Jesús A. Morlett-Chávez

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.

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.

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.

Zearalenone Induced Cytotoxicity and Oxidative Stress in Human Peripheral Blood Leukocytes

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin produced by a variety of Fusarium fungi that are commonly found in feed. ZEA cause reproductive disorders of farm animals and occasionally hyperoestrogenic syndromes of humans; also, has been shown to be hepatotoxic, genotoxic, hematotoxic and immunotoxic, evidence primarily based on studies with different biological models in vitro and in vivo in poultry and laboratory rodents. However, there is little knowledge about of the effects of ZEA in human leukocytes. Objective: The aim of this study was to investigate the cell viability and lipid peroxidation effects caused by ZEA in human peripheral blood leukocytes. Methods: Human leukocytes were exposed in vitro with ZEA in concentrations ranging from 10 to 80 μg/mL during 1 h of incubation. Cell viability was measured by staining with trypan blue and neutral red assays, and lipid peroxidation was evaluated by the amount of the lipid peroxidation product, malondialdehyde (MDA). Results: ZEA significantly decreased the cell viability in human leukocytes with concentrations ranging from 10 μg/mL to 80 μg/mL. Also, we found significant increases of MDA levels in the cells exposed to concentrations between 40 and 80 μg/mL. Conclusion: The data suggest that lipid peroxidation is involved in the citotoxicity of ZEA in human leukocytes.

High prevalence of diabetic retinopathy and lack of association with integrin α2 gene polymorphisms in patients with type 2 diabetes from Northeastern Mexico

Diabetic retinopathy (DR) is one of the primary causes of blindness in the working age population and is characterized by angiogenesis in the retina. Platelets have been suggested to be involved in the pathogenesis of diabetic microvascular complications. The integrin receptor for collagen/laminin, α2β1, mediates platelet primary adhesion to subendothelial tissues, which is an essential first step in thrombus formation. The gene encoding the α2 subunit of α2β1 integrin has ≥8 polymorphisms, including a BglII/NdeI restriction fragment length polymorphism. To explore the prevalence of DR in a population from Northeastern Mexico, unrelated, hospitalized patients who had received a diagnosis of type 2 diabetes mellitus (DM2) at least 10 years previously were recruited (n=177). DR was diagnosed in a masked manner by independent ophthalmologists using fundus images captured using a non-mydriatic retinal camera. A total of 121 patients with DM2 (68%) had some degree of DR development (DR patients), and 56 patients with DM2 (32%) did not exhibit any sign of DR (No-DR patients). The results showed that after 15 years of DM2 progression, there is an increased risk of DR (P=0.0497; odds ratio, 1.993). In addition, insulin therapy and family history of DM2 were significantly associated with DR. In order to detect a possible association between DR and BglII/NdeI α2 gene polymorphisms, a comparative cross-sectional study between DR and No-DR patients was conducted. The α2 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay. Statistical analysis revealed no association between BglII/NdeI genotypes and the development of DR in this group of patients. In conclusion, the present data indicate a high prevalence of DR in the Mexican population and suggest that the damage in DR is due to other factors, such as the duration of the DM2, and is not linked to BglII/NdeI α2 gene polymorphisms.

Gallic Acid as a Putative Antioxidant in Usage Against Liver Disease

Abstract Hepatitis C virus (HCV) infection causes high level of oxidative stress and hepatic inflammation primarily, and derives in different pathologies as liver cirrhosis, hepatic failure, and hepatocellular carcinoma (HCC). Currently available treatment is a combination of pegylated interferon-α and ribavirin, surgical resection, and liver transplantation. However, more than 50% of patients remain diseased and the treatment prior mentioned causes serious adverse effects and there is still no preventive vaccine available. Considering the limited treatment options, it remains the urgent need to find additional therapeutic agents. Chemoprevention is defined as the use of nontoxic substances and it has been considered as the alternative strategy to reduce the cancer development and carcinogenesis before invasion and metastasis occur. Polyphenols, such as ellagic acid, ellagitannins, and punicalagin and phenolic acids, such as gallic acid are abundant plant phenolic compounds and they are extracted from different fruit peels and seed. Those kinds of compounds are known to reduce the risk of disease and have shown to induce cytotoxicity in several cancer cell lines, including liver cell lines. In vitro and in vivo studies have shown that the GA has a positive effect on various liver diseases, such as hepatitis by HCV infection, hepatocellular carcinoma, steatosis, and fatty liver. Histopathology and serum biochemical studies in mice fed with a diet supplemented with GA, observed that GA protects against hepatic steatosis, obesity, hypercholesterolemia, and insulin resistance. This effect occurs in part through a reversing of the common high fat diet that caused disturbances to a range of metabolic pathways, including lipid metabolism, glucose metabolism (glycolysis and gluconeogenesis), and amino acids metabolism. On the other hand, GA also has in vitro effect in hepatocarcinoma cells that express nonstructural proteins of HCV reducing viral replication. Viral proteins expression and the levels of oxidative stress decreases the viability of liver cancer cells in cell culture. Some of the main action mechanisms are the activation of protein kinase ATM, dehydrogenase UDP-glucose, and NFκB pathway. In addition, when glutathione is depleted, induction of apoptotic, antiangiogenic effect, and inhibition of tumor cell proliferation is activated. For these reasons, the use of antioxidant compounds, particularly gallic acid, has been studied recently because it is a naturally abundant plant phenolic compound, it may be considered a tool as a biomarker, or alternative therapy in the prevention and treatment of liver diseases.

Some Mexican Plants Used in Traditional Medicine

In Mexico, there is an area known as semiarid region that is located in northern Mexico, and this region is rich in biodiversity (endemic flora and fauna). In the semiarid region of Mexico are more than 3500 species of plants that have been identified and used as natural alternatives to treat different ailments (digestive ailments, kidney problems, skin conditions, etc.). The use of plants for medicinal purposes was a common practice in Mexico before the arrival of the Spanish in the sixteenth century; although this knowledge was underestimated for a long time, now interest has reemerged in the use of plants as alternative remedies or traditional medicine. It is now known that the medicinal plant capacity is due to its biological properties, which are due to chemical compounds that are synthesized by the plant metabolisms, called phytochemicals. Phytochemicals are bioactive compounds that have important biological properties such as anticancer property, antimicrobial, antiparasitic, antioxidant, and interest in the recovery of these compounds has grown in recent years, in order to find natural alternatives to synthetic drugs, which they are used for different chronic conditions such as cancer.

Microbial diversity and biochemical profile of aguamiel collected from Agave salmiana and A. atrovirens during different seasons of year

AbstractAguamiel is a beverage produced by some Agave species that is consumed in its fresh or fermented form. Despite its uses and popularity, seasonal effects on its microbial and chemical profiles are unknown. In this study, using aguamiel collected from A. salmiana and A. atrovirens during different seasons, we identified microorganisms by sequencing the 16S and 18S rDNA genes and determined their chemical profiles. In total, 49 microbial strains were identified (38 bacteria and 11 yeasts). The highest richness and biodiversity were observed during winter and summer. Different lactic acid bacteria and yeast genera with potential industrial applications were identified, such as Acetobacter, Lactobacillus, Leuconostoc, and Clavispora. The analysis of the chemical profiles indicated the presence of maltooligosaccharides and fructooligosaccharides, which are associated with human health improvements, during spring in Agave aguamiel. Aguamiel can be used in the food industry due to its microbiological and chemical profiles.