples (2 µL) were analysed on TLC Silica gel 60 F254 (Merck, Darmstadt, Germany) with eluent system (acetonitrile/water – 17/3), developed two or three times and detected with thymol reagent (2 g thymol in 100 mL methanol with 5% sulphuric acid) as described previously (Praznik & Loeppert, 2016). A standard mixture containing glucose, fructose, sucrose and kestose (1 mg/ mL each) was applied as reference. Higher oligosaccharides were analysed using TLC as described above with an eluent system of 1-butanol/1-propanol/ethanol/water – 2/3/3/2, two times developed and detected with thymol reagent. For quantification, samples were applied in defined volume using Linomat (CAMAG, Muttenz, Switzerland), developed with the same solvent system and converted into digital data using UN-SCANIT version 4.1 software (Silk Scientific, Inc., Orem, Utah, USA).
3.
Results
3.1.
Fractionation using SEC
To elucidate the influence of dp of fructans on the growth kinetic of probiotic bacteria, the samples were separated into different dp fractions using preparative SEC. For the dp analysis of fraction and original samples, an HPAEC_PAD system being calibrated with definite fructan standards was applied (details not shown). The calculated average weight dp (dpw) and average number dp (dpn) are presented in Table 1. Dpn is in direct relationship with the molar concentration of the main population of fructan molecules at a certain dp in the samples as described in detail previously (Praznik et al., 2007). Inulin (dpn = 7) was separated into 5 fractions with a dpn ranging from 2 (F5) to 19 (F1) (Table 1). Agave fructan (dpn = 10) was
Table 1 – Average degree of polymerization (dpw, dpn) of the used fructans and fractions after preparative SEC; dpw and dpn values were calculated from the distribution of dps obtained by HPAEC-PAD analysis (analysis in duplicate).
Agave FOS (Metlos®) Agave fructan (Metlin®) AF1 AF2 AF3 Chicory FOS (Raftilose®) Chicory inulin (Raftiline®) F1 F2 F3 F4 F5
dpw
dpn
9 22 14 6 4 7 16 34 20 12 8 3
6 10 11 5 3 5 7 19 12 8 5 2
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separated into three fractions ranging from a dpn of 3 (AF3) to a dpn of 11 (AF1) (Table 1). To have more data points, the commercially available agave fructan Metlos® (dpn = 6) was also applied.
3.2.
Growth in the presence of different fructan samples
All applied fructans showed a significant growth enhancement on all tested strains, with differences depending on the probiotic strain as well as molecular structure and dp of fructan. In general, the lower the dp, the faster the induced growth of the probiotics. The correlation between growth induction and dp was strain dependent. L. paracasei CRL431 and L. acidophilus significantly grew with all fractions, also with higher dp. In fact, the growth of these two strains was promoted significantly in the presence of all tested fructan fractions with a slightly lower effect of the higher molecular AF1, dpn = 11 (Fig. 1A and B); L. rhamnosus GG could use all fructans, but grew fast and until a comparable OD600 of fructose only with AF3 (dpn = 3). AF2 (dpn = 5) and AF1 (dpn = 11) promoted growth with a significant
time delay and until a lower maximal OD600 (Fig. 1C); L. reuteri grew well with AF2 (dpn = 5) and AF3 (dpn = 3), but only slightly with AF1 (dpn = 11) (Fig. 1D); L. paracasei DSM20315 and B. animalis grew in the presence of AF3 (dpn = 3) similarly to the fructose standard (Fig. 1E–F). However, these strains could only slightly use AF2 (dpn = 5) and not significantly use AF1 (dpn = 11), indicating that they can use only low molecular mixed-type branched fructan sources well. L. paracasei DN114001 showed a similar fermentation behaviour as L. paracasei DSM20315, but in the presence of AF3 (dpn = 3) the maximal OD600 was significantly lower than that of fructose (Fig. 1G). The fermentation behaviour was similar for inulin. The development of L. paracasei CRL431 was promoted significantly in the presence of all tested inulin fractions with a slightly lower effect of the high molecular F1 (dpn = 19) (Fig. 2A); L. acidophilus grew well in the presence of all 5 inulin fractions. In the presence of F1 (dpn = 19), the development was significantly delayed compared to the lower molecular samples, but growth was still significant (Fig. 2B). L. rhamnosus GG could use all inulins, but the usage of inulin sources was highly dependent on the dp. The delay of growth enhancement compared
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Fig. 1 – Growth curve of (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001 in the presence of no sugar (control), 0.5% fructose, and 0.5% fractions from agave fructans with different dp (see Table 1) (n = 3).
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(E)
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Fig. 1 – (continued)
to fructose was highest for F1 (dpn = 19), followed by F2 (dpn = 12), F3 (dpn = 8), F4 (dpn = 5) and F5 (dpn = 2) as expected (Fig. 2C). L. reuteri grew well with F5 (dpn = 2) only, whereas the growth with all other fractions was significantly induced, but delayed and with a significantly lower maximal OD 600 (Fig. 2D). L. paracasei DSM20315 grew in the presence of F5 (dpn = 2) even faster than the fructose standard, and it could also metabolize F4 (dpn = 5) but with a significant delay and reached lower maximal OD600. However, L. paracasei DSM20315 could not use F1 (dpn = 19) to F3 (dpn = 8) (Fig. 2E). Similarly the development of B. animalis in the presence of inulin was highly dependent on the molecular weight. In fact, B. animalis could not use F1 to F3, whereas it grew very slowly with F4 (dpn = 5) and well with F5 (dpn = 2) (Fig. 2F). The growth of L. paracasei DN114001 was only significantly promoted with F5 (dpn = 2), but with a delay of 12 hours compared to fructose (Fig. 2G). The time after which the half maximal OD600 was reached (ET50) was lowest for glucose (ranging from 3 to 9 hours), followed by fructose (ranging from 5 to 11 hours) for all probiotic strains (Table 2). For three strains (L. acidophilus, L. rhamnosus GG and L. paracasei CRL431), the ET50 value could be determined for all fractions. As expected, the ET50 was dependent on the dp (Table 2A), ranging from 8 to 14 hours for fractions from agave fructans and 11 to 24 hours for fractions from inulin
for L. acidophilus. Interestingly for L. paracasei CRL431, the ET50 values were comparable for F2 to F5 from inulin and for AF2 and AF3 from agave fructan. Only the fraction with the highest dp AF1 led to a significantly higher ET50. For L. rhamnosus GG the ET50 values caused by fractions from inulin ranged from 20 to 33 h, and thus indicating a significantly delayed growth promotion. AF3 from agave fructan led to an ET50 of 6 hours, whereas the other fractions led to a significant delay of growth promotion with ET50 of 23 and 30 hours, respectively. AF3 from agave fructan and F4 and F5 from inulin exerted significant growth promotions of L. paracasei DSM20315, with ET50 values ranging from 8 to 12 hours. The fraction with the lowest dp from both fructans sources exerted a growth induction of B. animalis and L. paracasei DN114001, which resulted in ET50 values between 5 and 8 hours and 22 hours for F5 for L. paracasei DN114001. Additionally, the time of reaching 20% of the maximal OD600 value was determined to compare all samples, including those where no ET50 could be determined (Table 2B). As expected, the lower the dp of the fraction, the lower was the time of reaching the 20% value depending on the strain. Only for L. reuteri inulin of middle to high dp (F1 to F4) showed a similar value, thus indicating that in this range of dps the inulin molecules are used similarly efficiently. L. paracasei DN114001 did not reach
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Fig. 2 – Growth curve of (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001 in the presence of no sugar (control), 0.5% fructose, and 0.5% fractions from inulin with different dp (see Table 1) (n = 3).
the 20% value in the presence of either AF1 or AF2 from agave fructan or F1 to F4 from inulin. The results from the turbidity measurement were controlled by conventional culture methods on agar plates and correlated well (data not shown). On agar plates without carbohydrate (negative control), no bacteria grew at all, whereas on all other plates containing 1% glucose or fructans a significant growth corresponding to the respective turbidity was observed.
3.3. Influence of the degree of polymerization of fructan on growing induction The ET50 values or time of reaching the 20% value of the OD600 maximum caused by fructose were plotted against the dpn values of the fractions (Fig. 3). Due to a low growth induction for some strains, the dependency could only be determined for four strains. The correlation between dpn and ET50 or the time for reaching the 20% value was linear for L. acidophilus and L. paracasei CRL 431 in the presence of agave fructans and exponential for inulin (Fig. 3A and B). The values and increases were lower in the presence of agave fructans, confirming a
higher growth induction. An exception was the correlation of the ET50 of L. paracasei CRL 431 in the presence of higher molecular fractions of agave fructans. The ET50 of L. rhamnosus GG increased with the dpn of fructans exponentially, with lower ET50 values for the agave fructans but a higher increase of the ET50. The same dependency was found for the time after which the 20% value of the positive control was reached. For agave fructan, the dependency of dpn and time after which the 20% value was reached was in a logarithmic way, with a higher increase when compared to inulin, but with lower values at a dp below 7. The correlation of dpn and growth promotion of L. reuteri to 20% equivalent to fructose was significantly dependent in an exponential way for agave fructan and in a logarithmic way for inulin. For this strain the values and increase until a dpn of 12 were significantly lower for agave fructan, indicating a higher support for growth development.
