Adriana C. Flores-Gallegos

Comparative study of fungal strains for thermostable inulinase production

Fructose and fructo-oligosaccharides (FOS) are important ingredients in the food industry. Fructose is considered an alternative sweetener to sucrose because it has higher sweetening capacity and increases iron absorption in children, and FOSs are a source of dietary fiber with a bifidogenic effect. Both compounds can be obtained by enzymatic hydrolysis of inulin. However, inulin presents limited solubility at room temperature, thus, fructose and FOS production is carried out at 60°C. Therefore, there is a growing interest to isolate and characterize thermostable inulinases. The aim of this work was to evaluate the capacity of different fungal strains to produce potential thermostable inulinases. A total of 27 fungal strains belonging to the genera Aspergillus, Penicillium, Rhizopus, Rhizomucor and Thermomyces were evaluated for production of inulinase under submerged culture using Czapek Dox medium with inulin as a sole carbon source. Strains were incubated at 37°C and 200 rpm for 96 h. Crude enzyme extract was obtained to evaluate inulinase and invertase activity. In order to select the fungal strain with the highest thermostable inulinase production, a selection criterion was established. It was possible to determine the highest inulinase activity for Rhizopus microsporus 13aIV (10.71 U/mL) at 36 h with an optimum temperature of inulinase of 70°C. After 6 h at 60°C, the enzyme did not show any significant loss of activity and retained about 87% activity, while it only retains 57% activity at 70°C. According to hydrolysis products, R. microsporus produced endo and exo-inulinase.

Production of thermostable xylanase by thermophilic fungal strains isolated from maize silage

The search for microorganisms able to produce thermostable xylanases with high yield and characteristics desired for industrial applications has been strongly encouraged since such enzymes are widely used in large-scale processes. In the present study, thermophilic fungal strains able to grow at high temperatures (≥55 °C) were isolated from maize silage. The strains were molecularly identified and used for the production of extracellular xylanase by solid-state fermentation using corn cobs as support-substrate material. Species from the genera Rhizomucor and Aspergillus were identified among the isolated strains and these species demonstrated good ability to produce xylanase under solid-state fermentation conditions. Maximal values of enzymatic activity (824 U/g) and productivity (8.59 U/g.h) were obtained with Rh. pusillus SOC-4A (values per g dry weight of fermented medium). The xylanase produced by this fungus presented thermal stability at 75 °C, with maximum activity at 70 °C and pH 6.0, revealing, therefore, great potential for application in different areas.

Gene encoding inulinase isolated from Penicillium citrinum ESS and its molecular phylogeny.

Inulinase is an enzyme produced by plants and several microorganisms, including fungi, to hydrolyze the β-2,1 glycosidic linkages present in some oligosaccharides to produce fructose and glucose. This enzyme, in conjunction with invertases, levanases, and two types of 1-fructosyl transferases have been described as members of the glycosyl hydrolases (family 32), the most diverse group of enzymes used by microbes for biomass degradation. As being part of the same clan, they have common evolutionary origin sharing the most important functional characteristics. Recently, a xerophylic fungi strain isolated from Mexican semi-desert, Penicillium citrinum ESS has been reported as inulinase producer, which could have greater stability than other enzymes due to a metabolic machinery adapted to typical temperature changes in this region. To continue the understanding of action mechanisms of these enzymes and to establish evolutionary relationships within this family, in the present study, phylogenetic analyses were used to analyze amino acid sequences coding fungal and yeast glycoside hydrolases of family 32, including the new sequenced inulinase of P. citrinum ESS. It was possible to elucidate the action mechanism of fungal glycoside hydrolases in present study and to classify inulinase from P. citrinum ESS as an exo-inulinase on the basis of their amino acid sequence phylogenetic affinities.

Nucleotide and amino acid variations of tannase gene from different Aspergillus strains.

Tannase is an enzyme that catalyses the hydrolysis of ester bonds present in tannins. Most of the scientific reports about this biocatalysis focus on aspects related to tannase production and its recovery; on the other hand, reports assessing the molecular aspects of the tannase gene or protein are scarce. In the present study, a tannase gene fragment from several Aspergillus strains isolated from the Mexican semidesert was sequenced and compared with tannase amino acid sequences reported in NCBI database using bioinformatics tools. The genetic relationship among the different tannase sequences was also determined. A conserved region of 7 amino acids was found with the conserved motif GXSXG common to esterases, in which the active-site serine residue is located. In addition, in Aspergillus niger strains GH1 and PSH, we found an extra codon in the tannase sequences encoding glycine. The tannase gene belonging to semidesert fungal strains followed a neutral evolution path with the formation of 10 haplotypes, of which A. niger GH1 and PSH haplotypes are the oldest.

Native yeasts for alternative utilization of overripe mango pulp for ethanol production

Mango fruits (Mangifera indica L.) are highly perishable, causing postharvest losses and producing agroindustrial waste. In the present work, native yeasts were used to evaluate ethanol production in overripe mango pulp. The two isolated strains showed similar sequences in the 18S rDNA region corresponding to Kluyveromyces marxianus, being different to the data reported in the NCBI database. Values of up to 5% ethanol (w/v) were obtained at the end of fermentation, showing a productivity of 4g/l/day, a yield of up to 49% of ethanol and a process efficiency of 80%. These results represent a viable option for using the surplus production and all the fruits that have suffered mechanical injury that are not marketable and are considered as agroindustrial waste, thus achieving greater income and less postharvest losses.

