Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

Diversity of cultivable hydrogen-producing bacteria isolated from agricultural soils, waste water sludge and cow dung

This project aimed to study the diversity of cultivable hydrogen-producing bacteria, isolated from agricultural soils, waste water sludge and cow dung by analyzing 16S rRNA gene. Isolation performed anaerobically on nutrient agar using environmental samples as inoculum yielded 106 pure isolates. These isolates were tested for their capability to produce hydrogen. Then 16S rRNA gene of the 11 isolates having such ability were PCR amplified and subjected to restriction fragment length polymorphism (RFLP) analysis in order to group them into operational taxonomic units (OTUs). RFLP was evaluated for its ability to group the 1500-1600 bp PCR products into OTUs. Isolates were presumptively identified by analysis of partial 16S rRNA gene sequence data. Each OTU was found to be phylogenetically cohesive. The 11 isolates were put into 2 main groups. Group 1 composted of WS-2-2, AS_I-1 and AS_I-2 whose partial 16S rRNA genes showed similarity of 98-99% to members of the genus Paenibacillus. Group 2 composted of WS-4-2, Lao-1-2, WS-7-11, AS-1, AS-4, CD-5 and CD-6 having highest similarity of 98-99% to members of four genera in the family Enterobacteriacea. AS_I-2 which produced the highest amount of hydrogen gas of 2.70 ml had the highest 16S rRNA gene similarity of 99% to Paenibacillus polymixa.     

Enzymatic and molecular characteristics of Geotrichum candidum strains as a starter culture for malting

Geotrichum candidum yeasts are proposed as a starter culture during malting. They have a double positive effect on the process. They eliminate the fungal pathogenic microflora and improve the technological properties of the finished product, malt. Among published research little or no information can be found considering the biosynthesis of hydrolytic enzymes. Most of the data is related to the production of lipases, proteases and cellulases. This paper examines the enzymology of a number of G. candidum strains. The main focus within the evaluation process was placed on whole cell barley grain cultivation. Hydrolase, which is present in cell wall degradation, was found at a satisfactory level. All tested strains produced both β‐1,3‐glucanases and β‐1,4‐glucanases capable of hydrolysing barley β‐glucan and reducing the amount of this polysaccharide in the wort. Molecular analysis of the tested strains with the use of restriction fragment length polymorphism–polymerase chain reaction and randomly amplified polymorphic DNA confirmed not only the species affiliation, but also the genetic similarity between the tested strains. Copyright © 2014 The Institute of Brewing & Distilling     

Κ‐CN gene polymorphism in Nili‐ravi buffalo,Achai and Sahiwal cattle of Pakistan

Kappa‐casein (κ‐CN) is the subtype of casein protein, an important constituent of bovine milk protein. The current study was undertaken to investigate the genetic polymorphism in κ‐CN gene of Nili‐ravi buffalo, Achai and Sahiwal cattle of Pakistan using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) technique. The Nili‐ravi buffalo was found to be monomorphic (genotype BB only) for κ‐CN gene. Achai cattle were polymorphic for κ‐CN (having three genotypes AA, AB and BB) with a frequency of 0.70, 0.18 and 0.12, respectively, while in Sahiwal cattle, both the genotypes AA and AB were found with genotypic frequencies of 0.92 and 0.08, respectively. The presence of genotype BB in Achai cattle is surprising as it is absent in most of the cattle breeds worldwide.     

Differentiation between Aspergillus flavus and Aspergillus parasiticus from pure culture and aflatoxin-contaminated grapes using PCR-RFLP analysis of aflR-aflJ intergenic spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification.     

Identification of Aspergillus tubingensis strains responsible for OTA contamination in grapes and wine based on the acyl transferase domain of a polyketide synthase gene

Restriction digestion analysis of the acyl transferase (AT) domain sequences of a polyketide synthase (PKS) gene was tested as a rapid method to identify isolates of Aspergillus tubingensis from grapes. Restriction endonuclease digestion of PKS products using the endonucleases Bcc I, Hae III, Hpa II, Mbo I and Taq I distinguished five types of restriction fragment length polymorphism (RFLP). Ochratoxigenic isolates were only identified within RFLP-types I and III. The RFLP assay is proposed as a rapid and easy method to identify A. tubingensis isolates from grapes. Amino acid sequences of AT domains from representative A. tubingensis isolates of the RFLP types obtained were aligned and analysed using phylogenetic methods. A comparison was also made with reference strains of Aspergillus section Nigri . Most of the A. tubingensis strains clustered into two distinct groups Gr1 and Gr2 with the exception of two isolates that remained unclustered. These results support the intraspeficific variability within A. tubingensis species reported using other techniques.     

Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine

Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and beta-Proteobacteria.     

Predominant yeasts in the sourdoughs collected from some parts of Turkey

In the present study, a total of eight sourdough samples were collected from three different bakeries at two different times in Turkey. Also, laboratory-scale sourdough production was conducted by daily back-slopping for 7 days. Microbiological and chemical properties of the sourdoughs were investigated. Yeast species in the sourdoughs were identified by subjecting all presumptive yeast cultures to internal transcribed spacer region amplification of the 5.8S rRNA gene, restriction fragment length polymorphism analysis using Hae III, Hha I, and Hinf I endonucleases, and sequence analysis of the D1/D2 domain of the 26S rDNA gene. A total of seven profiles were determined according to the restriction fragments. Totally, 148 yeast isolates were identified at the species level (≥400 bp , 99% identity) as Saccharomyces cerevisiae (106), Kazachstania bulderi (11), Pichia fermentans (nine), Pichia membranifaciens (eight), Kazachstania servazzii (seven), Kazachstania unispora (four), and Hanseniaspora valbyensis (three). Although collected sourdoughs were produced without using baker's yeast, S. cerevisiae was the most frequently isolated yeast species. This can be related to the contamination of the bakery environment with commercial baker's yeast during the production of other bakery products. The pH and acidity levels of the collected sourdough samples ranged from 3.71 to 3.96 and 6.78 to 23.93 mL 0.1 N NaOH/10 g dough, respectively. Mean values of the content of maltose + sucrose, glucose, fructose, lactic acid, and acetic acid were 2.43, 1.57, 2.67, 7.30, and 1.40 g/kg, respectively. Due to the artisan and region-dependent handling of the sourdough, different biochemical patterns were observed among the collected samples.     

Using requirement-functional-logical-physical models to support early assembly process planning for complex aircraft systems integration

The assembly line process planning connects product design and manufacturing through translating design information to assembly integration sequence. The assembly integration sequence defines the aircraft system components installation and test precedence of an assembly process. This activity is part of the complex systems integration and verification process from a systems engineering view. In this paper, the complexity of modern aircraft is defined by classifying aircraft system interactions in terms of energy flow, information data, control signals and physical connections. At the early conceptual design phase of assembly line planning, the priority task is to understand these product complexities, and generate the installation and test sequence that satisfies the designed system function and meet design requirements. This research proposes a novel method for initial assembly process planning that accounts for both physical and functional integrations. The method defines aircraft system interactions by using systems engineering concepts based on traceable RFLP (Requirement, Functional, Logical and Physical) models and generate the assembly integration sequence through a structured approach. The proposed method is implemented in an industrial software environment, and tested in a case study. The result shows the feasibility and potential benefits of the proposed method.     

Genetic characterization of commercial Saccharomyces cerevisiae isolates recovered from vineyard environments

One hundred isolates of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 were recovered from spontaneous fermentations carried out with grapes collected from vineyards located close to wineries in the Vinho Verde wine region of Portugal. Isolates were differentiated based on their mitochondrial DNA restriction patterns and the evaluation of genetic polymorphisms was carried out by microsatellite analysis, interdelta sequence typing and pulsed-field gel electrophoresis (PFGE). Genetic patterns were compared to those obtained for 30 isolates of the original commercialized Zymaflore VL1 strain. Among the 100 recovered isolates we found a high percentage of chromosomal size variations, most evident for the smaller chromosomes III and VI. Complete loss of heterozygosity was observed for two isolates that had also lost chromosomal heteromorphism; their growth and fermentative capacity in a synthetic must medium was also affected. A considerably higher number of variant patterns for interdelta sequence amplifications was obtained for grape-derived strains compared to the original VL1 isolates. Our data show that the long-term presence of strain VL1 in natural grapevine environments induced genetic changes that can be detected using different fingerprinting methods. The observed genetic changes may reflect adaptive mechanisms to changed environmental conditions that yeast cells encounter during their existence in nature.