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PII: S0144-8617(18)30729-X
DOI: https://doi.org/10.1016/j.carbpol.2018.06.075
Reference: CARP 13749
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Design of a fermentation process for agave fructooligosaccharides production using
agave fructooligosaccharides
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Sandoval-González S.R., Jiménez-Islas, H., Navarrete-Bolaños, J.L.†
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Departamento de Ingeniería Bioquímica-Ciencias de la Ingeniería. Instituto Tecnológico de
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Celaya. Av. Tecnológico s/n. CP 38010. Celaya Gto. México. †Corresponding author: e-
mail: luis.navarrete@itcelaya.edu.mx.
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Highlights:
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processes is confirmed.
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Abstract
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performed enzyme kinetics studies to achieve agave fructans hydrolysis. The results
showed that under constant operating conditions (pH 7.7, 40 °C, 175 rpm of agitation, and
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0.005 VVM of aeration) results in the production of an enzymatic extract with 49.57 mg/L.
This enzymatic extract, when mixed with an agave fructans solution containing 37.8 g/L,
allowed us to obtain products with 18% more FOS content the original concentration. The
mass spectrum plot shows that the hydrolyzed product contains FOS with a degree of
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polymerization from 5 to 9 hexose units. These results are promising because they show
FOS production from agave and confirm that importance of using native strains in the
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design of directed fermentation processes.
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KEYWORDS: agave fructans, fructooligosaccharides production, endoinulinases,
Saccharomyces paradoxus.
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1. Introduction N
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Fructans are a heterogeneous mixture of fructose polymers linked by β-(2→1) bonds, in
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which fructosyl units are bound to a D-glucose residue at the terminal reducing end by an
α-(1→2) bond (Sánchez et al., 2008; de Oliveira et al., 2011; Singh et al., 2016). According
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to the type of linkage and structure, the fructans are classified into several categories,
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including inulins (lineal (2→1) link), levans (lineal (2→6) link), graminans (branched, with
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both β-(2→1) and β-(2→6) links), neo-inulin and neo-levan, formed from neo-kestose
(slightly branched, with both β-(2→1) and β-(2→6) links), and neo-fructans (highly
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branched, formed from neo-kestose with both β-(2→1) and β-(2→6) links) (Vijn &
Smeekens, 1999; van den Ende et al., 2004; Livingston et al. 2009; Santiago-García and
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López, 2009). Other fructans, with lineal structure and a (2→1) link, are known as
fructooligosaccharides (FOSs), the difference in the inulin being the length of the polymer
chain, which is shorter for FOSs, with a degree of polymerization from three to 10 fructose
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molecules, which apparently confers better technical applications and nutritional properties
than any other fructans on it, including inulin (Sanchez et al. 2008; Chen & Liu, 1996;
Santiago-García & López, 2009; Ávila-Fernández et al. 2011; Mutanda et al., 2014). Based
on the above, three different methods for FOS production have been developed: (1)
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extraction from certain foods or plant materials where FOSs are naturally present; (2)
enzymatic synthesis from sucrose; and (3) enzymatic hydrolysis of inulin. Of these, the
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extraction process from natural sources is not viable, due to low concentrations (Sangeetha
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et al., 2005; Sánchez et al., 2008; Dominguez et al., 2012; Mutanda et al., 2014; Zeng et al.,
2016; Singh et al., 2016; Xu et al., 2016). Enzymatic synthesis from sucrose, using
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enzymes with transfructosylation activity (i.e., β-fructosyltransferase (EC 2.4.1.9) and β-
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fructofuranosidase (EC 3.2.1.26), it is not viable either because the yields achieved are low,
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seemingly due to the presence of glucose, which inhibits the fructosyl-transfer reaction, and
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the high enzyme cost (Sangeetha et al., 2005; Mutanda et al., 2008; Dominguez et al., 2012;
Vega-Paulino & Zúniga-Hansen, 2012; Lorenzoni et al., 2014; Zeng et al., 2016). The
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(EC 3.2.1.7) activity (which randomly breaks down the internal β-D-(2→1) glycosidic
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option. This produces higher yields, which suggests that such enzyme preparations can be
used as viable alternatives for industrial FOS production (Singh et al., 2010, Singh et al.,
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using enzymes is heavily dependent on their cost, however, and often this process is
hampered by the lack of prolonged operational stability, and difficulties in recovering and
reusing the enzymes (Lorenzoni et al., 2015). Alternatives commonly used to overcome
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such technical drawbacks are enzyme immobilization (which is only justified if the enzyme
selected microorganisms because of their easy cultivation and high yields, which is the best
way to obtain low-cost enzymatic preparations (Cadena et al., 2010; Vega-Paulino &
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Zúniga-Hansen, 2012; Xu et al., 2016).
