Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria

Accepted Manuscript Title: Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria Authors: Artur Szwengiel, Ghomaka Lydia Nkongha PII: DOI: Reference:

S0144-8617(18)31362-6 https://doi.org/10.1016/j.carbpol.2018.11.029 CARP 14280

To appear in: Received date: Revised date: Accepted date:

1 September 2018 17 October 2018 9 November 2018

Please cite this article as: Szwengiel A, Nkongha GL, Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.11.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria Artur Szwengiel,a* Ghomaka Lydia Nkongha,a Department of Fermentation and Biosynthesis, Institute of Food Technology of Plant Origin, Poznań University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznań, Poland a

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*Corresponding author: Artur Szwengiel, Department of Fermentation and Biosynthesis, Institute of Food Technology of Plant Origin, Poznań University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznań, Poland e-mail: [email protected]

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e-mail:

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[email protected] (A. Szwengiel) [email protected] (G.L. Nkongha)

Highlights

The significant effects of levan depolymerization was determined using RSM.

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The optimal parameters of bacterial levan endohydrolysis were estimated.

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The molar mass distribution was significantly modified.

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The degree of polymerization of levan had an effect on bacterial growth.

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Abstract

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Levan is a fructan composed of β -(2, 6) linkages in its main chain. Its health properties,

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especially its prebiotic potential can be partially modified by changing its molar mass distribution. Given that native levan is rarely fermented by probiotic bacteria, especially lactic acid bacteria (LAB), levanoligosaccharides (LOS) were produced by mild acid hydrolysis. The response surface

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methodology (RSM) was applied to determine the optimum parameters for depolymerization. Gel permeation chromatography (GPC) was used to characterize the LOS produced and to show the differences between inulin and levan. The prebiotic potential of four fractions of LOS with different molar mass distributions was investigated. MRS (Mann Rogosa Sharpe) medium supplemented with the LOS were inoculated with bacterial strains and growth was monitored by measuring the turbidity of the cultures. The utilization of oligofructans was also confirmed by RP-UHPLC-UV-ESI-MS 1

(liquid chromatography coupled with mass spectrometry) measurements of LOS derivatized with 1phenyl-3-methyl-5-pyrazolone (PMP). It was observed that the degree of polymerization of LOS has an influence on the growth of the tested bacteria.

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Key words: levan, fructooligosaccharides, GPC, RSM,

1. Introduction

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Levan is a fructan consisting mainly of β -(2, 6)-linked fructosyl residues, occasionally containing β -(2, 1)-linked branches (Öner, Hernández, & Combie, 2016). This non-structural

polysaccharide is produced by several microorganisms and a few plant species (Hamdy et al., 2017;

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Matsuhira et al., 2014; Srikanth, Reddy, Siddartha, Ramaiah, & Uppuluri, 2015). It is not produced on

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an industrial scale because its large scale production is neither economical nor efficient (Öner et al.,

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2016). This lack of industrial production might account for the limited data available on the prebiotic

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effects of levan-type fructans. It has been observed that LOS can stimulate the growth of bacteria more efficiently than native levan (Mardo et al., 2017; Porras-Domínguez et al., 2014; Yamamoto et al.,

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1999). LOS can be produced by acid or enzymatic hydrolysis of levan (Marx, Winkler, & Hartmeier, 2000; Porras-Domínguez et al., 2014).

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Inulin in contrast to levan is the best studied prebiotic (Bosscher, Loo, & Franck, 2006; Choudhary et

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al., 2018; López-Molina et al., 2005; Meyer, 2015). It is a fructan with mainly β (2-1) fructosylfructose linkages (Shoaib et al., 2016). The industrial manufacture of inulin involves its extraction from plants, mostly chicory (Gholami, Raouf Fard, Saharkhiz, & Ghani, 2018). Microbial

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fructosyltransferases and endoinulinases are involved in fructooligosaccharides (FOS) production (Meyer, 2015). Another group of fructans are so-called mixed-type fructans; they are highly branched and contain significant amounts of β-2,1 and β-2,6 linkages (Benkeblia, 2013). Mixed-type fructans include graminan fructans found in cereals such as wheat, rye and barley (Verspreet et al., 2015) and agavins which are fructans present in the stem of agave plants (Mueller et al., 2016).

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Given that the beneficial effects of inulin have been extensively discussed and reviewed in the past, a brief overview of the beneficial effects of levan, LOS and mixed-type fructans is presented below. It has been reported that levan can regulate the level of serum cholesterol and triglycerides in rats (Belghith et al., 2012; Dahech et al., 2013; Yamamoto et al., 1999) and levels of serum triglycerides, body fat and body weight in humans (S. Kang, Jang, Lim, & Song, 2003). Levan has been observed to

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exhibit antitumor activity in vitro (Yoon, Yoo, Cha, & Gyu Lee, 2004) and in vivo in mice (Yoo, Yoon, Cha, & Lee, 2004). Recent studies confirm the antitumor activity of levan and its derivatives (Abdel-Fattah, Gamal-Eldeen, Helmy, & Esawy, 2012; Kazak Sarilmiser, Ates, Ozdemir, Arga, &

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Toksoy Oner, 2015). A number of studies indicate that levan possesses antioxidant activity in vitro (Abdel-Fattah et al., 2012) and in vivo (Belghith et al., 2012; Dahech et al., 2011, 2013). In vitro

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experiments and studies in animals have demonstrated the ability of levan and LOS to influence and stimulate the immune system increasing resistance to infection and reducing inflammation (Huang,

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Chang, Chang, Tseng, & Pan, 2015; Li & Kim, 2013; Rairakhwada et al., 2007; Xu et al., 2006).

