Microfiltration-mediated extraction of dextran produced by Leuconostoc mesenteroides SF3

Journal Pre-proof Microfiltration-mediated extraction of dextran produced by Leuconostoc mesenteroides SF3 ˜ ´ ˜ Elsa D´ıaz-Montes, Jorge Ya´ nez-Fern andez, Roberto Castro-Munoz

PII:

S0960-3085(19)30752-7

DOI:

https://doi.org/10.1016/j.fbp.2019.11.017

Reference:

FBP 1186

To appear in:

Food and Bioproducts Processing

Received Date:

11 August 2019

Revised Date:

21 November 2019

Accepted Date:

22 November 2019

˜ ´ ˜ R, Please cite this article as: D´ıaz-Montes E, Ya´ nez-Fern andez J, Castro-Munoz Microfiltration-mediated extraction of dextran produced by Leuconostoc mesenteroides SF3, Food and Bioproducts Processing (2019), doi: https://doi.org/10.1016/j.fbp.2019.11.017

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Microfiltration-mediated extraction of dextran produced by Leuconostoc mesenteroides SF3

Elsa Díaz-Montesa, Jorge Yáñez-Fernándeza*, Roberto Castro-Muñozb*

aUnidad

Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional,

b

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Av. Acueducto s/n Col. Barrio La Laguna, Ticoman, CP 07340, México City, México.

Tecnologico de Monterrey, Campus Toluca. Avenida Eduardo Monroy Cárdenas 2000

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San Antonio Buenavista, 50110 Toluca de Lerdo, Mexico. E-mail:

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[email protected] ; [email protected] .

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*Corresponding authors:

J. Yáñez-Fernández; E-mail address: [email protected]

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R. Castro-Muñoz; E-mail address: [email protected]; [email protected].

Highlights 

The dextran produced by Leuconostoc mesenteroides SF3 has been fully characterized.

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

An integrated MDF-MF system has been, for the first time, used for dextran recovery.



A comparison between solvent extraction and MDF-MF has been performed.



The use of the membrane technology promotes the reduction of ethanol consumption. 1

Abstract Recently, the production of metabolites from fermentation broths has been of great importance due to microorganisms are able to produce a wide variety of products and by-products; however, one of the challenging tasks is the extraction of such metabolites. The extraction with organic solvents is likely the most used methodology to recover the metabolites from the fermentation broths; which has been criticized according to the

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negative effect of using solvents on the environment. In this sense, physical separation

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methods, like membrane processes, have started to be involved in such recovery task. Thereby, we propose a membrane technology (i.e., microfiltration (MF)) for the

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separation of dextran produced by Leuconostoc mesenteroides SF3. Herein, a

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comparison between dextrans extracted with solvent extraction (i.e., ethanol) and integrated MF system is presented. The results revealed that the membrane process

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modified some of the physicochemical (e.g., hygroscopicity, solubility, water absorption capacity, and porosity) and morphological characteristics of the dextran. In addition, the

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MF process displayed a 33.5% dextran extraction yield with a saving of 75% in ethanol consumption for dextran precipitation.

Keywords: Leuconostoc mesenteroides; fermentation; dextran; Microfiltration;

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Diafiltration.

1. Introduction

Dextran is a polysaccharide produced extracellularly by lactic acid bacteria and it generally possess chains of D-glucose in α (1-6) bond with different branching (α (1-2), 2

α (1-3), or α (1-4)) (Abdel-Rahman et al., 2007; Schmid, 2018; Singleton et al., 2002). The type of dextran depends on the microbial strain because dextrans can differ in the length of the chains and the degree of branching. Herein, the main difference on their physicochemical properties and uses (Abdel-Rahman et al., 2007; Schmid, 2018), such as stabilizers and moisturizers (Heinze et al., 2006). The production of dextran is mainly carried out by facultative bacteria Leuconostoc mesenteroides, which is cultivated in a

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medium rich in sucrose (Garvie, 1984). When dealing with the separation and extraction

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of the dextran, the precipitation with polar solvents (e.g., ethanol and methanol) has been the most used technique. This polysaccharide is certainly insoluble in such

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alcohols (Sarwat et al., 2008; Song et al., 2010; Vettori et al., 2011).

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The production yields of dextran produced at lab scale using solvents are ranged from 2 up to 44 g L-1 depending on the initial substrate and the strain used (Aman et al., 2012;

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Behravan et al., 2003; Qader et al., 2005; Santos et al., 2000; Sarwat et al., 2008); however, the use of solvents leads to the implementation of additional methods for their

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removal and purification of the product. Today, the use of membrane technologies has considerably increased based on the advantages of such technologies, including energy savings, separation efficiency, and the quality of the obtained products (Cassano et al., 2014, 2011; Charcosset, 2006). Moreover, specific membrane processes, specifically

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those governed by pressure gradient, have become a friendly alternative to the environment (Castro-Muñoz, 2018; Castro-Muñoz et al., 2017). Over the last decade, these processes have been mainly involved in the treatment of agro-industrial waste, as well as the recovery of high-added value compounds from natural sources (Li and Chase, 2010; Vladisavljević et al., 2003). Particularly, microfiltration (MF) is indeed a 3

pressure-driven membrane process that uses membranes with a cut-off from 0.05 up to 10 µm, while its pressure requirements are between 0.1 and 2.0 bar (Castro-Muñoz et al., 2016b). MF is mainly applied for the removal and separation of non-soluble molecules and the retention of organic matter. In this way, it is suitable as a process of clarification, and thus considered as a pre-treatment process as well (Davey et al., 2016; Phanthumchinda et al., 2018). At this point, taking into the features of MF