3.4.
Degradation of fructans by probiotic strains
The time until glucose (1%) as sugar source was exhausted was quite different among the different strains, ranging from 6 to
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Fig. 2 – (continued)
24 hours. In detail no glucose could be detected in the media supernatant any more after 6 h (L. reuteri), 14 h (L. paracasei CRL 431, L. acidophilus), 18 h (L. rhamnosus GG, L. paracasei DN114001). And 24 h (L. paracasei DSM 20312, B. animalis) (data not shown). The degradation process of the fructans by probiotics was dependent on the strains and fructan structure. For some strains it was found to be a continuous cleavage of the oligosaccharides to disaccharides and monosaccharides before absorption by the cells. It can be clearly seen that mono- and disaccharides and the oligosaccharides with the lower dp are cleaved prior to oligosaccharides with higher dp. Other strains did not or hardly cleave the fructans extracellularly, so it is assumed that they are taken up before usage intracellularly. Strains that grow fast and with fractions from all dp led to a reduction of 1-kestose and oligosaccharides significantly faster than the other strains and can even use those with higher dp. These strains are L. paracasei CRL431 and L. acidophilus (Fig. 4A and B). They cut out monosaccharides from Raftilose®, Raftiline®, Metlos® and Metlin®, with the highest level of monosaccharides reached after 8–16 hours (data not shown). Strains with a delayed growth and low usage of fractions with high dp (Fig. 2), such as L. rhamnosus GG, B. animalis, and L. paracasei DN114001, reduced the amount of oligosaccharides in the medium slowly and hardly used oligosaccharides
with a dpn > 8 (Fig. 4C, 4F, and 4G). B. animalis separated fructose from Raftilose® and Raftiline®, as well as from Metlos® and Metlin®, extracellularly (data not shown). It slowly used the disaccharide and hardly used 1-kestose, indicating that fructose may be preferentially absorbed by the cells. L. paracasei (DN114001) and L. rhamnosus GG cut mono- or disaccharide from the fructans extracellularly (data not shown). Mono- and disaccharides are used fast, but 1-kestose was used slowly. Much higher amounts of disaccharides are formed from Raftilose® and Raftiline®, although the growth induced is comparable to Metlin® and Metlos®. Thus oligosaccharides from Metlos® and Metlin® may be taken up and used intracellularly. L. reuteri cleaves mono- and to a lower extent disaccharides from Raftilose®, Raftiline®, Metlos® and Metlin® before usage intracellularly (data not shown). L. reuteri does not use oligosaccharides with a dp > 3 in the first 24 hours, but can cleave the oligosaccharides up to a dp of 6 after 48 hours. This corresponds to the results of the growth curves, which show a very low, delayed growth in the presence of fractions with higher dp. L. paracasei DSM 20312 seems to cleave all oligosaccharides into mono- or disaccharides before absorption into the cell. Monosaccharides are used fast, but oligosaccharides with dp > 3 very slow (data not shown). Higher amounts of mono- and
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Table 2 – (A) Time [h] after which the half maximal OD600 is reached equivalent to medium effective time ET50 [h]; all samples are used at a final concentration of 0.5% (n = 3). (B) Time [h] after which 20% of the maximal OD600 value increase is reached compared to fructose (n = 3). B. animalis
L. acidophilus
L. paracasei ssp. paracasei DN 114001
(A) Glucose Fructose Fructan from agave AF1 AF2 AF3 Inulin from chicory F1 F2 F3 F4 F5 (B) Fructan from agave AF1 AF2 AF3 Inulin from chicory F1 F2 F3 F4 F5
5 5
L. reuteri
DSM20315
CRL 431
L. rhamnosus GG
9 9
8 9
7 10
8 11
3 9
6 6
nd nd 5
14 9 8
nd nd 8
nd nd 9
14 9 8
nd 9 8
30 23 6
nd nd nd nd 6
24 14 11 11 11
nd nd nd nd 22
nd nd nd 12 8
20 13 10 10 10
nd nd nd nd 7
33 27 22 22 20
4 3 4
11 7 5
nd nd 7
26 6 5
11 7 5
nd 7 6
27 18 4
6 3 3 3 3
21 10 8 9 8
nd nd nd nd 21
39 38 35 8 5
18 10 8 7 8
13 15 19 12 3
31 26 20 15 14
nd, not determinable.
disaccharides are formed from Raftilose® and Raftiline®, although the growth induced by all 4 fructan sources is comparable. Thus, we assume that oligosaccharides from Metlos® and Metlin® may be taken up and cleaved intracellularly.
4.
Discussion
Fructans exert a significant prebiotic effect on seven selected probiotic strains as determined based on a turbidity measurement and confirmed by conventional cultivation. In such a way, wrong positive results caused by precipitation of media components may be excluded. The prebiotic effect was dependent on the dp and structure of fructan. In fact, the lower the dp the faster the induced growth of the probiotics. The dependency of the dp on the growth promotion of the probiotic strains is highly strain dependent. Some strains grow in the presence of different dps and structural composition of fructans. As example L. paracasei CRL431 even used fructan sources of all dp ranges similarly efficiently. Some strains used only lower molecular fructans (L. paracasei DN114001 or B. animalis), whereas some grew with all fractions but much faster with lower molecular fructans (L. acidophilus, L. paracasei DSM20315, L. rhamnosus GG, L. reuteri). The results are consistent with a previous study where probiotics grew either independently on dp, only with low dp fructans or with all fructans, but better with those with lower dps (Velázquez-Martínez et al., 2014). In contrast to this previous
study, we did not find any probiotic strain in our study that did not grow at all in the presence of fructans as sole carbon source (Velázquez-Martínez et al., 2014). The preferred usage of inulintype fructans with low dps by B. animalis corresponds to a result of a previous study where only Raftilose® showed a bifidogenic effect in rats but not Raftiline® (Kleessen et al., 2001). Fraction 5 from inulin promotes growth of L. paracasei DSM20315 and L. reuteri even faster than the fructose standard, but slower than the glucose standard. Thus, this fraction may contain glucose and result in a faster growth promotion since those two strains can use glucose significantly better than fructose (Table 2). The ET50 values caused by all fructan fractions could only be determined for three strains because the growth promotion of the lower molecular samples was too low for some strains. In general, the ET50 value was lower the lower the dp of the fructan sample. For most strains, agave fructans led to lower values and increase of the ET50 than inulin. In general agave fructans of comparable dp were used more often and/or faster than inulin. This may be caused by the mixed-type structure of fructans with high branching characteristics. This structure leads to a more compact conformation and better solubility, thus being more easily absorbed by the cells and better accessible for the bacteria. Different degradation ways and patterns were found for inulin and agave fructan. Some strains cut monosaccharides extracellularly before usage in the cell, whereas other strains cleave into disaccharides. In case of L. paracasei CRL 431, L. paracasei (DN114001 and DSM 20312) and L. rhamnosus GG, no monosaccharide is formed and the amount of disaccharides
272
Journal of Functional Foods 24 (2016) 264–275
(A) ii)
i)
25
Inulin
Agave Fructan
ET50 [h]
20 R² = 0.8663 15 10
R² = 0.8812
OD600_20% [h]
25
20 15
R² = 0.8042
10 R² = 0.9918 5
5 0
5
10 dpn
15
0
20
5
10 dpn
15
20
(B) ii)
i)
20
25
ET50 [h]
OD600_20% [h]
R² = 0.8008
20
R² = 0.9063 15 10
15 R² = 0.8475 10 R² = 0.9926 5
5 0
10
20 dpn
30
0
40
(C)
5
10 dpn
15
20
ii)
i)
35
25
ET50 [h]
R² = 0.9063 15 10 5
OD600_20% [h]
30 R² = 0.8008
20
R² = 0.9434
25 20 15 10
R² = 0.9399
5 0
0
10
20 dpn
30
40
0
5
10 dpn
15
20
OD600_20% [h]
(D) 45 40 35 30 25 20 15 10 5 0
R² = 0.8847
R² = 0.9801
0
5
10 dpn
15
20
Fig. 3 – Correlation of the dpn with growth promotion: (A) L. acidophilus, (B) L. paracasei ssp. paracasei CRL431, (C) L. rhamnosus GG and (D) L. paracasei ssp. paracasei DSM 20312: (i) correlation of the dpn and the ET50; and (ii) correlation of the dpn and the time when 20% of the maximal OD600 value increase in the presence of fructose is reached. In case of agave fructans, the commercial products Metlos® and Metlin® were tested in parallel to obtain more than 3 different weight fractions. Grey circles represent agave fructan and black diamonds represent inulin.