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Agavins have been observed to stimulate the growth of LAB and Bifidobacteria bacteria during in

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vitro studies (Koenen, Cruz Rubio, Mueller, & Venema, 2016; Mueller et al., 2016). A reduction in

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food intake and body weight gain has been observed in rats and mice fed a diet supplemented with agavins (Márquez-Aguirre et al., 2016; Rendón-Huerta, Juárez-Flores, Pinos-Rodríguez, Aguirre-

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Rivera, & Delgado-Portales, 2012; Urías-Silvas et al., 2008). It has been reported that the health benefits of levan are produced directly through stimulation

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of the immune system or indirectly by the stimulation of the growth and metabolism of gut bacteria, including probiotics. The broad range of potential beneficial effects depends on the structure of the levan; the most important factor affecting its properties is its molar mass. Given that bacteria prefer

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oligosaccharides with lower DP but those with a higher DP are preferred in order to obtain direct stimulation of the human body, a product with balanced molar mass is desirable. The following hypothesis was tested: the relationship between the molar mass of levan and rate of bacterial growth. The optimum parameters required to produce disperse fractions of LOS by mild acid hydrolysis of levan were determined with the use RSM. The molar mass distribution and DP of the LOS fractions produced were calculated. The growth of bacteria in culture media supplemented 3

with the LOS and the influence of DP on the growth of the bacteria was investigated. The aspects of mild acid and enzymatic hydrolysis and the application of LOS with different DP have been discussed.

2. Materials and methods

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2.1 Materials The levan used in our experiments was enzymatically synthesized in our laboratory. Pullulan

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standards (Shodex, Japan), inulin from Dahlia tubers (Fluka, Switzerland), ethanol, HCl, NaOH, NaCl, glucose with puriss. p.a. grade (POCH S.A., Poland) L-cysteine, PMP and ammonium acetate (SigmaAldrich, USA) were used. The Man, Rogosa and Sharpe medium (MRS) from VWR International,

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Radnor, PA was used but the organic and inorganic components used to prepare the MRS media

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supplemented with LOS were bought in BTL (Poland) and POCH S.A. (Poland) respectively. Diethyl

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ether, methanol and acetonitrile were obtained from Sigma-Aldrich (USA).

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2.2 Levan synthesis

Levan was synthesized in reaction medium containing 15% sucrose using partially purified

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levansucrase from Bacillus subtilis DSM 347. The optimum transfructosylation parameters were pH 5.5 in the presence of 2.5 mM Mn2+ at 50oC as described previously (Szwengiel, Goderska, &

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Gumienna, 2016). Four volumes of 96% (v/v) ethanol were used to precipitate polymer from one volume of reaction medium at 4 oC throughout the night. The precipitate was washed twice with cold

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90% ethanol to remove glucose, fructose and unreacted sucrose. It was then lyophilized and stored in a desiccator.

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2.3 Levan depolymerization Acid depolymerization was performed to obtain LOS. The optimum concentration of HCl,

temperature and reaction time required to produce the LOS most suitable for use in fermentation trials were determined by central composite design (DOE) experiments and RSM analysis (Table 1). For each experiment, 5ml of a 10% levan solution and 5 ml of HCl were put in separate tubes. The tubes

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were placed in a water bath which had been heated to the temperature at which the hydrolysis would be performed. Once the levan solution and HCl had attained the temperature at which hydrolysis would be performed, HCl was mixed with the levan solution and the mixture heated for the required amount of time. At the end of the set time, the mixture was removed from the water bath and the reaction neutralized by titration with 0.1N NaOH to pH 7. The hydrolysates were then prepared for

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analysis by GPC. The production of bulk quantities of LOS was performed in triplicate after the optimum reaction

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conditions for LOS production had been determined. 450 mL of 10% levan solution and 450 mL of

0.07 M HCl (the effective concentration during depolymerization was 0.035 M), both heated to 75 oC or 85 oC were mixed together and heated for 50 minutes. At the end of 50 minutes the reaction was

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stopped by titration with 0.1N NaOH to pH 7. The solution was put in a 1000 mL flask and distilled

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water was added after neutralization. The end concentration of the LOS solution in the flask was 0.045

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g/mL. Part of the solution was directly used in fermentation trials. The other part was precipitated with

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96% (v/v) ethanol for 24 hours at 4 oC and then lyophilized. The freeze-dried product was used to prepare a 0.045 g/ml LOS solution. The LOS obtained directly after levan depolymerization and those

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obtained after precipitation and freeze-drying were analyzed by GPC.

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2.4 GPC analysis

GPC was performed using three aqueous Ultrahydrogel columns with a guard column (Waters,

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MA, USA). The mobile phase was water supplied at 0.6 mL/min flow rate. The samples (hydrolysates) were dissolved in water and filtered before analysis with 0.45 μm PTFE hydrophilic filters (Milliore, MA, USA). The separation was performed using the SEC (size exclusion

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chromatography) equipment (Malvern, TX, USA) with triple detection (Viscotek 305 TDA) which included a combination of light scattering (LS), viscometric (Vis) and refractive index (RI) detectors. The three detectors were calibrated with the use of a pullulan standard (113 000 g/mol) to measure the absolute molar mass of each sample. The fructose and pullulan standards in a range of 5900 to 708000 g/mol were used for conventional calibration when a single detector (RI) was employed to obtain the

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relative molecular mass. The detectors and columns were thermoregulated in the same oven at 55 oC. The calculations of average molar masses (Mn – number average molar mass, Mw – weight average molar mass, Mz – z-average molar mass), – dispersity index (Mw/Mn), intrinsic viscosity ([η]), viscosimetric radius (Rη), M-H (a) – exponent (a) in the Mark-Houwink equation were performed using the Omi SEC 4.7 software (Malvern, TX, USA).