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technology, there are current findings about its potentiality on recovering metabolites

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from fermentation broths (Díaz-Montes and Castro-Muñoz, 2019). In addition, there are variations in configuration of the processes using membranes, e.g., diafiltration, in which

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water is added to the feed tank of the retentate with the same feed flow rate as the

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permeate flux (Tejeda et al., 1995), allowing to keep the constant volume. This allows to wash the dissolved components and therefore reduce the phenomenon of fouling

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produced in the membranes; importantly, this approach contributes to improve the recovery rate of the permeate components (Grandison and Lewis, 1996; Scott and

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Hughes, 1996; Tejeda et al., 1995). This approach has been successfully used for the recovery of several compounds of fermentation broths, such as proteins, sugars, and dextrans (Scott and Hughes, 1996; Su et al., 2018). In this work, we propose MF system as a potential candidate for extracting dextran from a fermentation broth using a

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strain of L. mesenteroides. Furthermore, changes were evaluated on the physicochemical and morphological properties of the recovered dextran using MF, and dextran extracted using conventional solvent (i.e., ethanol) extraction method. Finally, the performance of the selected MF membrane was determined in terms of productivity, fouling index and recovery of hydraulic membrane permeability. 4

2. Materials and Methods 2.1. Dextran production by fermentation Leuconostoc mesenteroides subsp. mesenteroides SF3 (GenBank: KR362874) was isolated from aguamiel of Agave salmiana (Mexico City, Mexico) and it was grown on MRS agar for 20 h at 30 ± 1°C (Castro-Rodríguez et al., 2015). L. mesenteroides SF3

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was inoculated in a conical flask with 200 mL of MRS media (10% sucrose, for 20 h at

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30 ± 1°C) to a final concentration of 108 cells mL-1. The inoculum was transferred to the bioreactor with 1800 mL of MRS media (10% sucrose) and incubated for 20 hours at 30

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± 1°C. Once the fermentation is over, the biomass was eliminated by centrifugation (at

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4000 rpm for 20 min) and the supernatant (fermentation broth) was treated by the MF

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system.

2.2. Experimental set-up and procedures for the treatment of fermentation broth

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using MF system

2.2.1. Selection of the feed flow rate condition in MF membrane The specifications of the MF membrane are shown in Table 1. MF experiments were carried out in total recycle to find the optimal feed flow rate at 25 ± 1°C. Experimentally,

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the permeate was measured as a function of the time to find the axial feed flow rate (Qf) (A. Cassano et al., 2007). Herein, the optimal permeate flux was found at a feed flow 70 L h−1.

Table 1. Specifications of the MF membrane used for the recovery of dextran. 5

2.2.2. Microdiafiltration treatment in a batch diafiltration mode The fermentation broth was processed in a batch diafiltration mode (microdiafiltration, MDF; see Figure 1A) (Kovács and Czermak, 2013). The experiments were performed at different transmembrane pressures (TMP: 0, 0.4, 0.7, 1.0, 1.4, and 1.7 bar), 25 ± 1°C, and Qf of 70 L h-1; to identify the limiting TMP. The permeate flux (Jp) was

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gravimetrically measured as the change of permeate weight with time using a digital

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balance and its productivity was calculated using Eq. 1 (Kovács and Czermak, 2013;

ṁp A

(𝟏)

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Jp =

-p

Tejeda et al., 1995), as follows:

where ṁp is the mass flow (kg h-1) and A is the membrane area (m2). The results of Jp

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were expressed in kg m−2 h−1.

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Figure 1. General drawing of the integrated membrane system for the recovery dextran from the fermentation broth. A) microdiafiltration (MDF); B) microfiltration (MF).

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2.2.3. Microfiltration treatment in a batch concentration mode The second MF process was operated in a batch concentration mode (see Figure 1B). The MDF-Retentate was recirculated at 25 ± 1°C, Qf = 70 L h-1 and different TMP (0, 0.4, 0.7, 1.0, 1.4, and 1.7 bar) to identify the limiting TMP (Castro-Muñoz et al., 2015; Su et al., 2018), as shown in section 2.2.2. 6

Concentration configuration mode in the MF process was carried out according to the volumetric Jp (modified Eq. 1: L m−2 h−1) and the volume reduction factor (VRF), which is defined as the ratio between the initial feed volume and the final retentate volume (Castro-Muñoz and Yañez-Fernandez, 2015). VRF is denoted by the following Eq. 2 (Cao et al., 2018): VF VR

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(𝟐)

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VRF =

-p

where VF is the feed volume (L) and VR is the retentate volume (L).

2.3. Membrane properties: Water permeability, fouling index and cleaning

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efficiency

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2.3.1. Water permeability

Water permeability (Lp) was determined by the slope of the straight line obtained plotting the water flux (Jw) at different TMP values. Tests were carried out on total

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recycling mode at 25 ± 1°C, and Qf = 70 L h-1 The Lp was determined by Darcy’s law

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according to Eq. 3 and 4 (Castro-Muñoz et al., 2015; Su et al., 2018), as follows: Jw =

ṁp A

(𝟑)

Lp =

Jw ∆P

(𝟒)

where ṁp is the mass flow (kg h-1), A is the membrane area (m2), ΔP is the variation of TMP (bar) and Jw is expressed in kg m-2 h-1. 7

2.3.2. Fouling index The fouling of the membrane (If, %), after the filtration process (MDF-MF), was measured by means of Eq. 5 (Cassano et al., 2015; Su et al., 2018): Lp1 ) ∗ 100 Lp0

(𝟓)

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If = (1 −

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where Lp0 (kg m-2 h-1 bar-1) and Lp1 (kg m-2 h-1 bar-1) are the water permeabilities

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measured before and after MDF-MF process, respectively.