In the last decade, interest in studying the functional properties of agave fructans has
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increased due the high content present in agave plants, which compares favorably with the
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fructans content present in chicory and Jerusalem artichoke (major natural sources of
fructans) (Silver, 2006), and studies that have indicated that agave fructans have a potential
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prebiotic effect, similar to the one observed in established inulin-type prebiotics derived
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from chicory root (Santiago-García and López, 2009; Gomez et al., 2010; Márquez-Aguirre
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et al. 2013; Moreno-Vilet et al., 2014). These characteristics suggest that agave plants could
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also be an abundant raw material for FOS production. Based on the above, the enzymatic
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hydrolysis of agave fructans, using commercial endo-inulinase, has been performed, but
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due the complex structure of agave fructans, no hydrolysis products have been observed, or
they were not significant (Ávila-Fernández et al. 2011; Michel-Cuello et al., 2012;
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Huazano-García & López, 2017). Therefore, the production of FOSs from agave fructans
has been a technical challenge for the last few years. Nowadays, agave plants are exploited
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(pulque), and distilled (tequila and mezcal) beverages, with national and international
spontaneous fermentation in the previously prepared heart cavity of the agave plant
(Garcia-Aguirre et al., 2009). Studies have demonstrated that aguamiel contains some
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native strains that have capacity for inulinase synthesis with exo-inulinase activity, and that
in their chemical composition are found presents the FOS. Therefore it is probably that in
the aguamiel is found native strains with capacity to synthesize endoinulinases (Cruz et al.,
2006; Ortiz-Basurto et al., 2008; Lappe-Oliveras et al., 2008; Garcia-Aguirre et al., 2009).
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On the other hand, previous studies have shown that the use of native strains from
spontaneous fermentations can produce high yields in directed fermentation with the best
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quality features of the final products and carry out conversions with more efficiency
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because the strains are naturally adapted to those ecological niches (Navarrete-Bolaños et
al., 2012).
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On the basis of these observations, herein, a candidate native Saccharomyces isolate,
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obtained from aguamiel samples and identified by molecular detection, was used in the
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analysis of process variable parameters to maximize both the endo-inulinase synthesis and
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the enzymatic hydrolysis of agave fructans. Such knowledge is needed in order to optimize
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FOS production from agave fructans in a 3-L stirred-tank fermenter, so as to realize the
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industrial level.
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Mature 'hearts' or 'piñas' (plants without leaves) from six-year-old (on average) Agave
angustifolia, harvested from the Jalpilla locality of Comonfort Gto, Mexico, were cooked in
an autoclave at 90°C for 15 min, and pressed to obtain the agave juice containing the
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fructans. This was stored at -79°C until it was needed. Other pines were milled and the pulp
2.2 Aguamiel
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Comonfort Gto, Mexico were used for microbial ecology studies.