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2.5 Prebiotic potential of LOS A preliminary evaluation of the prebiotic potential of LOS was performed. Lactobacillus

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plantarum subsp. plantarum DSM 20174 from DSMZ Collection (Leibniz Institute, Potsdam,

Germany) and a commercial probiotic supplement (DOZ, Poland) containing Bifidobacterium

bulgaricus LDB01, Lactobacillus casei SD5213 were used.

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animalis lactis BS01, Lactobacillus acidophilus LA02, Streptococcus thermophilis FP4, Lactobacillus

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Dried bacterial cultures were activated by growing them in 50 mL of MRS medium (sterilized

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at 121 oC for 15 min) supplemented with 0.25% L-cysteine at 370C for 48 hours in anaerobic

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conditions in tightly closed conical flasks. After 48 hours, the cultures were centrifuged in 50 mL centrifuge tubes at 10000 g for 15 minutes at 4 oC. The supernatant was poured out and the bacterial

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cells were washed once with sterile 0.9% saline solution to remove nutrients before the fermentation trials.

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The fermentation trials were performed in bacteriological tubes containing 18 mL of

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concentrated, sterile (121 oC, 15 min) MRS* each and 2 ml of water (negative control) or 4.5% glucose solution (positive control) or 4.5% LOS solution after cold sterilization (0.22 µm PES membrane filters, Millipore, Ireland). The composition of the medium was calculated using the

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formula of DSMZ Collection. The MRS* was made of: 10 g/L peptone from casein, 5 g/L yeast extract, 1 g/L, Tween 80, 2 g/L K2HPO4, 5 g/L CH3COONa, 2 g/L (NH4) citrate, 0.2 g/L MgSO4·7H2O, 0.05 g/L MnSO4·H2O, 2.5 g/L L-cysteine. Meat extract and glucose were limited. The final concentration of LOS in tested media was 0.45%. Each tube was inoculated with the appropriate microorganisms. The cultures were added to obtain a turbidity of around 90% T (transmittance) which is equivalent to approximately 6 x 105 6

CFU/mL on the McFarland scale. The turbidity was measured using a Biolog turbidimeter (Biolog, CA, USA). The cultures were incubated at 37 oC under anaerobic conditions in tightly closed tubes. Growth was monitored by measuring the turbidity of the tubes at periodic time intervals over 72 hours. The pH of the cultures was measured directly after the end of fermentation using a pH meter (Elmetron CP-411, Poland).

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2.6 LOS derivatization LOS were derivatized with PMP using parameters described previously (Lattová, Snovida,

Perreault, & Krokhin, 2005; Stepan & Staudacher, 2011). An aqueous solution of LOS (10 µL) was

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mixed with 25 µL of 0.5 M methanolic PMP solution and 25 µL of 0.5 M NaOH. The mixtures were

incubated for 30 min at 70oC and then neutralized with 0.5 M HCl. Excess PMP reagent was removed

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by extraction. This involved the addition of 500 µL of diethyl ether to the mixtures, followed by vigorous mixing and removal of the organic layer after centrifugation at 3000g for 1 min. Extraction

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chromatography coupled with mass spectrometry.

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was performed 5 times. The derivatized samples before and after fermentation were analyzed by liquid

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2.7 RP-UHPLC-UV-ESI-MS analysis of derivatized LOS

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Reversed-phase (C18 column) ultra high-performance liquid chromatography electrospray ionization mass spectrometry (RP-UHPLC-UV-ESI-MS) analysis of derivatized LOS was performed.

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A Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific, Sunnyvale, CA, USA) coupled to a UltiMate 3000 Diode Array Detector (DAD) detector (Thermo Fisher Scientific, Sunnyvale, CA,

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USA) and a Bruker maXis impact ultra-high resolution orthogonal quadrupole-time-of-flight accelerator (qTOF) equipped with an ESI source and operated in the positive ion mode (Bruker Daltonik, Bremen, Germany) was used. The RP chromatographic separation was achieved with a

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Kinetex™ 1.7 µm C18 100 Å, LC column 100 × 2.1 mm (Phenomenex, Torrance, CA, USA). The mobile phase was composed of 8% acetonitrile in 5 mM ammonium acetate buffer (A) and 30% acetonitrile in 5 mM ammonium acetate buffer (B). The flow rate was 0.2 mL/min with a gradient elution of 0%–100% B over 60 minutes. The column temperature was set to 40 °C. The syringe and needle were washed before and after injection of each sample (water: methanol, 1:1 (v/v)). The carryover between samples was not observed. Detection was carried out at the wavelength of 245 nm 7

and DAD signals were collected in the range of 190 – 500 nm. The spectra for molecular ions [M+H]+ were extracted from full scan chromatograms (scan range: 80–1200 m/z). The ESI-MS settings were as follows: capillary voltage 4500 V, nebulizing gas 1.8 bar, and dry gas 9 L/min at 200 oC. The ESIMS system was calibrated using sodium formate cluster ions (Juo et al., 2014). The molecular ions were calculated using the rule that 1 mole of reducing saccharides reacts with 2

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moles PMP (Bai et al., 2015). The signals for hexose (DP1) and its oligomers (DPn) were detected in the mass spectrum: [DP1+H]+ = 511.219 m/z, [DP2+H]+ = 673.272 m/z, [DP3+H]+ = 835.324 m/z;

[DP4+H]+ = 997.377 m/z, [DP5+H]+ = 1159.430 m/z. Only a quality assessment was performed due to

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a lack of LOS standards. However the limit of quantification (LOQ) with UV detection has been

previously reported to be 0.26 µg/mL for glucose (DP1) and 0.15 µg/mL for isomatotriose (DP3) (Bai

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et al., 2015). Data was collected by otofCntrol 3.2 and computed using DataAnalysis 4.1 (Bruker Daltonik, Bremen, Germany).