2.3.3. Cleaning efficiency

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After the treatment of the fermentative broth using the MDF-MF process, the membrane

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was subjected to enzymatic cleaning (Ultrasil 67, Ecolab) at 1% (v/v; at 50 ± 1°C for 180 min), as modified procedure reported by Castro-Muñoz et al. (Castro-Muñoz et al., 2015). The cleaning efficiency was measured by the flux recovery ratio (FRR), which

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was calculated according to Eq. 6 (Faneer et al., 2017): Jw FRR = ( ) ∗ 100 Jw2

(𝟔)

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where Jw (kg m-2 h-1) and Jw2 (kg m-2 h-1) are the initial water flux before MDF-MF process and after enzymatic cleaning, respectively.

2.4. Dextran extraction by solvent precipitation

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The final extract (MF-Retentate) containing the dextran was precipitated using ethanol (1:1, v/v) and then stored at 4 ± 1°C for 72 h. The dextran was dried at 50 ± 1°C during 24 h and later pulverized with a milling device. As a control test, the fermentation broth was dissolved with ethanol (1/1, v/v), and it was stored for 72 h at 4 ± 1°C. The dextran was collected by decantation, diluted (with distilled water) and precipitated with cold ethanol (1:1, v/v) at 4 ± 1°C for 24 h; the

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decantation-dilution process was repeated three times, as reported by (Castro-

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Rodríguez et al., 2015; Sarwat et al., 2008). The dextran was dried at 50 ± 1°C for 24 h.

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Similarly the final product was pulverized with a milling device.

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2.5. Physicochemical parameters evaluated in fermentation broth 2.5.1. pH

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pH was measured by a laboratory pH meter (HI 98107, HANNA). It was calibrated with

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buffer solutions of pH 4 and 7.

2.5.2. Total solids content

Total solids content (TSC) was determined as the water loss using a drying oven (133000, Boekel Scientific). TSC calculated using Eq. 7 (AOAC, 1990; Valderrama-

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Bravo et al., 2013):

TSC = 100 − [(

Mi − Mf ) ∗ 100] Mi

(𝟕)

where Mi and Mf are the mass of samples before and after drying, respectively. Both measurements expressed in grams (g). 9

2.5.3. Reducing sugar Reducing sugars were determined by spectrophotometric technique. A sample (50 μL) was mixed with distilled water (950 μL) and DNS reagent (1000 μL), which was later heated in a water bath at 100 ± 1°C for 5 min. Afterwards, 1000 μL of distilled water were added into the samples. The measurements were done in a spectrophotometer

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(Lambda XLS, Perkin Elmer®) at 540 nm. The quantification was carried out by means

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of glucose curves and the results were expressed as glucose equivalents (g GE L-1)

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(Bello Gil et al., 2006).

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2.5.4. Viscosity

The viscosity was determined making measurements in a viscometer (DV-II +pro,

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2.6. Process yield

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Brookfield) with the needle S01, at 200 rpm and 25 ± 1°C.

The process yield was determined based on the relation of the dextran (g) obtained after drying to the contained in specific volume of the fermentation broth (L). Regarding, the ethanol consumption, the volume of used ethanol was measured in a laboratory test

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tube before each precipitation.

2.7. Identification of the dextran The identification of the polysaccharide (i.e., dextran) was done through by a microRAMAN spectrometer (HR800, LabRam), equipped with a He-Ne laser of nominal 10

power of 17.5 mW at 632.79 nm, aperture of 200 μm and 100 μm (hole and slit, respectively), an exposure time of 4 s and 8 accumulations, and using a 100x objective. Dextran linkages were determined by nuclear magnetic resonance (NMR) with a spectrometer (Bruker Ascend 750). For the analysis, dextran was diluted in D2O at 25°C, and the 1H NNR and 13C NMR spectra were recorded at a base frequency of 750 MHz. The percentages of the main linkages α (1-6) and branches linkage α (1-3) were

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calculated according to the relative intensities of anomeric carbons or protons (Bounaix

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2.8. Morphological characterization of dextran

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et al., 2009).

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Dried dextran sample was placed in a piece of aluminum and then examined in FE/SEM (JSM-7800F, JEOL) with lower electron detector (LED). The measurements were

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carried out at a working distance of 4.9-6.0 mm, electric potential difference of 1.5 kV.

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The images were recorded at different magnification.

2.9. Physicochemical parameters evaluated on dried dextran 2.9.1. Moisture content

The moisture content (H) was estimated by water loss using a drying oven (133000,

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Boekel Scientific). The H was calculated by using the Eq. 8 (AOAC, 1990; ValderramaBravo et al., 2013): H = [(

Mi − M f ) ∗ 100] Mi

(𝟖)

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where Mi and Mf are the mass of samples before and after drying, respectively. Both measurements expressed in grams (g).

2.9.2. Water activity The water activity was measured using a water activity meter (3-PRE Series, AquaLab).

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Triplicate samples were analyzed, and the mean was recorded.

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2.9.3. Hygroscopicity

The hygroscopicity was determined by the weight uptake. The sample was exposed to a

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expressed in g 100g-1 (Tonon et al., 2008).