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2.3 Strain isolation
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Samples of aguamiel were taken and inoculated on Petri dishes containing nutritive agar
(Difco, Detroit, MI) for bacteria, potato dextrose agar (Difco, Detroit, MI) and malt extract
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agar (MEA, Difco, Detroit, MI) for yeast and selective media formulated from aguamiel or
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agave extract, plus bacteriological agar at 1.5% (v/w). The inoculations were performed by
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taking 1 mL of aguamiel and spreading this amount onto the surface of each medium in the
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Petri dishes. These were then incubated at 28, 32, and 37°C for 24, 48, and 72 h. The
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colonies with different morphologies that developed were transferred to fresh agar media
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(nutritive, potato dextrose, and selective), and incubated again. The procedure was repeated
until pure cultures were obtained. To ensure the purity of the isolated colonies, during the
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whole isolation strategy, and at the end of every incubation period, the colonies developed
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Germany) methods, using staining techniques, and biochemical tests, using an API
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The pure bacteria and yeast strains that were isolated were cultured in potato dextrose (for
yeast) and nutritive (for bacteria) agar slants at 28°C for 48 h (representing the best
conditions for growth). Biomass samples were taken from each slant and transferred to 250
mL Erlenmeyer flasks containing 100 mL of nutritive broth (Difco, Detroit, MI) for
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bacteria, and potato dextrose broth (Difco, Detroit, MI) for yeast. These were mixed and
incubated at 28°C and 100 rpm for 48 h on a rotary shaker (model 4520, Forma Scientific,
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Marietta, OH) for culture propagation. The final product obtained from each Erlenmeyer
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flask propagation was centrifuged at 10,000 g (Hermle, model Z383). The supernatant
obtained was used as a source of extracellular enzyme, or crude enzymatic extract. All
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crude enzymatic extracts obtained were analyzed, according to the Bradford method
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(1976), to quantify the amount of synthesized enzyme. The endo-inulinase activity was
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measure by mixing 50 mL of each enzymatic extract with 100 mL of a fructans solution of
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80 g/L, and incubated on a rotary shaker at 28°C and 100 rpm for 24 h, followed by both
profile.
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Once the strain had been selected, molecular identification was performed, which included
ribosomal sequence analysis of the 5.8S rRNA gene and two ribosomal internal transcribed
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genomic DNA extraction from selected strains, according to the protocol proposed by
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Ausubel et al. (1999), using an InvitrogenTM column from the PureLinkTM Genomic DNA
set. For rRNA gene amplification, the DNA extracted was mixed with PCR supermix
PCR was performed under the following conditions: initial denaturalization at 95°C, for 5
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min, 35 cycles, with 1 min of denaturation at 95°C, 2 min of annealing at 50, 52, 54, 56,
and 58°C, 2 min of extension at 72°C, and a final extension for 10 min at 72°C. The PCR
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products were purified with the QIAquick® Gel Extraction Kit (QIAGEN® Group). Once
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purified, the products were sending to the Laboratorio Nacional de Genómica para la
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sequencing which was occurred using the Sanger technique on an Applied Biosystems
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Automated 3730xl DNA Analyzer (Applied Biosystems by Life Technologies, Carlsbad,
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CA, USA) and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Invitrogen). The
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sequence was compared with those reported on the National Center for Biotechnology
Information (NCBI) database using the 'Blasrx' algorithm for strain identification.
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considered: V1, acidity (expressed as pH) (temperature); V2, temperature (acidity); V3,
agitation; V4, aeration. These variables were singled out on the basis of their relationships
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with the biomass growth and metabolic activities (temperature and pH), and their
relationships with the heat and mass transfer limitations in the batch reactor (agitation and
aeration). Agitation and aeration are essentially collinear variables affecting the oxygen
transfer rate, which implicitly affects microbial growth, substrate transformation, and
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synthesis of the product. The feasible ranges for these variables were defined, on the basis
of a preliminary screening design (not shown), as being 3-7 for pH (V1), 25-35°C for
temperature (V2), 100-200 rpm for agitation (V3), and 0.5-1.5 vvm for aeration (V4), which
were used as boundaries of the search region to construct a central composite experimental
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design (Table 1). For this study, a 3-L stirred-tank bioreactor (AZ control bioreactor,
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fermentations, the starter inoculum concentration (1.0×106 cells/mL), volume medium (1.5
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L), and composition medium (Difco, PDB broth) were kept constant. The output functions
were both the enzyme quantity synthesized (measured as protein) (Y1), and biomass yield
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(Y2).