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2.8 Statistics

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The Kruskal-Wallis ANOVA and post-hoc comparisons of mean ranks of all pairs of groups were used for variables with non-normal distribution. The distribution of data was tested with Shapiro-Wilk W

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test. The standard DOE with resolution of cube V+, defined by factors/blocks/runs: 3/1/16 was used to

determine the optimum parameters for depolymerization. The factors: HCl concentration (x1, in range

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0.005–0.035 M), reaction temperature (x2, in range 40–90 oC), reaction time (x3, in range 10–117 min) were treated as independent variables. The ranges of the different factors were determined during

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preliminary studies to obtain products without high concentration of free hexoses after reaction. The Mw and Mw/Mn were dependent variables which describe levan depolymerisation. A Pareto chart of the standardized effect estimates was used to show statistically significant effects (p < 0.05). RMS was

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used to perform a regression analysis of the collected GPC data. The calculated models include linear (L), quadratic (Q) and interaction components. Statistical analysis was performed using Statistica software, Version 12 (StatSoft Inc., Tulsa, OK, USA).

3. Results

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3.1 Molar mass distribution of levan and inulin Levan was the polymer under investigation in this study. However, inulin as a well-known prebiotic was also analyzed by GPC to show the structural differences that exist between it and bacterial levan. The molar mass distribution and hydrodynamic parameters of the polymers were characterized. The results obtained using conventional calibration (CC) were compared with those

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from triple detection (TDA) (Table 1). Taking into account CC where pullulan standards were used, relative average molar masses were computed. The Mw of levan was 2.1 times higher than the Mw of

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inulin. The dispersity index of levan was also slightly higher than that of inulin. However TDA results showed that absolute Mw of levan is 6.7 higher than that of inulin. The difference between the average molar masses of inulin estimated with CC and TDA was statistically insignificant (p > 0.05) while the

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molar masses of levan estimated with CC and TDA were significantly different (p < 0.05). Provided

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that the hydrodynamic volume of the tested polymer and standards is the same for a given molar mass,

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CC and TDA yield identical results as observed for inulin. However the values measured for levan

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were different because its conformation and structure are not identical to those of the standards used during CC. The value of exponent (a) in the Mark-Houwink equation for levan being close to 0.084 is

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consistent with this fact. This value means that the molecules are very dense. The intrinsic viscosity and viscosimetric radius of inulin were twice lower than those of levan. This is consistent with CC

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results, i.e. a bigger size gives a lower retention volume and higher relative molar mass as a consequence. Given that the true molar mass of levan is much higher than that of inulin, the utilization

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of levan by bacteria can be greatly limited by the size of its molecules. Therefore the hypothesis that molar mass distribution is a crucial factor in the growth of bacteria on levan and LOS was tested.

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Table 1. Parameters of inulin and levan estimated using GPC with triple detection (TDA) and conventional calibration (CC) Detection method Parameter* Mn (Da) Mw (Da) Mz (Da)

CC** Inulin 3.32·103 a 4.18·103 a 5.00·103 a

levan 6.84·103 b 8.83·103 b 1.03·104 b

TDA** Inulin 3.68·103 a 4.41·103 a 5.49·103 a

levan 2.27·104 c 2.98·104 c 7.69·104 c

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Mw/Mn [η] (dL/g) Rη (nm) M-H (a)

1.19 a -

1.31 a -

1.20a 0.044a 1.4a 0.317b

1.30 a 0.085b 2.8b 0.084a

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*Mn – number average molar mass, Mw – weight average molar mass, Mz – z-average molar mass, Mw/Mn – dispersity index, [η] – intrinsic viscosity, Rη – viscosimetric radius, M-H (a) – exponent (a) in the Mark-Houwink equation **CC – the relative molar mass was obtained with a single concentration detector; TDA – the absolute molar mass was measured with a light scattering detector. In addition a concentration detector and viscometer were acting in concert. Different letters show significant differences in means (p < 0.05) between values in columns.

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3.2 Depolymerization of levan

The response surface methodology was used to determine the optimum concentration of acid (HCl), temperature and reaction time required for the effective depolymerization of levan, measured as

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Mw and Mw/Mn. The main purpose was to obtain the fraction of LOS most suitable for use in

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fermentation trials. It was assumed that effective depolymerization should yield LOS with lower molar

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mass (Mw and Mn), higher dispersity index (Mw/Mn) and minimal amounts of hexoses. The DOE

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consisted of 16 experiments (runs), 2 of which were central points (Table 2). The hydrolysates from the DOE runs were analyzed by GPC with CC because light scattering signals were too small and

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noisy to calculate reliable molar mass distribution. Even though the ranges of tested parameters were fitted during preliminary studies to perform depolymerization without extensive hydrolysis, in the 8th

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run, 43% of levan was hydrolyzed into hexoses. The recovery of levan was close to 100% for the other runs. The recovery of polymers was estimated (Table 2) from the signal of a refractometric detector.