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saturated solution of sodium chloride at 25 ± 1°C for 7 days, and the weight uptake was

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2.9.4. Solubility

The solubility (S) was determined according to the methodology described by Cone

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(Cone and Ashworth, 1947) and Sulieman et al. (Sulieman et al., 2014). Basically, the dextran (3 g) was suspended in distilled water (35 mL) during 1 min, and then kept resting for 15 min. Subsequently, the samples were re-agitated and followed by centrifugation (SOB-J40-16, SOLBAT) at 900 rpm for 15 min. The solids were re-diluted

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(40 mL) and centrifuged at similar conditions. The percentage of solubility was expressed as percentage based on Eq. 9, as follows: S = [(

Md − Ms ) ∗ 100] Md

(𝟗)

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where Md is the dextran mass and Ms is the solids mass. Both variables expressed in grams (g).

2.9.5. Absorption capacity The absorption capacity (in water and oil) was determined by the weight uptake. The sample (1 g) was mixed in 10 mL of water or oil, and later centrifuged at 4000 rpm for

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20 min; the solids were weighed and expressed in g g-1 (Anderson et al., 1970;

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Beuchat, 1977; Zambrano-Zaragoza et al., 2003).

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2.9.6. Density

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The density (apparent and packed) was determined by placing a specific amount of dextran powder in a graduated cylinder. The measurement of the volume before and

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2.9.7. Porosity

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after compacting the sample was recorded (Angelo and Subramanian, 2009).

The porosity (φ) between the dextran powders is given by the relation between the apparent and packed volume. The percentage of porosity was calculated by Eq. 10

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(Heinemann and Mittermeir, 2013), as follows: Ve φ = ( ) ∗ 100 Va

(𝟏𝟎)

where Ve and Va are the packed volume and the apparent volume, respectively. Both parameters are expressed in milliliters (mL).

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2.10. Statistical analysis The data are expressed as the means ± standard deviation. One-way analysis of variance (ANOVA) and correlation analysis were performed using the SAS statistical software (V9.4, SAS Institute S.A de C.V, Mexico). Tukey's multiple range tests were used to compare the means. Differences among the means of p<0.05 were considered

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significant.

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3. Results and discussion 3.1. Physicochemical composition of the fermentation broth

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Table 2 reports the physicochemical composition of the initial fermentation broth and

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the one treated by the integrated MF system. The suitable pH for the growth L. mesenteroides SF3 is 6.5. In principle, the initially culture medium was adjusted to such

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value; however, lactic acid is produced as a part of the main metabolic pathway of the bacteria and it directly influenced the pH decrease in the fermentation broth (pH value

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up to 3.61), which was maintained during the MDF-MF process. This result can be expected according to the cut-off of the MF membrane (i.e., 0.1 µm); in fact, the cut-off of the membrane is not enough to reject lactic acid (molecular weight 90.08 g/mol). The total solids content (11.16%) in the fermentation broth, including the dissolved

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components (e.g., sugars and dextran) and organic matter were not eliminated during the centrifugation. Moreover, MDF-Retentate (operated at 0.7 bar, 70 L h-1, 25°C) showed lower total solids content (3.36%) since in the diafiltration configuration diluted the retained components and the elimination of components with size smaller than the cut-off of the membrane. On the other hand, due to the concentration occurred during 14

the MF process in batch concentration mode, the MF-Retentate presented higher total solids content (6.17%) than the MDF-Retentate (3.36%). Reducing sugars (i.e., glucose and fructose) were the result of the hydrolysis of sucrose during the fermentation process. Initially, the fermentation broth had a content of 11.85 g GE L-1; which decreased by 98% during the MDF-MF process due to the permeation of glucose (MW = 180.16 g/mol) and fructose (MW = 180.16 g/mol).

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To date, it has been documented that the viscosity of the dextran around 15 cP (Sarwat

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et al., 2008). In principle, the viscosity of the fermentation broth (35.43 cP) is influenced by the dextran characteristics. Herein, the viscosity of the broth increased up to 267.33

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cP in MF-Retentate (VRF=2.45); this is due to the concentration of dextran during the

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MF process (operated at 0.9 bar, 70 L h-1, 25°C), as a result of the removal of water from the retained fraction. Importantly, this result provides an insight into the properties

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of gel formation of dextran.

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Table 2. Physicochemical composition of the fermentation broth before and after the MDF-MF process.

3.2. Microfiltration membrane performance during the treatment of the

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fermentation broth.

3.2.1. Microdiafiltration process Figure 2 shows the permeate flux of the fermentation broth during the MDF process. Initially, the permeate flux had a linear increase dependent on the TMP (from 0 up to 0.7 bar). At this, TMP the maximum permeate flux was about 13.71 kg m-2 h-1. In fact, the 15

permeate flux started to stabilize from 0.7 bar, reaching a steady-state with constant flux around 14 kg m-2 h-1. Generally, at this stage the permeate flux does not display any change as a function of the operating time. Moreover, the permeation rate does not depend on the driving force (Cassano et al., 2018), considering this pressure (i.e., 0.7 bar) as the limiting TMP.

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Figure 2. Permeate flux (Jp) behavior of the fermentation broth in the MDF process at

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different transmembrane pressure (TMP) (T = 25°C and Qf = 70 L h-1). Results

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expressed with the mean ± standard deviation for each sample (n = 3).