To define the ratio among the quantity of enzymatic extract to substrate to maximize the
FOS yield, an enzyme kinetics study was performed. The study began with exploratory
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assays of mixtures among an enzymatic extract (e.g., 50 mL) that had an enzyme
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concentration of 15.88 mg/L of total protein (on average), and substrate solutions (e.g., 25
mL) with different concentrations (from 60 to 200 g/L) at a ratio of 2:1 (enzyme
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volume:substrate volume). Once homogenized, the mixtures were incubated at 30°C and
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150 rpm for 192 h on a rotary shaker (Model 4520, Forma Scientific, Marietta, OH, USA),
taking samples each 24 h for FOS analysis. When a good approximation of the
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enzyme:substrate ratio had been reached, the initial reaction rate (V0) was measured to
determine the reaction time. With this information, the effects of varying the conditions of
the reaction were investigated, first for substrate solutions with different concentrations
mixed with an enzymatic extract containing a constant content of enzyme in a ratio of 2:1,
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followed for mixtures of different enzymatic extracts volume with protein constant
concentration (80 g/L) to obtain different (enzyme volume:substrate volume) ratios (from
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of enzymatic extract with 60 mL of substrate solution]). All final reaction samples were
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2.8 High performance liquid chromatography (HPLC) analysis
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To ensure that the enzymatic extracts had endo-inulinase activity, mixtures of enzymatic
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extracts with fructans solutions were filtered through Millipore membranes of 45-μm pore-
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diameter. Samples consisting of 20 μL of hydrolyzed extract were injected to the HPLC.
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The equipment used for the HPLC assays was modular equipment (Agilent Technologies
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1200 Series) that included a G1322 vacuum degasser, G1311 quaternary pump, G1329
detector, and the OpenLAB chromatography management system for computer control
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polymer profiles were obtained using a column of Prevail Carbohydrate ES 5µm, 250×4.6
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mm (Alltech, Grace Davison Discovery Sciences, Chicago IL, USA). The solvent elution
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was operated at 1.0 mL/min of a mixture containing acetonitrile (A) and water-ammonium
83%-A and 17%-B from 0 to 25 min, 73%-A and 27%-B from 25 to 40 min, 55%-A and
45%-B from 40 to 80 min, and 45%-A and 55%-B from 80 to 120 min. The separation was
performed at 40˚C, and the sugars were monitored using a refractive index detector
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concentration, using the relative percentage area covered in the chromatogram, was used to
calculate sugar concentrations. To establish the reference for the hydrolysis products, the
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USA).
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To determine the degree of polymerization for both the external standards and the samples
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enzymatically treated, a LC-MS coupled system (model LTQ XL, Thermo Scientific,
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Waltham MA, USA) was used and developed a method to quantify the carbohydrate
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distribution. This system offers a high capacity linear trap with two detectors, electrospray
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ionization, atmospheric pressure chemical ionization, and multiple molecular dissociation
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infusion at 180 μL per hour, with substrate concentrations from 0.6% (w/v), and recording
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the mass spectra in positive mode in a range of 100-2000 m/z. Data were acquired, and
Statistical analysis. The results from the experimental designs were analyzed using a
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probability distribution values for a confidence interval of 95% (α=0.05), and variable
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optimization, based on response surface methodology, which included data analysis using
least squares to construct mathematical models that described the relationship between the
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3. Results and discussion
Twenty (X1+X2+X3+.....+X20) pure cultures were isolated from the aguamiel. Seven (X2,
X6, X10, X11, X13, X15, and X17) presented characteristics of bacteria, and 13 (X1, X3, X4, X5,
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X7, X8, X9, X12 X14, X16, X18, X19, X20) of yeast. The enzymatic extract obtained from each
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one showed that nine (eight yeast: X1, X3, X4, X5, X7, X8, X12, X14, one bacterium: X15)
synthesized mainly enzymes with exo-inulinase activity, four (three yeast: X16, X19, X20,
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one bacterium: X6) synthesized mainly enzymes with endo-inulinase activity, two yeast (X9
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and X18) synthesized both exo- and endo-inulinase enzymes, and the others (X2, X10, X11,