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Values less than 100% indicate hydrolysis. Where a peak of hexoses appeared, this peak was not

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integrated. This was especially observed during the 8th run (Table 2).

Table 2. Design of central composite (response surface) experiment on levan depolymerization and recovery of polymer after incubation, factors/blocks/runs: 3/1/16, center points per block (C). Run 1 2 3 4 5

HCl* (M) 0.010 0.010 0.010 0.010 0.030

Temp. (ºC) 50 50 80 80 50

Time (min) 10 90 10 90 10

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6 7 8 9 10 11 12 13 14 15 16

0.030 0.030 0.030 0.005 0.035 0.020 0.020 0.020 0.020 0.020 (C) 0.020 (C)

50 80 80 65 65 40 90 65 65 65 65

90 10 90 50 50 50 50 0 117 50 50

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*the effective concentration during depolymerization = HCl concentration used/2 (1 volume of levan solution was mixed with 1 volume of acid solution) **calculation based on the signal of conventional dual-cell refractometer (dn/dc of levan in water is 0.0795)

3.3 Depolymerization parameters determination

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The standardized effects of reaction temperature and HCl concentration on the Mw of LOS produced is shown in Fig. 1B. The HCl concentration and reaction temperature had a negative linear

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and statistically significant effect (p < 0.05) on the Mw of the LOS. A significant negative interaction

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(p < 0.05) between the linear effects of temperature and HCl concentration was also observed. This

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correlates with the fitted surface model (Fig. 1A) which shows that as temperature and HCl

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concentration increases Mw decreases and vice versa. The calculated residuals were lower than 4.7%. The normal probability plot of the raw residuals showed a strong linear pattern (data not presented).

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This also confirms the accuracy of the fitted surface model for the effect of temperature and HCl concentration on Mw of LOS and normal distribution of residuals (Shapiro-Wilk W test, p > 0.05). The

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coefficients of multiple regression were computed as follows Mw = 7612.81 (p = 0.00) + 95965.03 (p = 0.03) x1 + 19.06 (p = 0.17) x2-1825.37 (p = 0.02) x1x2; R2 = 0.710, where x1 – HCl concentration, x2 – reaction temperature. The full model in which all effects were included (significant and insignificant)

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was also calculated (Mw = 5532.05 + 198550.06x1 - 1170478.67x12 + 58.95x2 - 0.17x22 + 4.36x3 + 0.1x32 - 2628.43x1x2 - 490.04x1x3 - 0.13x2x3, where: x1 – HCl concentration, x2 – reaction temperature, x3 – reaction time). The R2 was then equal 0.867 and the residuals were lower than 4.4%. Both models were validated experimentally and the differences between measured and predicted values (∆) were lower than 5.4%.

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The effect of temperature and HCl concentration on the dispersity (Mw/Mn) of LOS was also studied. A Pareto chart of the standardized effect estimates (Fig. 1D) shows that the concentration of HCl has a statistically significant (p < 0.05) positive linear effect on the dispersity of LOS produced. This effect was 30% greater than the linear effect of temperature. The HCl concentration also has a

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statistically significant (p < 0.05) positive but quadratic effect on the Mw/Mn of the LOS. These statistical relationships between HCl concentration and temperature on the Mw/Mn of LOS are

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consistent with the fitted surface model shown in Fig. 1C. The fitted surface model shows that Mw/Mn is high at low and high HCl concentrations, while it decreases with increasing temperature. The residuals (∆ < 5.2%) of the fitted surface model showed normal distribution which was confirmed

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with Shapiro-Wilk W test (p > 0.05). The regression coefficients were estimated as follows Mw/Mn =

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1.06 (p = 0.00) - 17.70 (p = 0.12) x1 + 686.78 (0.02) x12+0.01 (p= 0.02) x2; R2 = 0.722, where x1 – HCl

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concentration, x2 – reaction temperature. The reaction time did not have a statistically significant effect

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(p > 0.05) on Mw and Mw/Mn. The full model was also calculated (Mw/Mn = 2.25-60.64x1+698.37x12 0.02x2 + 0x22 - 0.01x3 + 0.00x32 + 0.63x1x2 + 0.08x1x3+0.00x2x3, where: x1 – HCl concentration, x2 –

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reaction temperature, x3 – reaction time); R2 = 0.956, the residues were lower than 4.4%. The

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experimental validation of models was performed; the ∆ was lower than 12%.

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Fig. 1. Results of response surface experiments on levan depolymerization (factors/blocks/runs: 3/1/16); A and C – response surface of Mw and Mw/Mn, respectively, as functions of temperature and HCl concentration, B and D – Pareto chart of variables which have significant effects on Mw and Mw/Mn, respectively. Pareto charts show standardized effects (vertical lines indicate statistically significant effects): L – linear, Q – quadratic, 1Lby2L – linear interaction between variable 1 and 2

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Analysis of the molar masses and dispersity indexes of the hydrolysates from the RSM

showed that the LOS fraction from run no 10 was the fraction most suitable for use in fermentation trials (this run is labeled in Fig. 1). The reaction conditions for this run resulted in the production of

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LOS with the lowest Mw and highest Mw/Mn observed in this study. In addition, recovery was close to 100%. For the purpose of this study, a high Mw/Mn was preferred since it implies the fraction has more short chained polymers compared to native levan. Furthermore as seen from the size of the hexose peak, the LOS fraction from experiment no 10 had a similar amount of hexose (retention volume 32.8 mL) as the unhydrolyzed levan sample (Fig. 2A).