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3.2.2. Microfiltration process

Figure 3 represents the permeate flux of the MDF-Retentate during the MF step. A

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similar behavior was observed compared to the filtration of the fermentation broth during the MDF step (see Figure 2). As explained previously, to maintain a continuous process

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the TMP selected, well known as limiting TMP, must be lower than the critical value, this means work under the critical zone in which the fouling effect is minimum. In this case, the limiting TMP was found out at 0.9 bar, which provided a permeation rate of 14.86 kg

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m-2 h-1.

Figure 3. Permeate flux (Jp) of the MDF-Retentate in the MF step as a function of the different transmembrane pressure (TMP) (T = 25°C and Qf = 70 L h-1). Results expressed with the mean ± standard deviation for each sample (n = 3).

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Once selected the operating conditions for the batch concentration mode, Figure 4 shows the permeate flux behavior and VRF of the MF membrane. Typically, permeate flux (Jp) usually decreases as a function of the VRF. This due to the concentration polarization phenomenon, membrane fouling, and concentration of solutes. For instance, during the first 60 min, the Jp decreased from 16.17 up to 11.31 L m-2 h-1, followed by a continuous decrease up to a permeate flux of 10.48 L m-2 h-1 (during an

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operating time of 150 min). Finally, at the end of the MF step (i.e., 240 min), the final

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productivity in terms of permeate flux of the process was about 8 L m-2 h-1.

At the same time the degree of concentration (VRF) in the MF-Retentate fraction was

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increasing.

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The permeate flux marked the end of the MF process because the first 180 min, the MF-Retentate concentration increased linearly (from 0 up to 2.35) and remained

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a maximum VRF of 2.45.

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constant, but the permeate flux was blocked by membrane fouling at 240 min, achieving

Figure 4. Time course of permeate volumetric flux (Jp; ■) and volume reduction factor (VRF: □) of the MF process for the MDF-Retentate (TMP = 0.9 bar, Qf = 70 L h-1, and T

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= 25°C). Results expressed with the mean ± standard deviation for each sample (n = 3).

During the filtration process, membranes tend to be affected and fouled by solutes presented in feed stream (i.e., fermentation broth). Definitely, the separation performance of any membrane-based technology depends on the physicochemical composition of the feed (Castro-Muñoz et al., 2016a; Cho et al., 2000). 17

Moreover, depending on the type of feed solution, the fouling phenomenon, defined as the accumulation of material on the membrane surface, can occur. The fouling can be reversible and irreversible, which may imply any of the different types of fouling mechanisms, e.g., complete or partial pore blocked, cake formation, and internal pore blocked (A Cassano et al., 2007; Choi et al., 2005). The MF membrane had an initial pure water flux of 645 kg m-2 h-1, (see Table 3), which decreased up to 58 kg m-2 h-1

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after the filtration of the MDF-retentate. This decrease in the initial water flux represents

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a decrease of about 90%), which is related to the water permeability (33.81 kg m-2 h-1 bar-1). After enzymatic cleaning, the water permeate flux was recovered up to 89.2%

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(576 kg m-2 h-1); which means that 10.8% of the initial permeability was not recovered.

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This may give an idea the presence of an irreversible fouling. Generally, incomplete recovery of the initial water permeability of the MF membrane may suppose from an

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internal pore blocking to a cake formation due to the large pore size, which can allow the penetration of particles/solutes in the internal structure of the membrane, and thus

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causing irreversible fouling (A. Cassano et al., 2007; Castro-Muñoz, 2019).

Table 3. Water permeability, fouling index and cleaning efficiency of the MF membrane

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(at TMP = 0.9 bar).

3.3. Comparison of the dextran production between membrane process and solvent extraction After the microfiltration-mediate extraction of the polysaccharide, a precipitation with solvents has been performed for the total recovery of the dextran. Some authors have 18

adapted the extraction protocol according to specific objectives; for example, Shamala and Prasad (Shamala and Prasad, 1995) used a common washing with ethanol (1:1, v/v), which was also reported by Holló and László (Holló and László, 1970). While other authors precipitated the dextran washing from 2 up to 4 times with ethanol (1:1 v/v) (Moosavi-Nasab et al., 2010; Sarwat et al., 2008). Thereby, in this work, the control tests were processed by washing 4 times with ethanol

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(1:1, v/v), to ensure the total precipitation of dextran. On the other hand, for the dextran

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separated by MF, one time washing with ethanol was only needed. Importantly,

according to Table 4, the use of the proposed membrane process allowed to reduce the

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use of ethanol by 75%; this is because the final volume of the membranes process

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(MDF-MF process), as a concentration step, needed less ethanol volume for the precipitation of the dextran. Moreover, the final yield of the MDF-MF process was

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around ~22 g L-1, higher value that the one obtained by the solvent extraction method (EtOH method: ~16 g L-1). Finally, it is important to mention that two advantages can be

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highlighted by using membrane process: i) higher yield of desired product, ii) less use of solvent for precipitation.

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Table 4. Comparison of the productivity of MDF-MF and solvent extraction protocols.

3.4. Identification of dextran It is well known that the composition of the carbohydrates includes glucoses in their structures. They present typical groups C-O-H; therefore, the RAMAN spectroscopy detected these groups in the dextran in terms of frequencies. According to Vasco et al. 19

(Vasko et al., 1971), the frequency between 700-1500 cm-1 is characteristic by carbohydrates; they also identified 28 vibrational frequencies related to dextran. Figure 5 compares the spectra of the dextran obtained by both methods (i.e., solvent extraction and MDF-MF process). In principle, both dextrans presented the same pattern which display the characteristic peaks at the specific frequencies mentioned by

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Vasco et al. (Vasko et al., 1971).