273
Journal of Functional Foods 24 (2016) 264–275
(A)
(B)
Raftilose
Metlos
Raftiline
100
100
80
80
80
60
60
60
40
40
20
20
20
0
0
0
0
12
24
48
0
12
24
48
(C)
Raftiline
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
%
40
Raftilose
Metlos
%
100
0
12
24
48
0
12
24
0 0
48
12
24
48
0
12
24
48
(D)
Raftiline
Raftilose 80
60
60
40
40
20
20
0
0 0
12 24 time [h]
Raftiline
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
%
80
Raftilose
Metlos
100
100
0
48
12
24
0
48
0
time [h]
12
24
48
0
0 0
12
24
0
48
12
24
48
(F)
(E)
Metlos
Raftilose
Raftiline
Raftilose
Raftiline
100
100
100
100
100
80
80
80
80
60
60
60
60
40
40
40
40
40
20
20
20
20
20
0
0
0
0
%
%
80
60
0
12
24
0
48
12
24
48
0
12
24
48
0
12
24
48
0 0
12
24
48
(G)
Raftilose
Raftiline 100
80
80
60
60
40
40
20
20
%
100
0
0 0
12 24 time [h]
48
0
12 24 time [h]
48
oligosaccharide dp10 oligosaccharide dp9 oligosaccharide dp8 oligosaccharide dp7 oligosaccharide dp6 oligosaccharide dp5 oligosaccharide dp4 kestose disaccharide monosaccharide
Fig. 4 – Degradation of fructo-oligosaccharides up to a dp of 10: (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001. For Raftiline® and Raftilose® all degradation patterns could be determined. However, for Metlos® only a few patterns could be quantified, and for Metlin® the samples could not be quantified at all due to the branched structure.
stays constant. This may indicate that the initially available disaccharide is not used or that it is continuously cleaved off in a comparable rate to its uptake by the cells. The formation of disaccharides is much lower for agave fructans, whereas the growth induction is comparable. Thus, agave fructan seems to be taken up to some extent by these strains without previous cleavage to smaller units. The uptake by the cells may be possible due to the branching structure and better solubility. The highest growth induction occurs after most of the oligosaccharides are cleaved to disaccharides and monosaccharides, and the growth stops after the carbohydrate source being used
by the respective strain is exhausted. In the medium, cultivated with strains which may take up the fructans without cleavage, the carbohydrate disappears continuously. The analysis of oligosaccharides in the medium shows the faster metabolization of oligosaccharides with lower dp for all strains. The growth induction dependent on dp corresponds well with the degradation of the oligosaccharides. In such a way, strains that cannot grow in the presence of fractions of oligomers with higher dp (as shown using Bioscreen) do not use the oligosaccharides as shown by TLC analysis of the medium. Strains with the highest growth induction even with
274
Journal of Functional Foods 24 (2016) 264–275
fractions containing fructans with high dp showed the fastest decrease of oligosaccharides in the medium, including the oligosaccharide fractions with the highest dp. The dependence of the degradation pattern on the probiotic strain is consistent with previous studies (Makras, Van Acker, & De Vuyst, 2005; Tsujikawa et al., 2013). It was shown that L. paracasei degraded inulin into mono- and disaccharide units before it is intracellularly used, whereas L. delbrueckii does not degrade it extracellularly but can take it up without prior cleavage (Tsujikawa et al., 2013). The different location of degradation of fructans was shown before. In such a way, Bacteroides uses FOS faster than B. longum, which may be caused by the localization of the enzymes for cleavage being intracellular in Bifidobacteria and extracellular in Bacteroides. Thus various bacterial strains prefer different chain lengths of fructans (Van der Meulen, Makras, Verbrugghe, Adriany, & De Vuyst, 2006). Another substrate preference was shown for L. paracasei ssp. Paracasei, which uses free fructose and glucose first, followed by FOS and inulin. In case of this strain, the degradation of the larger chain lengths of inulin occurs so fast that fructose and kestose are accumulated before they can be metabolized (Makras et al., 2005). Furthermore, a combination of fructans with low and high dp would be favourable to have a fast and long lasting growth induction of the probiotic strains. A mixture of short- and longchain fructans is also preferable for a high production of shortchain fatty acids (Stewart et al., 2008).
5.
Conclusions
Inulin-type fructan from chicory and mixed-type fructan from agaves exerted a significant prebiotic effect as shown for seven probiotic strains using turbidity measurement and confirmation by cultivation on agar plates. The growth enhancement was higher with fructans of lower polymerization degree and/ or distinct branching characteristics. The determination of the kinetic of the growth induction is of importance, since a combination of fructans with low and higher dp may be preferred to induce the growth of probiotics fast, but long lasting until distal colon regions. Inulin-type unbranched fructans are more often cleaved extracellularly into mono- or disaccharides prior to fermentation, whereas mixed-type fructans are taken up by some bacterial strains and cleaved intracellularly. An intracellular cleavage is preferred because only the respective probiotics may use the fructan source. In case of an extracellular cleavage, the formed mono- and disaccharides may be used by other strains as well. Our findings on the effectivity on the prebiotic functionality dependent on structure, dp and cleaving mechanism may contribute significantly for an improved usage of pre- and probiotics in the functional food industry or as pharmaceuticals.
Conflict of interest The authors declare that there is no conflict of interest.
Acknowledgement Many thanks to Prof. W. Kneifel from the University of Natural Resources and Life Sciences, Vienna, for providing several bacterial strains.
REFERENCES
Capitán-Cañadas, F., Ortega-González, M., Guadix, E., Zarzuelo, A., Suárez, M., de Medina, F., & Martínez-Augustin, O. (2014). Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4. Molecular Nutrition & Food Research, 58, 1098–1110. dos Reis, S., da Conceição, L., Rosa, D., dos Santos Dias, M., & do Carmo Gouveia Peluzio, M. (2015). Mechanisms used by inulin-type fructans to improve the lipid profile. Nutrición Hospitalaria, 31, 528–534. Gibson, G., & Roberfroid, M. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition, 125, 1401–1412. Goh, Y. J., Lee, J. H., & Hutkins, R. W. (2007). Functional analysis of the fructo-oligosaccharide utilization operon in Lactobacillus paracasei 1195. Applied and Environmental Microbiology, 73, 5716– 5724. Ito, H., Takemura, N., Sonoyama, K., Kawagishi, H., Topping, D., Conlon, M., & Morita, T. (2011). Degree of polymerization of inulin-type fructans differentially affects number of lactic acid bacteria, intestinal immune functions, and immunoglobulin A secretion in the rat cecum. Journal of Agricultural and Food Chemistry, 59, 5771–5778. Kleessen, B., Hartmann, L., & Blaut, M. (2001). Oligofructose and long-chain inulin: Influence on the gut microbial ecology of rats associated with a human faecal flora. The British Journal of Nutrition, 86, 291–300. Koenen, M. E., Cruz Rubio, J. M., Mueller, M., & Venema, K. (2016). The effect of agave fructan products on the activity and composition of the microbiota determined in a dynamic in vitro model of the human proximal large intestine. Journal of Functional Foods 22, 201–210. Márquez-Aguirre, A., Camacho-Ruiz, R., Arriaga-Alba, M., Padilla-Camberos, E., Kirchmayr, M., Blasco, J., & González-Avila, M. (2013). Effects of Agave tequilana fructans with different degree of polymerization profiles on the body weight, blood lipids and count of fecal Lactobacilli/ Bifidobacteria in obese mice. Food & Function, 4, 1237–1244. Makras, L., Van Acker, G., & De Vuyst, L. (2005). Lactobacillus paracasei subsp. paracasei 8700:2 degrades inulin-type fructans exhibiting different degrees of polymerization. Applied and Environmental Microbiology, 71, 6531–6537. Moreno-Vilet, L., Garcia-Hernandez, M. H., Delgado-Portales, R. E., Corral-Fernandez, N. E., Cortez-Espinosa, N., Ruiz-Cabrera, M. A., & Portales-Perez, D. P. (2014). In vitro assessment of agave fructans (Agave salmiana) as prebiotics and immune system activators. International Journal of Biological Macromolecules, 63, 181–187. Mueller, M., Viernstein, H., Loeppert, R. & Praznik, W. (2016). Growth of selected probiotic strains with fructans from chicory and agaves. Agro Food Industry Hi Tech 27, 40–42. Pompei, A., Cordisco, L., Raimondi, S., Amaretti, A., Pagnoni, U. M., Matteuzzi, D., & Rossi, M. (2008). In vitro comparison of the prebiotic effects of two inulin-type fructans. Anaerobe, 14, 280– 286. Praznik, W., & Loeppert, R. (2016). Structure of fructooligosaccharides from leaves and stem of Agave tequilana
Journal of Functional Foods 24 (2016) 264–275
Weber, var. azul. Carbohydrate Research, 381, 64–73. Article in preparation. Praznik, W., Löppert, R., & Huber, A. (2007). Analysis and molecular composition of, fructans from different plant sources. In N. Shiomi, N. Benkeblia, & S. Onodera (Eds.), Recent advances in fructooligosaccharide research (pp. 93–117). Kerala, India: Research Signpost. Roberfroid, M., & Delzenne, N. (1998). Dietary fructans. Annual Review of Nutrition, 18, 117–143. Santiago-García, P., & López, M. (2014). Agavins from Agave angustifolia and Agave potatorum affect food intake, body weight gain and satiety-related hormones (GLP-1 and ghrelin) in mice. Food & Function, 5, 3311–3319. Stewart, M. L., Timm, D. A., & Slavin, J. L. (2008). Fructooligosaccharides exhibit more rapid fermentation than long-chain inulin in an in vitro fermentation system. Nutrition Research, 28, 329–334. Tsujikawa, Y., Nomoto, R., & Osawa, R. (2013). Difference in degradation patterns on inulin-type fructans among strains of Lactobacillus delbrueckii and Lactobacillus paracasei. Bioscience of Microbiota, Food and Health, 32, 157–165. van de Wiele, T., Boon, N., Possemiers, S., Jacobs, H., & Verstraete, W. (2007). Inulin-type fructans of longer
275
degree of polymerization exert more pronounced in vitro prebiotic effects. Journal of Applied Microbiology, 102, 452– 460. van Loo, J., Coussement, P., de Leenheer, L., Hoebregs, H., & Smits, G. (1995). On the presence of inulin and oligofructose as natural ingredients in the western diet. Critical Reviews in Food Science and Nutrition, 35, 525–552. Van der Meulen, R., Makras, L., Verbrugghe, K., Adriany, T., & De Vuyst, L. (2006). In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Applied and Environmental Microbiology, 72, 1006–1012. Velázquez-Martínez, J., González-Cervantes, R., HernándezGallegos, M., Mendiola, R., Aparicio, A., & Ocampo, M. (2014). Prebiotic potential of Agave angustifolia Haw fructans with different degrees of polymerization. Molecules: A Journal of Synthetic Chemistry and Natural Product Chemistry, 19, 12660– 12675. [electronic resource]. Vogt, L., Meyer, D., Pullens, G., Faas, M., Smelt, M., Venema, K., Ramasamy, U., Schols, H., & De Vos, P. (2015). Immunological properties of inulin-type fructans. Critical Reviews in Food Science and Nutrition, 55, 414–436.