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Experiment no 10 (HCl concentration: 0.035M, temperature: 65 oC; time: 50 mins) was repeated and three additional temperatures (75, 85 and 95 oC) were tested with the purpose of determining if the Mw and Mw/Mn of the LOS fraction produced could be improved, that is a lower Mw and higher Mw/Mn without a significant increase in hexose content. The GPC chromatogram of the products from the four different reactions is shown in Fig. 2A. Increasing the reaction temperature caused a decrease in Mw as

by a broadening of the peak in the retention volume range from 25.8 to 32.2 mL.

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seen by the main peak shifting towards the right and an increase in Mw/Mn of the LOS fraction as seen

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There was a very high level of hydrolysis at 95 oC as seen from the high hexose peak of the product

(Fig. 2A). Of the four products, the one from hydrolysis at 75 oC was considered to be the best given that its molar mass is lower than that of the LOS fraction obtained at 65 oC while its hexose content is

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similar to that of the unhydrolyzed levan (Table 3). The second best is the hydrolysate from hydrolysis

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at 85 oC. However its concentration of hexoses was three times higher than that detected in the

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experiment at 75 oC.

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Bulk quantities of LOS were produced for use in the fermentation trials. LOS were produced by acid hydrolysis of levan at two temperatures: 75 oC and 85 oC. For each experiment, part of the

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hydrolysate was additionally precipitated with ethanol (96%), freeze-dried and then used to make a solution for use in fermentation. The other part was used directly in fermentation. Thus four different

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fractions of LOS were obtained. The GPC chromatograms of the four LOS fractions shows

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depolymerized levan obtained at 75 and 85 oC, before and after precipitation (Fig. 2A and Fig. 2B respectively). It is clear that hexoses were removed after precipitation but the molar mass distribution of LOS was changed also because small oligomers were not precipitated efficiently (right side of

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peak).

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Fig. 2 GPC profiles: A – native levan and samples after depolymerization at 0.035 M HCl during 50 min at four temperatures, B, C – samples of levan after depolymerization at 75 and 85 oC, respectively, or depolymerization and precipitation (prec.) with ethanol. Hexoses were eluated at 32.5 mL. The molar masses, dispersity index and hexose content of the LOS fractions are shown in

Table 3. The fractions were named following a specific pattern: fraction name/reaction temperature.

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The abbreviation “prec.” was added to the name of the fractions which were precipitated. LOS/65 and LOS/75 have the highest dispersity index making them the most heterogeneous fractions. Their hexose content is similar to that of the unhydrolyzed levan, which is the control sample. The Mw and Mn of LOS/75 (prec.) is higher than that of LOS/75 and it does not contain free hexoses. The same was observed for the Mn of LOS/85 (prec.) and LOS/85. LOS/95 has the lowest Mn, Mw and DP but its

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hexose content is greater than the LOS content. LOS/65 and LOS/95 were not precipitated because only the LOS obtained from hydrolysis at 75 and 85 oC were used for fermentation trials. The DP of samples after precipitation was higher, showing that the efficiency of precipitation of short chain oligomers was low. This is a drawback despite the fact that precipitation removed free hexoses which can give false positive growth during fermentation trials. Generally, the efficiency of precipitation was

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45% for LOS/75 (prec.) and only14% for LOS/85 (prec.).

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Table 3. Molar mass distribution, degree of polymerization and recovery of levan after depolymerization at 0.035 M HCl during 50 min at four temperatures and precipitation (prec.) with ethanol. Sample/Temp. (oC)*

Mn (Da)

Mw (Da)

Mw/Mn DP = Mn/162 DP = Mw/162 Hexose** (mg/g)

Control LOS/65 (DOE, noo 10) LOS/75 LOS/75 (prec.) LOS/85 LOS/85 (prec.) LOS/95

6.84·103 a 4.20·103 b 2.50·103 c 4.10·103 b 1.50·103 e 2.19·103 c 9.40·102 e

8.83·103 a 7.30·103 b 4.20·103 d 5.59·103 c 2.30·103 e 2.83·103 e 1.20·103 f

1.3c 1.8a 1.7a 1.4b 1.5b 1.5b 1.2c

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54a 45b 26d 35c 14e 17e 7f

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42a 26b 15c 25b 9d 13c 6d

10b 12b 20c 0a 73d 0a 515e

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Mn – number average molar mass, Mw – weight average molar mass, Mw/Mn – dispersity index, DP – degree of polymerization, different letters show significant differences in means (p < 0.05) between values in rows *control sample is the native product without depolymerization; levan depolymerized at 75 and 85 oC was analyzed with GPC directly or after precipitation (prec.) with four volumes of 96% (v/v) ethanol and dissolution with water after freeze-drying, LOS/65 is the product obtained after run noo 10 of design experiment **recorded in GPC chromatogram, the calculation based on signal of conventional dual-cell refractometer (dn/dc of fructose in water is 0.1378), milligrams of hexose per 1 g of fructan

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3.4 Degree of polymerization effect on the growth of bacteria The growth of selected cultures was monitored in culture media containing LOS obtained by

acid hydrolysis of levan during 72 hours (Fig. 3). Preliminary studies showed that native inulin and

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levan was not utilized by the bacterial strains: their growth curves in media containing inulin or levan was the same as that in culture media without sugars. The aim of this study was to determine whether Lactobacillus plantarum subsp. plantarum DSM 20174 or microorganisms from a commercial probiotic mixture could grow in culture media containing levanoligosaccharides: LOS/75, LOS/75 (prec.), LOS/85 and LOS/85(prec.). It was observed that bacteria grew in media supplemented with