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Figure 5. RAMAN spectra of dextran obtained from fermentation with L. mesenteroides

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SF3.

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Dextran is composed of a linear chain of glucoses (D-glucopyranoses) linked by α (1-6) bonds that cause repetitive structures of isomaltoses (α-D-glucopyranose-(1-6)-D-

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glucopyranose). Similarly, they may have branches of D-glucopyranoses attached to the linear chain with links α (1-2) (kojibiose: α-D-glucopyranose-(1-2)-D-glucopyranose), α

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(1-3) (nigerose: α-D-glucopyranose-(1-3)-D-glucopyranose), or α (1-4) (maltose: α-Dglucopyranose-(1-4)-D-glucopyranose (Sidebotham, 1974). In such a way, the 1H and 13C

spectra allow knowing the type of bonds present in the common molecule. For

instance, Figure 6 shows the 1H spectra of the dextrans biosynthesized by L.

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mesenteroides SF3, which were actually extracted by both methods. In both 1H spectra, the dextran produced by the SF3 bacteria showed typical bonds of carbon 1 α (1-6) and α (1-3) with anomeric signals centered at a chemical change of 4.9 and 5.2 ppm, respectively; the peak at 4.7 ppm corresponds to HDO (Bounaix et al., 2009; Maina et al., 2008); while the peaks of the bulk proton region in the range from 3.0 up to 4.0 ppm 20

correspond to protons of hydrogen from carbon 2 up to 6 (Bounaix et al., 2009; Gil et al., 2008; Han et al., 2014). Some works have reported that the 1H spectra for dextrans extracted from different strains of L. mesenteroides (e.g., B-512F, BD1710, FT045B, KIBGE-IB22, and BA08), in which the main peaks correspond to bonds α (1-6) (greater proportion) and α (1-3) (less proportion). Such results are in agreement with the literature (Gil et al., 2008; Han

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et al., 2014; Lule et al., 2016; Siddiqui et al., 2014; Vettori et al., 2012).

Figure 6. 1H NMR spectra of dextrans produced by L. mesenteroides SF3 (recorded at

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750 MHz in D2O at 25°C).

The relative intensities of the anomeric peaks (see Figure 6) allowed determining in

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each dextran the proportion of the α (1-6) and α (1-3) bonds, for example, it was found that dextran extracted using ethanol extraction presented about 77.35% of α (1-6)

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bonds, while the one extracted using the integrated membrane process presented 88.18% (Table 5). The difference between the proportions of the bonds is due to the dehydration of the dextran molecule because of the addition of ethanol. In aqueous solutions (i.e., the fermentation broth or the MF-Retentate) the dextran molecules are

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held together through the water by hydrogen bonds; however when ethanol is added, the water has greater affinity with it and that causes the breakdown of the hydrogen bonds with the dextran and the precipitation of it (Lubambo et al., 2013). Nevertheless, the chains of greater molecular weight are the first to precipitate, and the short chains remain dispersed in the ethanol extract to be eliminated by decantation (as 21

in the MDF-MF process); but to efficiently remove scattered short chains, it is require continuous washings (as in the conventional method) (Lule et al., 2016); which results in a final precipitate with a more homogeneous size (Gupta et al., 2005). Han et al. (Han et al., 2014) and Gil et al. (Gil et al., 2008) reported that the proportion of the bonds present in the molecule influences the solubility in most of the dextrans from L. mesenteroides when they are re-diluted; thereby, a greater percentage (> 90%)

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of α (1-6) bonds may promote better solubility.

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Table 5. Branching composition of the dextran produced by L. mesenteroides SF3.

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Apart from the chemical similarities in the presence of α (1-6) bonds , the dextrans also presented similarities between D-glucopyranose and ramifications in C3 (Sidebotham,

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1974; Wishart et al., 2007), but differences in proportions for each dextran; however, these results allowed to generate a chemical structure model of the dextran molecule,

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as shown in Figure 7.

Figure 7. Proposed molecular structural model of the dextran produced by L.

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mesenteroides SF3.

The 13C spectra of the dextrans (Figure 8) showed the same peaks, which were identified representing the anomeric carbon (C1) in the range of 95-100 ppm. In particular, the peak identified at ~99 ppm comprises with α (1-3) bond that is involved in the links of the dextran ramifications; while the peak identified at 97.77 ppm 22

corresponds to the α (1-6) bond. The peaks identified in the region 70-75 ppm refer to the carbons C2, C3, C4, and C5, and the signal identified at ~65 ppm is corresponding to the C6, implying signals from the carbons that make up the linear structure of dextran (Gil et al., 2008; Shukla et al., 2011). Seymour et al. (Seymour, 1979) and Shukla et al. (Shukla et al., 2011) stated that the dextran from a strain of L. mesenteroides present two types of D-glucopyranosyl

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of the branches in the anomeric carbons with bonds (1-3).

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residues, 3-mono-O-substituted and 3,6-di-O-substituted, which represent the position

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Figure 8. 13C NMR spectra of dextrans produced by L. mesenteroides SF3 (recorded at

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750 MHz in D2O at 25°C).

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3.5. Morphology of dextran

Figure 9 shows the morphological structure of dextrans extracted by solvent extraction

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and MDF-MF process. It can be seen that the particle sizes of the dextran, which was extracted with ethanol, were about 12-600 μm, being irregular (top-left micrograph). While the dextran, obtained by membranes, presented smaller sizes (e.g., 12-250 μm) with more homogeneous distribution (lower-left micrograph).