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X13, X15, and X17) did not exhibit enzyme inulinase activity. Seemingly, the strains that
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synthesized either exo- and/or endo-inulinase activity acted synergistically with each other,
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and the degree of synergy must affect the level of fructose formation, and the sweetness of
the aguamiel. Probably, the endo-inulinase is the first enzyme to act in the agave fructans
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hydrolysis, to produce a wide range of fructo- and/or oligosaccharides, which are converted
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short processing time. The strains that did not show enzyme inulinase activity are probably
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found in ecological partnerships, as commensals or parasites, that do not alter the stability
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of the fructanolitic microorganisms, but they must have a specific objective or contribution
to the aguamiel, such as quality features, or acting in the fermentation process that converts
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sugars into ethanol to produce pulque, a traditional Mexican alcoholic beverage, produced
from the fermentation of the fresh sap (aguamiel) extracted from several species of Agave
(maguey) plants that grow on the central Mexico plateau. Evaluation of the enzymatic
extract that contained the endo-inulinase activity showed that the enzymatic extract from
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the X16 strain presented the highest enzyme content, achieved the most agave fructans
Once the strain X16 was identified as the best culture for endo-inulinase synthesis and
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maximum FOS yield from agave fructans hydrolysis, we proceeded with the strain
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identification. The results of the nucleotide sequencing analysis, which was deposited in
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99.8% identity score (0% expectancy), a strain that is the closest known relative of the
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well-known S. cerevisiae, which has also been shown to be a strain with possibilities for
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application in the wine industry, but has never been shown as a endo-inulinase-producer
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strain (Orlic et al., 2007).
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Once S. paradoxus was selected as the best native strain for endo-inulinase production, an
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11 3 35 100 1,5 5,36
12 5 20 150 1.0 17,60
13 9 30 150 1.0 2.00
14 7 35 200 1,5 32,90
15 7 25 200 0,5 28,81
16 5 30 150 2.0 15,47
17 5 30 50 1.0 19,52
18 3 35 200 0,5 22,59
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19 3 35 100 0,5 1.50
20 3 25 100 0,5 10,92
21 7 35 200 0,5 41,79
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22 5 30 150 0.0 21,49
23 1 30 150 1.0 1.20
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24 3 25 200 0,5 1.80
25 5 40 150 1.0 27,25
26 3 35 200 1,5 13,82
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The results (last column of Table 1) revealed that, based on the analysis of variance, only
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the pH variable exhibited a significant effect within their defined limits (p<0.05) for both
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output functions. The other variables that were not significant suggested the presence of an
inflection point, which could be a maximum or a minimum. Therefore, the results were
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used to construct a second-order polinomial model, using the least squares method, to
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describe the relationship that exists among the independent variables (temperature,
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agitation and aeration) and dependent variables (endo-inulinase synthesis: YP), by obtaining
− 0.00007𝑉3 𝑉4 − 3.88𝑉42
The model was also analyzed based on the analysis of variance, which did not show a
statistically significant relationship (p<0.05) between the output function and the process
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parameters among the defined limits; however, when the models were used to construct
response plots, these showed the presence of a maximum, which suggests the presence of a
global optimum (Figure 1). Therefore, on the basis of this model, the location of the
optimum that maximizes the in situ endoinulinase production was accurately computed via
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derivation of the second-order model, and the solution of the resulting set of linear
equations:
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𝜕𝑌1
= 19.92 – 2(1.4)𝑉1 + 0.17𝑉2 – 0.028𝑉3 − 2.41𝑉4
𝜕𝑉1
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𝜕𝑌1
= −2.18 + 0.17𝑉1 + (2)(0.001)𝑉2 + 0.031𝑉3 + 0.027𝑉4
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𝜕𝑉2
𝜕𝑌1
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= −0.077 – 0.028𝑉1 + 0.013𝑉2 – (2)(0.0006)𝑉3 − 0.00007𝑉4
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𝜕𝑉3
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𝜕𝑌1
= 5.45 – 2.41𝑉1 + 0.27𝑉2 – 0.00007𝑉3 − (2)(3.88)𝑉4
𝜕𝑉4
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Figure 1. Graphical representations of the second-order polynomial model that show the
variable values that maximizes the endoinulinase synthesis.