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LOS and glucose: the transmittance of these media after incubation was lower than that of the control medium which was not supplemented with sugar. The growth intensity was of the order: control (negative) < LOS/75 (prec.) < LOS/85 (prec.) < LOS/75 < LOS/85 < Glc (positive sample with glucose). The lower growth intensity in media supplemented with LOS after precipitation suggests that precipitation with ethanol resulted in the removal of low molar mass chains which are easily

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fermentable. This theory was confirmed by measurements of pH, where pH values were inversely related to growth intensity, i.e. pH value was the lowest in medium supplemented with glucose (5.1 ±

0.2) and the highest in control sample (6.4 ± 0.2). The simple linear regression showed that 75% of pH

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changes in tested media can be explained by changes in transmittance. This supports the hypothesis that the molar mass distribution of LOS influences their utilization by bacteria. The composition of

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microorganisms was not determined after incubation of the commercial probiotic mixture. However it can be said with certainty that the mixture has lower nutrient requirements than Lactobacillus

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plantarum subsp. plantarum DSM 20174 because the transmittance of the negative control medium

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containing it was 30% lower than that containing Lactobacillus plantarum subsp. plantarum DSM

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20174. A comparison of the growth curves Glc and LOS/85 for the probiotic mixture, (Fig. 3B) shows

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that bacterial growth was slower in media containing LOS than that containing glucose. The intensity of growth in both media was the same after 30 hours of incubation. However during the first 30 hours,

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the change in transmittance in the Glc medium was more intense than in LOS/85. This suggests that the rate of enzymatic hydrolysis of LOS was lower than the rate at which the probiotic mixture utilized

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the carbon source.

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Fig. 3 Percent transmittance of cultures during 72 hours of incubation at 37 oC in MRS media. A – growth curves of Lactobacillus plantarum subsp. plantarum DSM 20174; B – growth curves of commercial mixture of microorganisms (DOZ S.A., Poland) containing Bifidobacterium animalis lactis BS01, Lactobacillus acidophilus LA02, Streptococcus thermophilis FP4, Lactobacillus bulgaricus LDB01 and Lactobacillus casei SD5213. MRS medium was supplemented with levan after depolymerization (LOS) at 75 and 85 oC, or after depolymerization and precipitation (prec.) with ethanol. The control (negative) medium was not supplemented with sugars and the positive control medium (Glc) contained glucose. Additional measurements were performed to prove that the tested cultures metabolize LOS.

The RP-UHPLC-UV-ESI-MS determination of derivatized LOS was performed to compare the profile of oligosaccharides in the culture media before and after fermentation. The UV chromatograms are shown in Fig. 4. The qualification of saccharides position on UV chromatogram with DP ranging from 18

1 to 5 hexoses units was confirmed by ESI-MS. However the signal for DP5 molecular ion [M+H]+was very poor. Given that the intensity of LOS peaks after 24 hours of growth is much lower than the same peaks before inoculation, it can be concluded that the tested cultures utilized LOS in the

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tested media.

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Fig. 4. RP-UHPLC-UV chromatograms of growth media supplemented with LOS before incubation (0h) and after incubation ((24h). LOS were derivatized with 1-phenyl-3-methyl-5-pyrazolone, DP – degree of polymerization.

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4. Discussion

Preliminary studies showed that the tested strains did not grow in media supplemented with

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native inulin or levan. Agavins have been observed to be fermented faster than inulin type fructans of similar DP (Mueller et al., 2016). This effect was attributed to the high branching of the agavins producing a more compact molecule with higher solubility than the linear inulin-type fructans and thus

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better availability to the bacteria. Our GPC results showed a large difference between the hydrodynamic parameters of the tested inulin and levan, a difference which has an effect on the compactness of molecules and their solubility. Nevertheless the molar mass of the tested levan seemed to be the most important factor limiting its utilization by bacteria. This result is consistent with the report of previous studies where it was observed that some Lactobacillus strains could ferment FOS but not inulin (Mei, Carey, Tosh, & Kostrzynska, 2011; 19

Mueller et al., 2016). Kang et al. (2003) reported that L. plantarum KCTC and P. pentosaceus KCTC could not utilize a high molecular weight levan (6·107 Da). In a recent study, 5 levan samples were observed to stimulate the growth of two probiotic lactobacillus species (Hamdy et al., 2017) however the molar masses of the levans were not determined so a comparison cannot be made with the results of our study. In a study by Yamamoto et al. (1999), the strain L. acidophilus could not grow on a low

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molecular weight levan (6000 Da). However in another study another strain L. paracasei LIAM was observed to grow in culture medium supplemented with a levan having molecular weight of 8300 Da

(Porras-Domínguez et al., 2014). This difference could be due to strain specificity in the utilization of

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prebiotics by probiotics. Mei et al. (2011) found that two P. pentosaceus strains differed in their

utilization of inulin. One strain P. pentosaceus (FRP 243) was unable to grow on inulin while the other

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P. pentosaceus (FRP 244) utilized inulin. It has been suggested that the molar mass of levan and not

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their linkage type determines their utilization by bacteria (Marx et al., 2000).