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Dextran extracted with ethanol included particles with rough, smooth, and porous surfaces (top-middle micrograph), while the one extracted with membranes presented particles with porous surfaces only (lower-middle micrograph). Also, the pores in the dextran particles extracted with ethanol had large sizes (>5 μm) and irregular shapes

23

(top-right micrograph). On the other hand, the pores of the dextran particles obtained by membranes had circular shapes with sizes over 10 μm (lower-right micrograph). Commonly, the morphological structure (e.g., pore size, shape) tends to depend on the extraction conditions (e.g., pH, temperature, and humidity). The porosity may play an important role for the compactness of the polysaccharide and the stability of the gel structure when applying any force (Purama et al., 2009).

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In polysaccharides, the presence of a greater number of pores is due to the formation of

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amorphous regions by the branches present in the structure (Whistler, 1973). These areas favor the water absorption and retention due to the molecular-water interaction,

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promoting higher solubility of the molecule (Shukla et al., 2011). At this point, the

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dextran extracted by the membrane system showed higher solubility compared to the

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one extracted by solvent protocol (see Table 6).

Figure 9. SEM images of dextrans obtained from fermentation with L. mesenteroides

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SF3.

3.6. Physicochemical properties of the dextran Table 6 reports the physicochemical characterization of both extracted dextrans. The

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moisture (in the range from 23%) and water activity (in the range from 0.2 to 0.3) are directly related to the shelf-life of food products (Quek et al., 2007). Ebadat (Ebadat, 2013) stated that the content of moisture in the powders should generally be lower than 5%, and thus they can be stored for a long time. In addition to this, products with a water activity lower than 0.6 are considered stable (Quek et al., 2007), this is because 24

most of the bacteria need a water activity between 0.6 and 0.8 for their physiological and metabolic activities (Beuchat et al., 2013). Regarding the hygroscopicity, the dextran extracted with ethanol (2.3 g 100g-1) was greater than the one extracted with membranes, e.g., 1.3 g 100g-1. It is likely that the membrane process allowed the removal of any glucose residues, as well as other components with low molecular weight; which may have hydrophilic nature (Koga et al.,

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2007). This phenomenon has been studied by Cocinero et al. (Cocinero et al., 2009),

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who described the interactions that occur between the hydrophilic groups of molecules and water.

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The solubility, defined as the chemical property of a solid to be dissolved in a solvent

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(e.g., water), is an important parameter in the development of new powder-type products. In general, both dextrans displayed solubility over 50%. According to Guo et

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al. (Guo et al., 2017) both parameters (i.e., solubility and hygroscopicity) in polysaccharides are related to specific factors, such as dimension, linearity and the

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presence of functional groups. Furthermore, low molecular weight polysaccharides, with branching, or with charged groups, tend to have a higher solubility and water adsorption capacity.

On the other hand, with the density (apparent and packed) values did not show any

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statistical difference both dextrans. However, porosity has shown a remarkable change, e.g. the dextran, extracted by MDF-MF process, showed higher porosity (16%) than the extracted one with ethanol (12%). Such differences could be related to the variations on particle size, size distribution, particle shape, and surface area (Angelo and Subramanian, 2009). 25

Table 6. Physicochemical characterization of dextran.

4. Conclusions In this work, a MDF-MF integrated process has been, for the first time, proposed as an alternative for a simultaneous extraction and recovery of dextran from the fermentation

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broth. The microfiltration-mediated extraction was compared to a common solvent

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extraction protocol in terms of product yield, and physicochemical properties of the

dextran. In this way, a successful extraction with a final yield (~22%) was obtained by

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using the integrated membrane process, such approach resulted in less ethanol

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requirement for the final dextran precipitation, e.g., 75% ethanol saving. Moreover, the effect of both extraction procedures on the physicochemical properties of the dextran

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was studied. Through the analysis of the physicochemical properties, the dextran (extracted by membranes) meets most of the physicochemical parameters considered

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in the selection of polysaccharides in the food industry (as thickening, gelling, stabilizing, emulsifying, and water-binding agents). This study can be considered as a starting point for merging different membrane technologies for the recovery of high-

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added value products from complex systems (e.g., fermentation broths).

Conflict of Interest The authors declare no conflict of interest.

26

Acknowledgment The authors thank to the Consejo Nacional de Ciencia y Tecnología (CONACyT No. 593731) and the Instituto Politécnico Nacional (SIP 20180365/20195542) for the financing granted, as well as the Centro de Nanociencias y Micro y Nanotecnologías of

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the Instituto Politécnico Nacional (CNMN-IPN) for their collaboration.

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en un Extruído de Cascarilla de Maíz con Base en Avena. Inf. Tecnológica 14, 37–40.

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Figure 1. General drawing of the integrated membrane system for the recovery dextran from the fermentation broth. A) microdiafiltration (MDF); B) microfiltration (MF).

36

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18 16

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12 10

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Jp (kg m-2 h-1)

14

8 6

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4 0 0.2

0.4

0.6

0.8 1.0 TMP (bar)

1.2

1.4

1.6

1.8

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0.0

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2

Figure 2. Permeate flux (Jp) behavior of the fermentation broth in the MDF process at different transmembrane pressure (TMP) (T = 25°C and Qf

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= 70 L h-1). Results expressed with the mean ± standard deviation for each sample (n = 3).