Available online at www.sciencedirect.com
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Growth of selected probiotic strains with fructans from different sources relating to degree of polymerization and structure Monika Mueller *, Jacqueline Reiner, Lisa Fleischhacker, Helmut Viernstein, Renate Loeppert, Werner Praznik Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
A R T I C L E
I N F O
A B S T R A C T
Article history:
Fructans as prebiotics are an important factor in the functional food industry due to their
Received 17 December 2015
beneficial effect on health. However, the influence of structure and degree of polymeriza-
Received in revised form 7 April
tion (dp) on their prebiotic effect is not fully elucidated so far. Unbranched inulin-type (β-
2016
2,1 linked) fructans from chicory and branched mixed-type (β-2,1 and β-2,6 linked) fructans
Accepted 10 April 2016
from agaves were separated into fractions with different dps using preparative size exclu-
Available online
sion chromatography, and the growth curves of selected probiotic strains were determined. All fructans exerted a significant growth enhancement, being higher with lower dp and with
Keywords:
branching. Lactobacillus acidophilus and L. paracasei CRL431 quickly used fractions indepen-
Fructans
dent of the dp, whereas other strains (i.e. L. reuteri) did not use or only slowly used high dp
Prebiotics
fractions. Most strains cleaved fructans into smaller units before uptake into the cells. Our
Probiotics
findings may contribute significantly to the development of improved prebiotic formulations. © 2016 Published by Elsevier Ltd.
Agave Chicory Degree of polymerization
1.
Introduction
Due to their beneficial effects on health, fructans as prebiotics are an important factor in the functional food industry. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health (Gibson & Roberfroid, 1995). Fructans are unbranched or branched fructose oligo- or polymers, either β-2,1-linked inulin-type, β-2,6linked levan-type or β-2,1 and β-2,6-linked mixed-type, and
indigestible for the human gut (Roberfroid & Delzenne, 1998). Fructose polymers are accumulated by a great variety of plants, including composites (e.g. chicory, Jerusalem artichoke), liliales (e.g. garlic, onion), asparagales (asparagus) or agavaceae (div. agave species) (Praznik & Loeppert, 2016; Praznik, Löppert, & Huber, 2007; van Loo, Coussement, de Leenheer, Hoebregs, & Smits, 1995). Only a few fructan-containing plants are currently used in the functional food industry, including chicory (Cichorium intybus), Jerusalem artichoke (Helianthus tuberosus) and agave (Agave tequilana). Chicory and Jerusalem artichoke – as composites – contain inulin-type fructan being predominantly
* Corresponding author. Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. Tel.: +43 1 4277 55414. E-mail address: [email protected] (M. Mueller). http://dx.doi.org/10.1016/j.jff.2016.04.010 1756-4646/© 2016 Published by Elsevier Ltd.
Journal of Functional Foods 24 (2016) 264–275
unbranched. Fructan from agaves possesses a mixed-type structure containing β-2,1 and β-2,6 linkages with branching characteristics (Praznik & Loeppert, 2016; Roberfroid & Delzenne, 1998). Until now the scientific focus was more concentrated on unbranched inulin-type fructan isolated from chicory and Jerusalem artichoke and not so much on the effect of mixedtype branched fructan from agaves. Fructan can exert its beneficial effect via direct or indirect mechanisms (Vogt et al., 2015). Indirect mechanisms involve a stimulation of the growth of probiotic bacteria and can be caused by their fermentation products such as short chain fatty acids. A direct effect was suggested for inulin-type fructans on lipid profile (dos Reis, da Conceição, Rosa, dos Santos Dias, & do Carmo Gouveia Peluzio, 2015) or on immunomodulation via activation of Toll-like receptors, nucleotide oligomerization domain containing proteins, C-type lectin receptors, or galectins, eventually inducing pro- and anti-inflammatory cytokines (Capitán-Cañadas et al., 2014; Vogt et al., 2015). A direct effect for agave fructooligosaccharide (FOS) on metabolic parameters (Márquez-Aguirre et al., 2013) and as immunomodulator (Moreno-Vilet et al., 2014) was suggested as well. The dp of fructans has a major impact on the kinetics of fermentation by probiotic bacteria and thus on the beneficial effect. For most probiotic strains, inulin-type fructans with lower dp lead to an earlier growth of lactobacilli and bifidobacteria than those with higher dp. Longer chain inulins, however, showed a more pronounced prebiotic effect affecting not only probiotics in the proximal colon, but also in the distal colon (Ito et al., 2011; Pompei et al., 2008; Stewart, Timm, & Slavin, 2008; van de Wiele, Boon, Possemiers, Jacobs, & Verstraete, 2007). Furthermore, the range of dp from inulin-type fructan has an impact on the short chain fatty acid profile, in such a way that increase of butyrate can only be found in faecal samples from rats fed with inulin of high dp (Kleessen, Hartmann, & Blaut, 2001). For mixed-type branched fructans from agave, a similar dependency was shown. Probiotic strains prefer fructan sources with different dps and grow only or faster with fructans of low dp (Velázquez-Martínez et al., 2014; Mueller et al, 2016). The effect of fructans from agave and chicory on food intake and weight gain was shown to be dependent on the dp and structure of fructan as well. In fact, agave fructan had a significant effect on weight reduction and on the increased secretion of peptides involved in appetite regulation, whereas inulin from chicory did not significantly show such an effect (Santiago-García & López, 2014). Recently we showed the dependency of dp and structure of fructan on the formation of short chain fatty acids playing a major role in the gut’s health. Lactate and butyrate production was higher from fructans with lower dp. Branched fructan with high dp led to a higher butyrate formation than unbranched fructans with high dp (Koenen, Cruz Rubio, Mueller, & Venema, 2016). An elucidation of the degradation pattern of fructans may help to explain the ability of probiotics to use fructans with different dp. Inulin-type fructans may be degraded either extracellularly or taken up by the bacteria and metabolized intracellularly (Tsujikawa, Nomoto, & Osawa, 2013). The presence of fructanase in the cell wall of several probiotic strains was found previously. The expression of the enzyme was en-
265
hanced using inulin as sole carbon source for Lactobacillus paracasei, even more than for FOS or fructose (Goh, Lee, & Hutkins, 2007). However, the influence of mixed-type branched fructans on the induction of fructanase expression was not shown so far. In general the influence of structure and dp on the prebiotic effectivity of fructan for developing of probiotic strains is not fully elucidated yet. Thus, we compared the growth enhancement of selected probiotic strains on different samples of dps from unbranched chicory inulin and agave fructan with mixed-type and branching characteristics. In this manner we obtained information about degradation of low and high molecular fructans during bacteria development and allowed an evaluation of their metabolism.
2.
Materials and methods
2.1.