Analytical Methods for Pectin Methylesterase Activity Determination: a Review

Pectin methylesterase is an enzyme with an important in vivo role in plants as well as in food industry. This enzyme catalyzes the hydrolysis of methyl ester bounds in pectin which is one of the main components of cell wall in plants, producing methanol and free carboxylic groups. The effect of pectin methylesterase in food quality has been extensively studied, producing desirable effects in texture improvement as well as undesirable effects in some beverages. Likewise, the low methoxyl pectin produced by this enzyme has characteristics that contribute to formulate best quality food products. Pectin methylesterase is a ubiquitously enzyme that presents multiple isoforms, but is not only present in plants; it is also found in fungi, bacteria, and yeast, which have specific chemical and physical characteristics. The latter makes the task of analyzing the wide variety of these enzymes with its specific characteristics difficult. Based on this enzyme relevance and the aforementioned, multiple methods have been developed in order to evaluate pectin methylesterase activity with different research objectives. In this paper, the importance of the enzyme as well as advantages and drawbacks of the different methods will be discussed besides applications and evolution of these will be mentioned. Additionally, this paper will improve the understanding of the systems used in pectin methylesterase activity analysis.

Plant Growth-Promoting Microbial Enzymes

Abstract Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms that colonize the rizosphere and roots of many plant species. They confer beneficial effects to plants by a variety of mechanisms, including synthesis of phytohormones, solubilization of minerals, production of siderophorus, and increases in nutrient uptake, leaf area, chorophyll, and soluble leaf protein content. Moreover, these microorganisms can also produce antioxidant enzymes to protect plants from environmental stresses that lead to the generation of reactive oxygen species, which cause cell damage. Within PGPRs, actinomycetes play an important role not only for their plant growth-promoting traits but also for secreting a wide range of antimicrobial products and enzymes that play a critical role in maintaining soil ecology and fertility. In this chapter, aspects concerning the production systems of these enzymes will be approached as well as their application in agriculture.

Enzymes for Fructooligosaccharides Production: Achievements and Opportunities

Abstract Microbial enzymes find application in enzymatic reactions for the production of fructooligosaccharides (FOS). Demands for an alternative healthy sweetener and multifunctional FOS have prompted investigators to explore microorganisms for microbial enzyme production as well as to develop bioprocesses for the production of high-fructose syrup and oligosaccharides based on transfructosylation of sucrose or hydrolysis of inulin. Fructosyltransferases have been characterized in several molds, yeasts, and a few bacteria. Recently, there has been a growing interest in finding novel enzyme producers by using agroindustrial media in both submerged and solid-state fermentation. This review discusses current knowledge on production, properties, and applications of microbial enzymes involved in the production of fructooligosaccharides and considers recent advances in the field. It also describes a comprehensive and illustrative analysis on basic and applied aspects of fungal enzymes, different substrates of microbial growth, and fundamental biotechnological and catalytic aspects to illustrate the potential of these biocatalysts in the food and bioprocessing industries.

Hydrolases of Halophilic Origin With Importance for the Food Industry

Abstract Currently, some enzymes used in the food industry do not have the same activity when they are exposed to extreme pH, high temperatures, low water activity, etc., Recently, there has been increased interest in finding enzymes with resistance to aggressive conditions to generate efficient and clean processes. Enzymes are used to reduce viscosity, improve extractions, perform bioconversions, produce separations, change functions, and modify flavors, among other uses. Enzymes produced by halophilic microorganisms are produced under high salt concentration environments, have a high percentage of amino acid acidic-residues, and low levels of lysine as well as high contents of hydrophobic residues, aspartic, and glutamic acid and a low content of aliphatic residues. Hydrolases act mainly on four families of molecules (carbohydrates, lipids, proteins, and nucleic acids). Some of these enzymes are amylases, proteases, pectinases, lipases, and DNAses, among others. In this chapter, we discuss the main discoveries on these enzymes as well as their properties, applications, and future trends.

Biocontrol as an Efficient Tool for Food Control and Biosecurity

Abstract Currently, the conservation and provision of security in the ecological environment have gained high importance, which it is framed in the term “Biosecurity.” This term integrates the care and maintenance of both flora and fauna and the natural habitats. The preventive measures where biosecurity must be a guarantee for all forms of life, and therefore the risks and damages caused, should be minimized as far as possible. In any process where biological, physical, or chemical agents are used, there is always a latent risk, so it is extremely important to know the risk factors and find a way of prevention. In recent times, biological threats are one of the challenges that has gained much attention, mainly due to the increased use of biological agents in a number of industrial sectors such as food production, agriculture, production of value-added compounds, etc. Therefore, this chapter will address the importance of standards and rules of biosafety, as well as the different strategies and specific characteristics of control in food production in traditional ways, including relevant information on emerging trends and alternatives of the conservation of these.