37

of ro -p 0.2

0.4

0.6

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0.0

0.8 1.0 TMP (bar)

lP

Jp (kg m-2 h-1)

18 16 14 12 10 8 6 4 2 0

1.2

1.4

1.6

1.8

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Figure 3. Permeate flux (Jp) of the MDF-Retentate in the MF step as a function of the different transmembrane pressure (TMP) (T = 25°C and Qf =

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70 L h-1). Results expressed with the mean ± standard deviation for each sample (n = 3).

38

18

2.8

16

of 2.6

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Jp (L m-2 h-1)

12 10

6

1.8 1.6 1.4

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4

0

0

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2

30

60

90

120

150

2.2 2.0

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8

2.4

VRF

14

1.2 1.0

180

210

240

Operating time (min)

Figure 4. Time course of permeate volumetric flux (Jp; ■) and volume reduction factor (VRF: □) of the MF process for the MDF-Retentate (TMP =

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0.9 bar, Qf = 70 L h-1, and T = 25°C). Results expressed with the mean ± standard deviation for each sample (n = 3).

39

EtOH method

MDF-MF process

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800

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600 400

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RAMAN intensity

1000

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200 0

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1200

300

600

900

1200

1500

Frequency (cm-1)

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Figure 5. RAMAN spectra of dextran obtained from fermentation with L. mesenteroides SF3.

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of ro -p re lP ur na Jo Figure 6. 1H NMR spectra of dextrans produced by L. mesenteroides SF3 (recorded at 750 MHz in D2O at 25°C).

41

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Figure 7. Proposed molecular structural model of the dextran produced by L. mesenteroides SF3.

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of ro -p re lP ur na Jo Figure 8. 13C NMR spectra of dextrans produced by L. mesenteroides SF3 (recorded at 750 MHz in D2O at 25°C).

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Figure 9. SEM images of dextrans obtained from fermentation with L. mesenteroides SF3.

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Table 1. Specifications of the MF membrane used for the recovery of dextran. CFP-1-E-4MA

Manufacturer

GE Healthcare Bio-Sciences Corporation

Cut-off (µm)

0.1

Membrane surface area (cm 2)

420

Membrane material

Polysulfone

Configuration

Hollow fiber cartridges

Operating pH

2-13

Max. operating temperature (°C)

80

Max. operating pressure (bar)

1.7

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Type

45

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Table 2. Physicochemical composition of the fermentation broth before and after the MDF-MF process. Fermentation broth

MDF-Retentate*

MF-Retentate**

pH

3.61 ± 0.24a

3.76 ± 0.12a

3.70 ± 0.00a

Total solids content (%)

11.16 ± 0.25a

3.36 ± 0.31c

6.17 ± 0.09b

Reducing sugars (g GE L-1)

11.85 ± 1.61b

1.36 ± 0.54a

0.29 ± 0.00a

Viscosity (cP)

35.43 ± 6.46b

28.67 ± 3.71b

267.33 ± 16.17a

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Determination

*MDF process was operated to TMP = 0.7 bar, Qf = 70 L h-1, T = 25°C. **MF process was operated to TMP = 0.9 bar, Qf = 70 L h-1, T = 25°C.

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Results expressed with the mean ± standard deviation for each sample (n = 3). Different letters in each

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row indicate a significant difference (p < 0.05) according to the Tukey's test.

46

Parameter

Value

h-1)

645.09 ± 47.28

Jw1 (kg m-2 h-1) Jw2 (kg

m-2

58.29 ± 2.42

h-1)

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Jw (kg

m-2

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Table 3. Water permeability, fouling index and cleaning efficiency of the MF membrane (at TMP = 0.9 bar).

576.00 ± 50.43 374.25 ± 27.43

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Lp (kg m-2 h-1 bar-1) Lp1 (kg m-2 h-1 bar-1)

33.81 ± 1.41

If (%)

90.95 ± 0.29

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FRR (%)

89.24 ± 1.28

Results expressed with the mean ± standard

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deviation for each sample (n = 3).

47

Yield (g L-1)

16.12 ± 0.84b

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Table 4. Comparison of the productivity of MDF-MF and solvent extraction protocols.

EtOH consumption (L / batch)

4.00 ± 0.00a

0.90 ± 0.00b

Dextran

Determination

MDF-MF process 22.48 ± 0.45a

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EtOH method

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Results expressed with the mean ± standard deviation for each sample (n = 3). Different letters in each row indicate a significant difference (p < 0.05) according to the Tukey's

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test.

48

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Table 5. Branching composition of the dextran produced by L. mesenteroides SF3. Branching ratio (%) Dextran

α (1-3)

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α (1-6) 77.35

22.65

MDF-MF process

88.18

11.82

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EtOH method

49

Dextran

Determination

EtOH method

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Table 6. Physicochemical characterization of dextran.

MDF-MF process

3.38 ± 1.02a

Water activity

0.30 ± 0.08a

0.21 ± 0.07a

Hygroscopicity (g 100g-1)

2.38 ± 0.56a

1.39 ± 0.15b 72.08 ± 0.87a

7.45 ± 0.48b

10.65 ± 0.40a

1.43 ± 0.17a

1.70 ± 0.08a

Apparent

0.73 ± 0.04a

0.71 ± 0.06a

Packed

0.83 ± 0.04a

0.85 ± 0.07a

12.22 ± 1.39b

16.37 ± 1.01a

Water

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Oil

Porosity (%)

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Density (g cm-3)

2.42 ± 0.37a

55.87 ± 0.87b

Solubility (%) Absorption capacity (g g-1)

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ro

Moisture (%)

Results expressed with the mean ± standard deviation for each sample (n = 3). Different letters in each

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row indicate a significant difference (p < 0.05) according to the Tukey's test.

50