Materials
Yeast extract was obtained from Oxoid (Hampshire, UK). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MA, USA). The source for inulin-type chicory fructan was Raftiline® or Raftilose® (Orafti, Oreye, Belgium) and for mixedtype agave fructans Metlin® or Metlos® (Nekutli, Guadalajara, Mexico). The prebiotic effect of fructan was tested on seven selected probiotic strains, namely Lactobacillus paracasei ssp. paracasei CRL 431 (ATCC 55544), Lactobacillus paracasei ssp. paracasei DN114001, Lactobacillus paracasei ssp. paracasei (DSM20315), Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus reuteri (ATCC 55730), Lactobacillus acidophilus LA-5 (DSM 13241) and Bifidobacterium animalis ssp. lactis BB12 (DSM 15954). Man-Rogosa-Sharpe (MRS) liquid medium was prepared by dissolving 10 g of peptone from casein, 8 g of meat extract, 4 g of yeast extract, 1 g di-potassium-hydrogen phosphate, 2 g Tween 80, 2 g of di-ammonium-hydrogencitrate, 5 g of sodium acetate, 0.2 g of magnesium sulphate, 0.5 g cysteine hydrochloride, and 0.04 g manganese sulphate per litre. The medium was autoclaved and stored at 4 °C until further usage. MRS plates were prepared with the same components as MRS broth and additionally agar (Sigma Aldrich).
2.2.
Determination of growth curves of probiotic strains
Growth curves were derived using Bioscreen C (Oy Growth Curves Ab Ltd, Helsinki, Finland) based on a turbidity measurement as indicated by an increased OD600 value. A fresh overnight culture was prepared and cell density was determined. The cell amount for a final starting OD600 of 0.1 was calculated. The bacteria were washed three times in phosphate buffered saline (PBS) supplemented with 0.5 g/L cysteine hydrochloride and resuspended in MRS-medium without carbohydrate source. The negative control was incubated without sugar and the samples in the presence of 1% carbohydrates in honeycomb plates with a final volume of 200 µL. The plates were incubated at 37 °C for 48 hours and the OD600 was measured every hour after mixing by shaking for 15 sec. All samples were tested in triplicates at various concentrations, and the average was calculated and plotted in the graphs.
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Journal of Functional Foods 24 (2016) 264–275
The turning point of the growth curve corresponding to the time after which the medium of the maximal OD600 increase was reached was determined using TableCurve2D software (Systat Software Inc., San Jose, CA, USA). The growth effects obtained by turbidity measurements using Bioscreen were controlled using conventional cultivation on agar plates. A fresh overnight culture was prepared and the OD600 was diluted to 10−3, 10−4 and 10−5 and spread onto an agar plate containing 1% carbohydrates. Cells were incubated at 37 °C for 48 hours under anaerobic conditions and the colonies formed were counted.
2.3. Preparative size exclusion chromatography (SEC) of fructans Agave fructan and inulin were separated into three to five fractions with different dps by means of preparative SEC. Samples were prepared by dissolving 400 mg of fructan (Metlin® or Raftiline®) in 2 mL bi-distilled water and dissolved under heating. The column system consisted of Biogel P-2 (fine, 45–90 µm, MW range 100–1800; 90 × 2.5 cm) and Biogel P-4 (extra fine, <45 µm, MW range 800–4000; 89 × 2.5 cm) in series (gel: Bio Rad Laboratories, Inc.) as described previously (Praznik & Loeppert, 2016). As eluent sterile, bi-distilled water was used at a flow rate of 30–36 mL/h performed by peristaltic pump; the system was connected to an RI-detector (Knauer, Berlin, Germany), fraction collector and recorder (Healthcare, Co). The injected volume of sample was 2 mL, the elution volume was 800 mL, and the fraction volume was 8–9 mL (25 min). To obtain appropriate quantities, identical fractions from several runs were pooled together, freeze dried, and stored under dry condition.
2.4. Analysis of the oligo- and polysaccharides of fructan by means of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) The distribution of dps of fructan fractions was determined using an IC-3000 ion chromatography system (Dionex, Thermo Scientific) with a guard column (4 × 50 mm) and an analytical column (4 × 250 mm) of CarboPac Pa1 (applied method modified from Thermo Scientific application note 67 and CarboPac combined Product Manual; Praznik & Loeppert, 2016). The modified eluent system consisted of solvent A (0.2 M sodium hydroxide), solvent B (0.1 M sodium hydroxide) plus 0.7 M sodium acetate, solvent C (water), and solvent D (0.5 M sodium hydroxide). Samples (10 µL) were separated using a multi-stepgradient starting with equilibration using 0.1 M sodium hydroxide for 10 min, followed by an increase of sodium acetate to 21 mM for 15 min. From min 25 to 90, A was decreased from 48.5 to 15%, B was increased from 3 to 70%, and C was decreased from 48.5 to 15%. Finally the column was washed for 10 min using 0.2 M sodium hydroxide. During separation the column oven temperature was set to 30 °C and sugars were detected using an electrochemical detector (PAD-detector; Dionex). The number-average degree of polymerization (dpn) and the weight-average degree of polymerization (dpw) of fructan fractions were calculated from the distribution of dps using CPCwin software (a.h. group, uni-graz).
2.5.
Degradation analysis of fructans
The degradation patterns of the fructan samples by the probiotic strains were determined using thin layer chromatography (TLC). Bacteria were incubated at 37 °C, in the presence of 1% glucose or 1% of the respective fructan samples in MRS medium starting with an OD600 of 0.1. Every 2 hours samples were collected, centrifuged and stored at −20 °C until further analysis. Samples (2 µL) were analysed on TLC Silica gel 60 F254 (Merck, Darmstadt, Germany) with eluent system (acetonitrile/water – 17/3), developed two or three times and detected with thymol reagent (2 g thymol in 100 mL methanol with 5% sulphuric acid) as described previously (Praznik & Loeppert, 2016). A standard mixture containing glucose, fructose, sucrose and kestose (1 mg/ mL each) was applied as reference. Higher oligosaccharides were analysed using TLC as described above with an eluent system of 1-butanol/1-propanol/ethanol/water – 2/3/3/2, two times developed and detected with thymol reagent. For quantification, samples were applied in defined volume using Linomat (CAMAG, Muttenz, Switzerland), developed with the same solvent system and converted into digital data using UN-SCANIT version 4.1 software (Silk Scientific, Inc., Orem, Utah, USA).
3.
Results
3.1.
Fractionation using SEC
To elucidate the influence of dp of fructans on the growth kinetic of probiotic bacteria, the samples were separated into different dp fractions using preparative SEC. For the dp analysis of fraction and original samples, an HPAEC_PAD system being calibrated with definite fructan standards was applied (details not shown). The calculated average weight dp (dpw) and average number dp (dpn) are presented in Table 1. Dpn is in direct relationship with the molar concentration of the main population of fructan molecules at a certain dp in the samples as described in detail previously (Praznik et al., 2007). Inulin (dpn = 7) was separated into 5 fractions with a dpn ranging from 2 (F5) to 19 (F1) (Table 1). Agave fructan (dpn = 10) was
Table 1 – Average degree of polymerization (dpw, dpn) of the used fructans and fractions after preparative SEC; dpw and dpn values were calculated from the distribution of dps obtained by HPAEC-PAD analysis (analysis in duplicate).
Agave FOS (Metlos®) Agave fructan (Metlin®) AF1 AF2 AF3 Chicory FOS (Raftilose®) Chicory inulin (Raftiline®) F1 F2 F3 F4 F5
dpw
dpn
9 22 14 6 4 7 16 34 20 12 8 3
6 10 11 5 3 5 7 19 12 8 5 2
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Journal of Functional Foods 24 (2016) 264–275
separated into three fractions ranging from a dpn of 3 (AF3) to a dpn of 11 (AF1) (Table 1). To have more data points, the commercially available agave fructan Metlos® (dpn = 6) was also applied.
3.2.
Growth in the presence of different fructan samples
All applied fructans showed a significant growth enhancement on all tested strains, with differences depending on the probiotic strain as well as molecular structure and dp of fructan. In general, the lower the dp, the faster the induced growth of the probiotics. The correlation between growth induction and dp was strain dependent. L. paracasei CRL431 and L. acidophilus significantly grew with all fractions, also with higher dp. In fact, the growth of these two strains was promoted significantly in the presence of all tested fructan fractions with a slightly lower effect of the higher molecular AF1, dpn = 11 (Fig. 1A and B); L. rhamnosus GG could use all fructans, but grew fast and until a comparable OD600 of fructose only with AF3 (dpn = 3). AF2 (dpn = 5) and AF1 (dpn = 11) promoted growth with a significant
time delay and until a lower maximal OD600 (Fig. 1C); L. reuteri grew well with AF2 (dpn = 5) and AF3 (dpn = 3), but only slightly with AF1 (dpn = 11) (Fig. 1D); L. paracasei DSM20315 and B. animalis grew in the presence of AF3 (dpn = 3) similarly to the fructose standard (Fig. 1E–F). However, these strains could only slightly use AF2 (dpn = 5) and not significantly use AF1 (dpn = 11), indicating that they can use only low molecular mixed-type branched fructan sources well. L. paracasei DN114001 showed a similar fermentation behaviour as L. paracasei DSM20315, but in the presence of AF3 (dpn = 3) the maximal OD600 was significantly lower than that of fructose (Fig. 1G). The fermentation behaviour was similar for inulin. The development of L. paracasei CRL431 was promoted significantly in the presence of all tested inulin fractions with a slightly lower effect of the high molecular F1 (dpn = 19) (Fig. 2A); L. acidophilus grew well in the presence of all 5 inulin fractions. In the presence of F1 (dpn = 19), the development was significantly delayed compared to the lower molecular samples, but growth was still significant (Fig. 2B). L. rhamnosus GG could use all inulins, but the usage of inulin sources was highly dependent on the dp. The delay of growth enhancement compared
(B)
(A)
1,4
1,2
1,2
1,0
1,0
OD600
OD600
0,8
0,6
0,4
0,8 0,6 0,4
0,2
0,2 0,0
0,0 0
10
20
30
0
40
10
20
30
40
30
40
time [h]
time [h]
(D)
(C)
1,2
1,8 1,6
1,0 1,4
0,8
OD600
OD600
1,2 1,0 0,8 0,6
0,6
0,4
0,4
0,2 0,2 0,0
0,0 0
10
20
30
time [h]
40
0
10
20
time [h]
Fig. 1 – Growth curve of (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001 in the presence of no sugar (control), 0.5% fructose, and 0.5% fractions from agave fructans with different dp (see Table 1) (n = 3).