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Given that marked bacterial growth was not observed in levan-supplemented culture media,

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the decision was made to hydrolyze the levans into LOS. In the study by Kang and Jang (2005), when a levan proved to be an unsuitable substrate for lactic acid bacteria, it was hydrolysed to LOS by 0.4

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M sulfuric acid for 3 min at 95 oC. The products were then intensively fractionated by ethanol precipitation and chromatography. The lactic acid bacteria were able to ferment LOS with DP between

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2 and 6. Unfortunately the recovery of LOS was not determined in that study, therefore the industrial application of depolymerization parameters is not possible. The use of such small oligosaccharides in

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vivo could be also limited because moderate hydrolysis of β-linked fructans by gastric acid was reported (Mardo et al., 2017; Yamamoto et al., 1999). In our study four fractions of LOS with different DP were produced by mild acid hydrolysis. The products were disperse which leads us to assume that

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new oligosaccharides will appear in vivo after digestion by gastric juice while part of the oligofructan will be hydrolyzed into fructose at the same time. Depolymerization using optimum reaction conditions significantly reduced the average molar mass of native levan and enabled the production of LOS without the need for additional fractionation steps. The great advantage of these LOS fractions is that they only contain trace amounts of fructose since endohydrolysis of levan was observed. Acid

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hydrolysis of Zymomonas mobilis levan in a microwave oven has been reported. The reaction time was reduced and LOS with DP of 4 to 14 were produced. However a significant amount of fructose was present in the product of the reaction (reaction time of 3 minutes) which had the highest conversion of levan to oligosaccharides (de Paula, Pinheiro, Lopes, & Calazans, 2008). It has been shown that endolevanase from Bacillus licheniformis can produce LOS with DP of 2 to 8, and yielding levanbiose

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as the final product after long reaction times (Porras-Domínguez et al., 2014). The enzymatic hydrolysis of levan by endolevanase is an effective method of levan depolymerization. We believe that the method of levan depolymerisation described in this study, using mild reaction conditions is just as

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effective and competitive.

The four fractions of LOS stimulated the growth of the tested strains. The utilization of the

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different fractions was strain specific, an observation consistent with previously reported results. The

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DP (Mw/162) of the studied LOS fractions increased in the order LOS/85 = LOS/85 (prec.) < LOS/75

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< LOS/75 (prec.) (Table 3). Mueller et al. (2016) reported that Lactobacillus strains fermented inulin-

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type and agavins with lower DP faster than those with a higher DP. Stewart and Slavin (2008) showed that human faecal bacteria fermented FOS faster than inulin. Therefore it was expected that bacterial

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growth rate will follow the order LOS/85 > LO/S85 (prec.) > LOS75 > LOS75 (prec.). However this was not the pattern observed. The Lactobacillus plantarum subsp. plantarum DSM 20174 and the

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probiotic mix grew fastest on LOS/85 followed by LOS/75. The slowest growth was observed in medium supplemented with LOS/75 (prec.) followed by LOS/85 (prec.). This proves that during

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precipitation a crucial LOS fraction was lost and DP calculated as Mn/162 (Table 3) should be considered because it characterized the right side of the peak in GPC results. If the following DP (Mn/162) order is taken into account: LOS/85 < LOS/85 (prec.) = LOS/75 < LOS/75 (prec.), the

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pattern of growth observed correlates with the DP order. LAB ferment prebiotics into short chain fatty acids leading to a drop in the pH of the culture media (Verbeke et al., 2015). In this study a decrease in pH resulting from fermentation was observed, however it was smaller than pH decreases previously reported (S. A. Kang et al., 2005; Mei et al., 2011). In a study by Mei et al., (2011), the decrease in the pH of the culture media caused by the 21

fermentation of FOS by LAB was similar to that caused by the fermentation of glucose by the same bacteria. However, the decrease in pH resulting from the fermentation of glucose was greater in that study than in this one (2.6 - 2.9 vs 1.01 - 1.44). The difference in the composition of the culture media might explain the difference in the pH changes. The MRS medium used by Mei et al., (2011) did not contain sodium acetate which is a buffering agent, while the MRS medium in this study contained

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sodium acetate. The presence of sodium acetate likely accounts for the smaller than expected decreases in the pH of the LOS-supplemented culture media in which growth occurred. Fecal bacteria utilizing levan produce mainly acetate (Adamberg et al., 2015) which is a short chain fatty acid

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(SCFA). The SCFA are weaker acids than lactic acid and so the effect of the buffering agent would be stronger on SCFA than on lactic acid.

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Analysis of the LOS profile before and after fermentation by RP-UHPLC-UV-ESI-MS confirmed that

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the tested bacterial strains utilized oligosaccharides with DP of 2-14. This data and GPC results also

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confirm that this method of depolymerization yields LOS with low amounts of hexose and can be an

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alternative to enzymatic hydrolysis for which highly active endo-levanase BT1760 was used (Mardo et al., 2017). BT1760 could only hydrolyze a maximum of 10% of levan (5 g/L out of 50 g/L) into LOS

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(DP: 1 to 13) and there was a significant release of fructose. This data also shows the great potential of Bacteroides thetaiotaomicron, mammalian gut commensal to cleave levan. It follows that our products

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(LOS) can stimulate different genera of microbiota because of the dispersity of the different fractions.

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This hypothesis has to be tested in the future.

Conclusion

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Mild acid hydrolysis resulted in the reduction of the molar mass of levans and the production

of disperse LOS fractions without the release of great amounts of fructose. The LOS exhibited a high prebiotic potential: they stimulated the growth of the tested bacterial strains. It was proved that the degree of polymerization has a significant effect on the growth of the bacteria. The utilization of prebiotics by probiotics is strain specific. Native levan and the fractions of LOS produced can be

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mixed to obtain a product with a broader molar mass distribution. Such a product will have new functional properties.

Acknowledgement

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This study was financially supported by the National Centre of Science, Poland (Project No. N N312

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214438).

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