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The solution of the model, based on solving the sets of linear equations, are V1 = 7.7, V2 =
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40, V3 = 175, and V4=0.005, which are the optimum values (pH=7.7, temperature=40°C,
indicated that the optimum conditions for endo-inulinase synthesis maximization are pH
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7.7, 40°C temperature, 175 rpm of agitation, and 0.005 VVM of aeration level. The
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optimum value of pH found inside among average values present in the aguamiel, from
which the S. paradoxus was isolated, the temperature value is consistent with the semi-
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desert areas of Mexico, where a great diversity of agave plants grow, and the optimum
values for agitation and aeration are low because they are only required to maintain a
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Confirmation assays, using the optimal culture medium under optimal process conditions,
were performed, the results yielded 49.57 mg of total protein/L (average), which compares
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favorably against the predicted model value (47.4 mg of total protein/L), and represent
three times more synthesized enzyme compared to those obtained at level shake flask using
the same culture medium (15.88 mg of total protein/L mg of total protein/L). These yield
results confirm the suitability of the optimization process parameters for the design of
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efficient processes, one of the larger obstacles associated with the enzymatic hydrolysis in
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3.4 Studies of enzymatic kinetics
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Once the enzymatic extracts with endo-inulinase activity had been obtained, a strategy
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experiment for agave fructans hydrolysis was performed. The exploratory assays to find an
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adequate enzyme-substrate reaction mixture showed, in general, an increased FOS yield
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directly proportional to the substrate concentration among the defined limits for both
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substrate and enzyme concentration, but the solution of agave fructans with a concentration
of 80 g/L, mixed with an enzymatic extract containing 15.88 mg of total protein/L showed
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the best FOS yield. For this initial ratio, the experimental assays to evaluate both the
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enzyme concentration effect, and the substrate concentration, revealed that the best ratio
extract, with a content of 10.5 mg of total protein/L and 100 mL of agave fructans solution
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at 8% (w/v).
The analysis of the hydrolyzed products, via LC-MS, showed that they contained 18% (on
average) more FOS compared to the control (natural samples), with a degree of
polymerization (DP) of preferably five to nine hexose units, and a low, and not significant,
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content of free hexoses (Figure 2). These results confirm that the native strain S. paradoxus
of exo-inulinase and invertase, which is another quality advantage of the enzymatic extract
because it limits the presence of free hexoses and sucrose, which is a quality requisite of the
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final product.
Figure 2. Mass spectrum plot that show the mass-to-charge ratio for samples without
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enzyme treatment (upper) and the hydrolyzed product by the enzymatic extract of S.
paraduxus (lower).
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4. Conclusions
The importance of this study is that it shows an alternative method of FOS production,
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based on enzymatic hydrolysis. Steps were described for designing an overall A. fructans
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enzymatic hydrolysis process for FOS production. This new product may have a huge
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prebiotic potential as new branched FOSs, and give rise to a many new application studies
inexpensive, renewable, and abundant raw material, and the enzyme can be produced in
situ, the FOS production process developed in this study is inexpensive, and simple to scale
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up to industrial production. The efficiency achieved up to now is low, however, and this is
needed, and to complement the global process design, which would probably increase the
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FOS production. In this manner, the first integral approach to the design of a
achieved. Finally, the results confirm that native strains are the best for use in evolving
from spontaneous to directed fermentation because they are naturally adapted to realize a
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specific function in a specific ecosystem, so as to maintain the equilibrium. Specifically, the
results show S. paradoxus, a native strain of the fermented agave plant, to be a new
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producer strain of endo-inulinase, which can be used for production of agave FOSs, and
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probably for FOS production from inulin because it has a simpler structure.
Acknowledgements
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The authors acknowledge the Tecnológico Nacional de México for financing the research
(5713.16-P).
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Conflict of interest
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There is no known actual or potential conflict of interest including any financial, personal
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or other relationships with other people or organizations associated with this work.
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