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(E)
(F) 0,8
1,2
0,7
1,0
0,6
OD600
OD600
0,8
0,6
0,4
0,5 0,4 0,3
0,2
0,2 0,1
0,0 0
10
20
30
40
0
10
time [h]
20
30
40
time [h]
(G) 1,8 1,6 1,4
OD600
1,2 control Fructose AF1 AF2 AF3
1,0 0,8 0,6 0,4 0,2 0,0 0
10
20
30
40
time [h]
Fig. 1 – (continued)
to fructose was highest for F1 (dpn = 19), followed by F2 (dpn = 12), F3 (dpn = 8), F4 (dpn = 5) and F5 (dpn = 2) as expected (Fig. 2C). L. reuteri grew well with F5 (dpn = 2) only, whereas the growth with all other fractions was significantly induced, but delayed and with a significantly lower maximal OD 600 (Fig. 2D). L. paracasei DSM20315 grew in the presence of F5 (dpn = 2) even faster than the fructose standard, and it could also metabolize F4 (dpn = 5) but with a significant delay and reached lower maximal OD600. However, L. paracasei DSM20315 could not use F1 (dpn = 19) to F3 (dpn = 8) (Fig. 2E). Similarly the development of B. animalis in the presence of inulin was highly dependent on the molecular weight. In fact, B. animalis could not use F1 to F3, whereas it grew very slowly with F4 (dpn = 5) and well with F5 (dpn = 2) (Fig. 2F). The growth of L. paracasei DN114001 was only significantly promoted with F5 (dpn = 2), but with a delay of 12 hours compared to fructose (Fig. 2G). The time after which the half maximal OD600 was reached (ET50) was lowest for glucose (ranging from 3 to 9 hours), followed by fructose (ranging from 5 to 11 hours) for all probiotic strains (Table 2). For three strains (L. acidophilus, L. rhamnosus GG and L. paracasei CRL431), the ET50 value could be determined for all fractions. As expected, the ET50 was dependent on the dp (Table 2A), ranging from 8 to 14 hours for fractions from agave fructans and 11 to 24 hours for fractions from inulin
for L. acidophilus. Interestingly for L. paracasei CRL431, the ET50 values were comparable for F2 to F5 from inulin and for AF2 and AF3 from agave fructan. Only the fraction with the highest dp AF1 led to a significantly higher ET50. For L. rhamnosus GG the ET50 values caused by fractions from inulin ranged from 20 to 33 h, and thus indicating a significantly delayed growth promotion. AF3 from agave fructan led to an ET50 of 6 hours, whereas the other fractions led to a significant delay of growth promotion with ET50 of 23 and 30 hours, respectively. AF3 from agave fructan and F4 and F5 from inulin exerted significant growth promotions of L. paracasei DSM20315, with ET50 values ranging from 8 to 12 hours. The fraction with the lowest dp from both fructans sources exerted a growth induction of B. animalis and L. paracasei DN114001, which resulted in ET50 values between 5 and 8 hours and 22 hours for F5 for L. paracasei DN114001. Additionally, the time of reaching 20% of the maximal OD600 value was determined to compare all samples, including those where no ET50 could be determined (Table 2B). As expected, the lower the dp of the fraction, the lower was the time of reaching the 20% value depending on the strain. Only for L. reuteri inulin of middle to high dp (F1 to F4) showed a similar value, thus indicating that in this range of dps the inulin molecules are used similarly efficiently. L. paracasei DN114001 did not reach
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(B)
(A)
1,2
1,4 1,2
1,0
1,0
OD600
OD600
0,8 0,8 0,6
0,6
0,4 0,4 0,2
0,2
0,0
0,0 0
10
20
30
0
40
10
20
30
40
30
40
time [h]
time [h]
(D)
(C)
1,2
1,8 1,6
1,0 1,4
0,8
OD600
OD600
1,2 1,0 0,8
0,6
0,4
0,6 0,4
0,2 0,2
0,0
0,0 0
10
20
30
0
40
10
20
time [h]
time [h]
Fig. 2 – Growth curve of (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001 in the presence of no sugar (control), 0.5% fructose, and 0.5% fractions from inulin with different dp (see Table 1) (n = 3).
the 20% value in the presence of either AF1 or AF2 from agave fructan or F1 to F4 from inulin. The results from the turbidity measurement were controlled by conventional culture methods on agar plates and correlated well (data not shown). On agar plates without carbohydrate (negative control), no bacteria grew at all, whereas on all other plates containing 1% glucose or fructans a significant growth corresponding to the respective turbidity was observed.
3.3. Influence of the degree of polymerization of fructan on growing induction The ET50 values or time of reaching the 20% value of the OD600 maximum caused by fructose were plotted against the dpn values of the fractions (Fig. 3). Due to a low growth induction for some strains, the dependency could only be determined for four strains. The correlation between dpn and ET50 or the time for reaching the 20% value was linear for L. acidophilus and L. paracasei CRL 431 in the presence of agave fructans and exponential for inulin (Fig. 3A and B). The values and increases were lower in the presence of agave fructans, confirming a
higher growth induction. An exception was the correlation of the ET50 of L. paracasei CRL 431 in the presence of higher molecular fractions of agave fructans. The ET50 of L. rhamnosus GG increased with the dpn of fructans exponentially, with lower ET50 values for the agave fructans but a higher increase of the ET50. The same dependency was found for the time after which the 20% value of the positive control was reached. For agave fructan, the dependency of dpn and time after which the 20% value was reached was in a logarithmic way, with a higher increase when compared to inulin, but with lower values at a dp below 7. The correlation of dpn and growth promotion of L. reuteri to 20% equivalent to fructose was significantly dependent in an exponential way for agave fructan and in a logarithmic way for inulin. For this strain the values and increase until a dpn of 12 were significantly lower for agave fructan, indicating a higher support for growth development.
3.4.
