Elsevier

Carbohydrate Polymers

Volume 198, 15 October 2018, Pages 94-100
Carbohydrate Polymers

Design of a fermentation process for agave fructooligosaccharides production using endo-inulinases produced in situ by Saccharomyces paradoxus

https://doi.org/10.1016/j.carbpol.2018.06.075Get rights and content

Highlights

  • A methodology to evolve from spontaneous to directed fermentation is shown.

  • A stepwise methodology to designing feasible fermentation processes is proposed.

  • The importance of using native strains in the design of directed fermentation processes is confirmed.

  • Saccharomyces paradoxus as promising strain for producing endo-inulinases is proposed.

Abstract

Saccharomyces paradoxus, a native microorganism of the aguamiel, was used successfully for endoinulinase synthesis for agave fructooligasaccharide (FOS) production. We optimized the fermentation parameters to maximize the enzyme synthesis, and we 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 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 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 design of directed fermentation processes.

Introduction

Fructans are a heterogeneous mixture of fructose polymers linked by β-(2→1) bonds, in which fructosyl units are bound to a D-glucose residue at the terminal reducing end by an α-(1→2) bond (Sáchez, Guio, Garcia, Silva, & Caicedo, 2008; Braz de Oliveira et al., 2011; Singh, Singh, & Kennedy, 2016). According to the type of linkage and structure, the fructans are classified into several categories, including inulins (lineal (2→1) link), levans (lineal (2→6) link), graminans (branched, with 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 branched, formed from neo-kestose with both β-(2→1) and β-(2→6) links) (Livingston, Hincha, & Heyer, 2009; Santiago-García & Lopez, 2009; Van-den-Ende, De Coninck, & Van-Laere, 2004; Vijn & Smeekens, 1999). 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 molecules, which apparently confers better technical applications and nutritional properties than any other fructans on it, including inulin (Ávila-Fernández, Galicia-Lagunas, Rodríguez-Alegría, Olvera, & López-Munguía, 2011; Chen & Liu, 1996; Mutanda, Mokoena, Olaniran, Wilhelmi, & Whiteley, 2014; Sáchez et al., 2008; Santiago-García & Lopez, 2009). Based on the above, three different methods for FOS production have been developed: (1) 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 extraction process from natural sources is not viable, due to low concentrations (Dominguez et al., 2012; Mutanda et al., 2014; Sáchez et al., 2008; Sangeetha, Ramesh, & Prapulla, 2005; Singh et al., 2016; Xu, Zheng, Xu, Yong, & Ouyang, 2016; Zeng et al., 2016). Enzymatic synthesis from sucrose, using enzymes with transfructosylation activity (i.e., β-fructosyltransferase (EC 2.4.1.9) and β-fructofuranosidase (EC 3.2.1.26), it is not viable either because the yields achieved are low, seemingly due to the presence of glucose, which inhibits the fructosyl-transfer reaction, and the high enzyme cost (Dominguez et al., 2012; Lorenzoni, Aydos, Klein, Rodrigues, & Hertz, 2014; Mutanda, Wilhelmi, & Whiteley, 2008; Sangeetha et al., 2005; Vega-Paulinoa & Zúniga-Hansena, 2012; Zeng et al., 2016). The enzymatic hydrolysis of inulin, using commercial enzymatic solutions with endo-inulinase (EC 3.2.1.7) activity (which randomly breaks down the internal β-D-(2→1) glycosidic linkages, producing a mixture of FOSs, containing β-D-Fru(1→2)-[β-D-Fru(1→2)-]n, where n = 1–9, and α-D-Glu(1→2)-[β-D-Fru(1→2)-]n, where n = 2–9) is seemingly the best option. This produces higher yields, which suggests that such enzyme preparations can be used as viable alternatives for industrial FOS production (Nguyen, Rezessy-Szabó, Czukor, & Hoschke, 2011; Singh & Singh, 2010; Singh et al., 2016). The commercial exploitation of industrial biotransformations 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 such technical drawbacks are enzyme immobilization (which is only justified if the enzyme is expensive, or is inactivated under reaction conditions), or in situ enzyme production by 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-Paulinoa & Zúniga-Hansena, 2012; Xu et al., 2016).

In the last decade, interest in studying the functional properties of agave fructans has increased due the high content present in agave plants, which compares favorably with the 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 prebiotic effect, similar to the one observed in established inulin-type prebiotics derived from chicory root (Gomez, Tuohy, Gibson, Klinder, & Costabile, 2010; Márquez-Aguirre et al., 2013; Moreno-Vilet et al., 2014; Santiago-García & Lopez, 2009). These characteristics suggest that agave plants could also be an abundant raw material for FOS production. Based on the above, the enzymatic hydrolysis of agave fructans, using commercial endo-inulinase, has been performed, but due the complex structure of agave fructans, no hydrolysis products have been observed, or they were not significant (Ávila-Fernández et al., 2011; Huazano-García & López, 2015; Michel-Cuello et al., 2012). Therefore, the production of FOSs from agave fructans has been a technical challenge for the last few years. Nowadays, agave plants are exploited for the production of Mexican indigenous nonalcoholic (aguamiel), alcoholic nondistilled (pulque), and distilled (tequila and mezcal) beverages, with national and international recognition (Lappe-Oliveras et al., 2008). Aguamiel is the sugary sap obtained by spontaneous fermentation in the previously prepared heart cavity of the agave plant (García-Aguirre et al., 2009). Studies have demonstrated that aguamiel contains some 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-Guerrero, Olvera, García-Garibay, & Gómez-Ruiz, 2006; García-Aguirre et al., 2009; Lappe-Oliveras et al., 2008; Ortiz-Basurto et al., 2008). 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 quality features of the final products and carry out conversions with more efficiency because the strains are naturally adapted to those ecological niches (Navarrete-Bolaños, 2012).

On the basis of these observations, herein, a candidate native Saccharomyces isolate, obtained from aguamiel samples and identified by molecular detection, was used in the analysis of process variable parameters to maximize both the endo-inulinase synthesis and the enzymatic hydrolysis of agave fructans. Such knowledge is needed in order to optimize FOS production from agave fructans in a 3-L stirred-tank fermenter, so as to realize the ultimate goal of an efficient FOS production process, which can be scaled up to an industrial level.

Section snippets

Agave juice

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 fructans. This was stored at −79 °C until it was needed. Other pines were milled and the pulp fraction was also stored at −79 °C until used.

Aguamiel

Samples of aguamiel obtained from traditional rural producers of Jalpilla locality of

Microbial ecology studies

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, X7, X8, X9, X12 X14, X16, X18, X19, X20) of yeast. The enzymatic extract obtained from each 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, one bacterium: X6) synthesized mainly enzymes

Conclusions

The importance of this study is that it shows an alternative method of FOS production, based on enzymatic hydrolysis. Steps were described for designing an overall A. fructans enzymatic hydrolysis process for FOS production. This new product may have a huge prebiotic potential as new branched FOSs, and give rise to a many new application studies in the alimentary and pharmaceutical industries. In addition, because agaves represent an inexpensive, renewable, and abundant raw material, and the

Conflict of interest

There is no known actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations associated with this work.

Acknowledgement

The authors acknowledge the Tecnológico Nacional de México for financing the research (5713.16-P).

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