Degradation of fructans by probiotic strains
The time until glucose (1%) as sugar source was exhausted was quite different among the different strains, ranging from 6 to
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(F)
(E) 1,2
1,0
1,0
0,8
OD600
OD600
0,8
0,6
0,6
0,4
0,4
0,2
0,2
0,0
0,0 0
10
20
30
40
0
10
time [h]
20
30
40
time [h]
(G) 1,8 1,6 1,4
OD600
1,2 control Fructose F1 F2 F3 F4 F5
1,0 0,8 0,6 0,4 0,2 0,0 0
10
20
30
40
time [h]
Fig. 2 – (continued)
24 hours. In detail no glucose could be detected in the media supernatant any more after 6 h (L. reuteri), 14 h (L. paracasei CRL 431, L. acidophilus), 18 h (L. rhamnosus GG, L. paracasei DN114001). And 24 h (L. paracasei DSM 20312, B. animalis) (data not shown). The degradation process of the fructans by probiotics was dependent on the strains and fructan structure. For some strains it was found to be a continuous cleavage of the oligosaccharides to disaccharides and monosaccharides before absorption by the cells. It can be clearly seen that mono- and disaccharides and the oligosaccharides with the lower dp are cleaved prior to oligosaccharides with higher dp. Other strains did not or hardly cleave the fructans extracellularly, so it is assumed that they are taken up before usage intracellularly. Strains that grow fast and with fractions from all dp led to a reduction of 1-kestose and oligosaccharides significantly faster than the other strains and can even use those with higher dp. These strains are L. paracasei CRL431 and L. acidophilus (Fig. 4A and B). They cut out monosaccharides from Raftilose®, Raftiline®, Metlos® and Metlin®, with the highest level of monosaccharides reached after 8–16 hours (data not shown). Strains with a delayed growth and low usage of fractions with high dp (Fig. 2), such as L. rhamnosus GG, B. animalis, and L. paracasei DN114001, reduced the amount of oligosaccharides in the medium slowly and hardly used oligosaccharides
with a dpn > 8 (Fig. 4C, 4F, and 4G). B. animalis separated fructose from Raftilose® and Raftiline®, as well as from Metlos® and Metlin®, extracellularly (data not shown). It slowly used the disaccharide and hardly used 1-kestose, indicating that fructose may be preferentially absorbed by the cells. L. paracasei (DN114001) and L. rhamnosus GG cut mono- or disaccharide from the fructans extracellularly (data not shown). Mono- and disaccharides are used fast, but 1-kestose was used slowly. Much higher amounts of disaccharides are formed from Raftilose® and Raftiline®, although the growth induced is comparable to Metlin® and Metlos®. Thus oligosaccharides from Metlos® and Metlin® may be taken up and used intracellularly. L. reuteri cleaves mono- and to a lower extent disaccharides from Raftilose®, Raftiline®, Metlos® and Metlin® before usage intracellularly (data not shown). L. reuteri does not use oligosaccharides with a dp > 3 in the first 24 hours, but can cleave the oligosaccharides up to a dp of 6 after 48 hours. This corresponds to the results of the growth curves, which show a very low, delayed growth in the presence of fractions with higher dp. L. paracasei DSM 20312 seems to cleave all oligosaccharides into mono- or disaccharides before absorption into the cell. Monosaccharides are used fast, but oligosaccharides with dp > 3 very slow (data not shown). Higher amounts of mono- and
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Table 2 – (A) Time [h] after which the half maximal OD600 is reached equivalent to medium effective time ET50 [h]; all samples are used at a final concentration of 0.5% (n = 3). (B) Time [h] after which 20% of the maximal OD600 value increase is reached compared to fructose (n = 3). B. animalis
L. acidophilus
L. paracasei ssp. paracasei DN 114001
(A) Glucose Fructose Fructan from agave AF1 AF2 AF3 Inulin from chicory F1 F2 F3 F4 F5 (B) Fructan from agave AF1 AF2 AF3 Inulin from chicory F1 F2 F3 F4 F5
5 5
L. reuteri
DSM20315
CRL 431
L. rhamnosus GG
9 9
8 9
7 10
8 11
3 9
6 6
nd nd 5
14 9 8
nd nd 8
nd nd 9
14 9 8
nd 9 8
30 23 6
nd nd nd nd 6
24 14 11 11 11
nd nd nd nd 22
nd nd nd 12 8
20 13 10 10 10
nd nd nd nd 7
33 27 22 22 20
4 3 4
11 7 5
nd nd 7
26 6 5
11 7 5
nd 7 6
27 18 4
6 3 3 3 3
21 10 8 9 8
nd nd nd nd 21
39 38 35 8 5
18 10 8 7 8
13 15 19 12 3
31 26 20 15 14
nd, not determinable.
disaccharides are formed from Raftilose® and Raftiline®, although the growth induced by all 4 fructan sources is comparable. Thus, we assume that oligosaccharides from Metlos® and Metlin® may be taken up and cleaved intracellularly.
4.
Discussion
Fructans exert a significant prebiotic effect on seven selected probiotic strains as determined based on a turbidity measurement and confirmed by conventional cultivation. In such a way, wrong positive results caused by precipitation of media components may be excluded. The prebiotic effect was dependent on the dp and structure of fructan. In fact, the lower the dp the faster the induced growth of the probiotics. The dependency of the dp on the growth promotion of the probiotic strains is highly strain dependent. Some strains grow in the presence of different dps and structural composition of fructans. As example L. paracasei CRL431 even used fructan sources of all dp ranges similarly efficiently. Some strains used only lower molecular fructans (L. paracasei DN114001 or B. animalis), whereas some grew with all fractions but much faster with lower molecular fructans (L. acidophilus, L. paracasei DSM20315, L. rhamnosus GG, L. reuteri). The results are consistent with a previous study where probiotics grew either independently on dp, only with low dp fructans or with all fructans, but better with those with lower dps (Velázquez-Martínez et al., 2014). In contrast to this previous
study, we did not find any probiotic strain in our study that did not grow at all in the presence of fructans as sole carbon source (Velázquez-Martínez et al., 2014). The preferred usage of inulintype fructans with low dps by B. animalis corresponds to a result of a previous study where only Raftilose® showed a bifidogenic effect in rats but not Raftiline® (Kleessen et al., 2001). Fraction 5 from inulin promotes growth of L. paracasei DSM20315 and L. reuteri even faster than the fructose standard, but slower than the glucose standard. Thus, this fraction may contain glucose and result in a faster growth promotion since those two strains can use glucose significantly better than fructose (Table 2). The ET50 values caused by all fructan fractions could only be determined for three strains because the growth promotion of the lower molecular samples was too low for some strains. In general, the ET50 value was lower the lower the dp of the fructan sample. For most strains, agave fructans led to lower values and increase of the ET50 than inulin. In general agave fructans of comparable dp were used more often and/or faster than inulin. This may be caused by the mixed-type structure of fructans with high branching characteristics. This structure leads to a more compact conformation and better solubility, thus being more easily absorbed by the cells and better accessible for the bacteria. Different degradation ways and patterns were found for inulin and agave fructan. Some strains cut monosaccharides extracellularly before usage in the cell, whereas other strains cleave into disaccharides. In case of L. paracasei CRL 431, L. paracasei (DN114001 and DSM 20312) and L. rhamnosus GG, no monosaccharide is formed and the amount of disaccharides
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(A) ii)
i)
25
Inulin
Agave Fructan
ET50 [h]
20 R² = 0.8663 15 10
R² = 0.8812
OD600_20% [h]
25
20 15
R² = 0.8042
10 R² = 0.9918 5
5 0
5
10 dpn
15
0
20
5
10 dpn
15
20
(B) ii)
i)
20
25
ET50 [h]
OD600_20% [h]
R² = 0.8008
20
R² = 0.9063 15 10
15 R² = 0.8475 10 R² = 0.9926 5
5 0
10
20 dpn
30
0
40
(C)
5
10 dpn
15
20
ii)
i)
35
25
ET50 [h]
R² = 0.9063 15 10 5
OD600_20% [h]
30 R² = 0.8008
20
R² = 0.9434
25 20 15 10
R² = 0.9399
5 0
0
10
20 dpn
30
40
0
5
10 dpn
15
20
OD600_20% [h]
(D) 45 40 35 30 25 20 15 10 5 0
R² = 0.8847
R² = 0.9801
0
5
10 dpn
15
20
Fig. 3 – Correlation of the dpn with growth promotion: (A) L. acidophilus, (B) L. paracasei ssp. paracasei CRL431, (C) L. rhamnosus GG and (D) L. paracasei ssp. paracasei DSM 20312: (i) correlation of the dpn and the ET50; and (ii) correlation of the dpn and the time when 20% of the maximal OD600 value increase in the presence of fructose is reached. In case of agave fructans, the commercial products Metlos® and Metlin® were tested in parallel to obtain more than 3 different weight fractions. Grey circles represent agave fructan and black diamonds represent inulin.
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(A)
(B)
Raftilose
Metlos
Raftiline
100
100
80
80
80
60
60
60
40
40
20
20
20
0
0
0
0
12
24
48
0
12
24
48
(C)
Raftiline
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
%
40
Raftilose
Metlos
%
100
0
12
24
48
0
12
24
0 0
48
12
24
48
0
12
24
48
(D)
Raftiline
Raftilose 80
60
60
40
40
20
20
0
0 0
12 24 time [h]
Raftiline
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
%
80
Raftilose
Metlos
100
100
0
48
12
24
0
48
0
time [h]
12
24
48
0
0 0
12
24
0
48
12
24
48
(F)
(E)
Metlos
Raftilose
Raftiline
Raftilose
Raftiline
100
100
100
100
100
80
80
80
80
60
60
60
60
40
40
40
40
40
20
20
20
20
20
0
0
0
0
%
%
80
60
0
12
24
0
48
12
24
48
0
12
24
48
0
12
24
48
0 0
12
24
48
(G)
Raftilose
Raftiline 100
80
80
60
60
40
40
20
20
%
100
0
0 0
12 24 time [h]
48
0
12 24 time [h]
48
oligosaccharide dp10 oligosaccharide dp9 oligosaccharide dp8 oligosaccharide dp7 oligosaccharide dp6 oligosaccharide dp5 oligosaccharide dp4 kestose disaccharide monosaccharide
Fig. 4 – Degradation of fructo-oligosaccharides up to a dp of 10: (A) L. paracasei ssp. paracasei CRL431, (B) L. acidophilus, (C) L. rhamnosus GG, (D) L. reuteri, (E) L. paracasei ssp. paracasei DSM 20312, (F) B. animalis, and (G) L. paracasei ssp. paracasei DN114001. For Raftiline® and Raftilose® all degradation patterns could be determined. However, for Metlos® only a few patterns could be quantified, and for Metlin® the samples could not be quantified at all due to the branched structure.
stays constant. This may indicate that the initially available disaccharide is not used or that it is continuously cleaved off in a comparable rate to its uptake by the cells. The formation of disaccharides is much lower for agave fructans, whereas the growth induction is comparable. Thus, agave fructan seems to be taken up to some extent by these strains without previous cleavage to smaller units. The uptake by the cells may be possible due to the branching structure and better solubility. The highest growth induction occurs after most of the oligosaccharides are cleaved to disaccharides and monosaccharides, and the growth stops after the carbohydrate source being used
by the respective strain is exhausted. In the medium, cultivated with strains which may take up the fructans without cleavage, the carbohydrate disappears continuously. The analysis of oligosaccharides in the medium shows the faster metabolization of oligosaccharides with lower dp for all strains. The growth induction dependent on dp corresponds well with the degradation of the oligosaccharides. In such a way, strains that cannot grow in the presence of fractions of oligomers with higher dp (as shown using Bioscreen) do not use the oligosaccharides as shown by TLC analysis of the medium. Strains with the highest growth induction even with
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fractions containing fructans with high dp showed the fastest decrease of oligosaccharides in the medium, including the oligosaccharide fractions with the highest dp. The dependence of the degradation pattern on the probiotic strain is consistent with previous studies (Makras, Van Acker, & De Vuyst, 2005; Tsujikawa et al., 2013). It was shown that L. paracasei degraded inulin into mono- and disaccharide units before it is intracellularly used, whereas L. delbrueckii does not degrade it extracellularly but can take it up without prior cleavage (Tsujikawa et al., 2013). The different location of degradation of fructans was shown before. In such a way, Bacteroides uses FOS faster than B. longum, which may be caused by the localization of the enzymes for cleavage being intracellular in Bifidobacteria and extracellular in Bacteroides. Thus various bacterial strains prefer different chain lengths of fructans (Van der Meulen, Makras, Verbrugghe, Adriany, & De Vuyst, 2006). Another substrate preference was shown for L. paracasei ssp. Paracasei, which uses free fructose and glucose first, followed by FOS and inulin. In case of this strain, the degradation of the larger chain lengths of inulin occurs so fast that fructose and kestose are accumulated before they can be metabolized (Makras et al., 2005). Furthermore, a combination of fructans with low and high dp would be favourable to have a fast and long lasting growth induction of the probiotic strains. A mixture of short- and longchain fructans is also preferable for a high production of shortchain fatty acids (Stewart et al., 2008).
5.
Conclusions
Inulin-type fructan from chicory and mixed-type fructan from agaves exerted a significant prebiotic effect as shown for seven probiotic strains using turbidity measurement and confirmation by cultivation on agar plates. The growth enhancement was higher with fructans of lower polymerization degree and/ or distinct branching characteristics. The determination of the kinetic of the growth induction is of importance, since a combination of fructans with low and higher dp may be preferred to induce the growth of probiotics fast, but long lasting until distal colon regions. Inulin-type unbranched fructans are more often cleaved extracellularly into mono- or disaccharides prior to fermentation, whereas mixed-type fructans are taken up by some bacterial strains and cleaved intracellularly. An intracellular cleavage is preferred because only the respective probiotics may use the fructan source. In case of an extracellular cleavage, the formed mono- and disaccharides may be used by other strains as well. Our findings on the effectivity on the prebiotic functionality dependent on structure, dp and cleaving mechanism may contribute significantly for an improved usage of pre- and probiotics in the functional food industry or as pharmaceuticals.
Conflict of interest The authors declare that there is no conflict of interest.
Acknowledgement Many thanks to Prof. W. Kneifel from the University of Natural Resources and Life Sciences, Vienna, for providing several bacterial strains.
REFERENCES
Capitán-Cañadas, F., Ortega-González, M., Guadix, E., Zarzuelo, A., Suárez, M., de Medina, F., & Martínez-Augustin, O. (2014). Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4. Molecular Nutrition & Food Research, 58, 1098–1110. dos Reis, S., da Conceição, L., Rosa, D., dos Santos Dias, M., & do Carmo Gouveia Peluzio, M. (2015). Mechanisms used by inulin-type fructans to improve the lipid profile. Nutrición Hospitalaria, 31, 528–534. Gibson, G., & Roberfroid, M. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition, 125, 1401–1412. Goh, Y. J., Lee, J. H., & Hutkins, R. W. (2007). Functional analysis of the fructo-oligosaccharide utilization operon in Lactobacillus paracasei 1195. Applied and Environmental Microbiology, 73, 5716– 5724. Ito, H., Takemura, N., Sonoyama, K., Kawagishi, H., Topping, D., Conlon, M., & Morita, T. (2011). Degree of polymerization of inulin-type fructans differentially affects number of lactic acid bacteria, intestinal immune functions, and immunoglobulin A secretion in the rat cecum. Journal of Agricultural and Food Chemistry, 59, 5771–5778. Kleessen, B., Hartmann, L., & Blaut, M. (2001). Oligofructose and long-chain inulin: Influence on the gut microbial ecology of rats associated with a human faecal flora. The British Journal of Nutrition, 86, 291–300. Koenen, M. E., Cruz Rubio, J. M., Mueller, M., & Venema, K. (2016). The effect of agave fructan products on the activity and composition of the microbiota determined in a dynamic in vitro model of the human proximal large intestine. Journal of Functional Foods 22, 201–210. Márquez-Aguirre, A., Camacho-Ruiz, R., Arriaga-Alba, M., Padilla-Camberos, E., Kirchmayr, M., Blasco, J., & González-Avila, M. (2013). Effects of Agave tequilana fructans with different degree of polymerization profiles on the body weight, blood lipids and count of fecal Lactobacilli/ Bifidobacteria in obese mice. Food & Function, 4, 1237–1244. Makras, L., Van Acker, G., & De Vuyst, L. (2005). Lactobacillus paracasei subsp. paracasei 8700:2 degrades inulin-type fructans exhibiting different degrees of polymerization. Applied and Environmental Microbiology, 71, 6531–6537. Moreno-Vilet, L., Garcia-Hernandez, M. H., Delgado-Portales, R. E., Corral-Fernandez, N. E., Cortez-Espinosa, N., Ruiz-Cabrera, M. A., & Portales-Perez, D. P. (2014). In vitro assessment of agave fructans (Agave salmiana) as prebiotics and immune system activators. International Journal of Biological Macromolecules, 63, 181–187. Mueller, M., Viernstein, H., Loeppert, R. & Praznik, W. (2016). Growth of selected probiotic strains with fructans from chicory and agaves. Agro Food Industry Hi Tech 27, 40–42. Pompei, A., Cordisco, L., Raimondi, S., Amaretti, A., Pagnoni, U. M., Matteuzzi, D., & Rossi, M. (2008). In vitro comparison of the prebiotic effects of two inulin-type fructans. Anaerobe, 14, 280– 286. Praznik, W., & Loeppert, R. (2016). Structure of fructooligosaccharides from leaves and stem of Agave tequilana
Journal of Functional Foods 24 (2016) 264–275
Weber, var. azul. Carbohydrate Research, 381, 64–73. Article in preparation. Praznik, W., Löppert, R., & Huber, A. (2007). Analysis and molecular composition of, fructans from different plant sources. In N. Shiomi, N. Benkeblia, & S. Onodera (Eds.), Recent advances in fructooligosaccharide research (pp. 93–117). Kerala, India: Research Signpost. Roberfroid, M., & Delzenne, N. (1998). Dietary fructans. Annual Review of Nutrition, 18, 117–143. Santiago-García, P., & López, M. (2014). Agavins from Agave angustifolia and Agave potatorum affect food intake, body weight gain and satiety-related hormones (GLP-1 and ghrelin) in mice. Food & Function, 5, 3311–3319. Stewart, M. L., Timm, D. A., & Slavin, J. L. (2008). Fructooligosaccharides exhibit more rapid fermentation than long-chain inulin in an in vitro fermentation system. Nutrition Research, 28, 329–334. Tsujikawa, Y., Nomoto, R., & Osawa, R. (2013). Difference in degradation patterns on inulin-type fructans among strains of Lactobacillus delbrueckii and Lactobacillus paracasei. Bioscience of Microbiota, Food and Health, 32, 157–165. van de Wiele, T., Boon, N., Possemiers, S., Jacobs, H., & Verstraete, W. (2007). Inulin-type fructans of longer
275
degree of polymerization exert more pronounced in vitro prebiotic effects. Journal of Applied Microbiology, 102, 452– 460. van Loo, J., Coussement, P., de Leenheer, L., Hoebregs, H., & Smits, G. (1995). On the presence of inulin and oligofructose as natural ingredients in the western diet. Critical Reviews in Food Science and Nutrition, 35, 525–552. Van der Meulen, R., Makras, L., Verbrugghe, K., Adriany, T., & De Vuyst, L. (2006). In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Applied and Environmental Microbiology, 72, 1006–1012. Velázquez-Martínez, J., González-Cervantes, R., HernándezGallegos, M., Mendiola, R., Aparicio, A., & Ocampo, M. (2014). Prebiotic potential of Agave angustifolia Haw fructans with different degrees of polymerization. Molecules: A Journal of Synthetic Chemistry and Natural Product Chemistry, 19, 12660– 12675. [electronic resource]. Vogt, L., Meyer, D., Pullens, G., Faas, M., Smelt, M., Venema, K., Ramasamy, U., Schols, H., & De Vos, P. (2015). Immunological properties of inulin-type fructans. Critical Reviews in Food Science and Nutrition, 55, 414–436.