scholarly journals Comprehensive Vitamer Profiling of Folate Mono- and Polyglutamates in Baker’s Yeast (Saccharomyces cerevisiae) as a Function of Different Sample Preparation Procedures

Metabolites ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 301
Author(s):  
Lena Gmelch ◽  
Daniela Wirtz ◽  
Michael Witting ◽  
Nadine Weber ◽  
Lisa Striegel ◽  
...  

Folates are a group of B9 vitamins playing an important role in many metabolic processes such as methylation reactions, nucleotide synthesis or oxidation and reduction processes. However, humans are not able to synthesize folates de novo and thus rely on external sources thereof. Baker’s yeast (Saccharomyces cerevisiae) has been shown to produce high amounts of this vitamin but extensive identification of its folate metabolism is still lacking. Therefore, we optimized and compared different sample preparation and purification procedures applying solid phase extraction (SPE). Strong anion exchange (SAX), C18 and hydrophilic–lipophilic-balanced (HLB) materials were tested for their applicability in future metabolomics studies. SAX turned out to be the preferred material for the quantitative purification of folates. Qualification of several folate vitamers was achieved by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-ToF-MS) measurements and quantification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) applying stable isotope dilution assays (SIDAs). The oxidation product s-pyrazino-triazine (MeFox) was included into the SIDA method for total folate determination and validation. Applying the best protocol (SAX) in regard to folate recovery, we analyzed 32 different vitamers in different polyglutamate states up to nonaglutamates, of which we could further identify 26 vitamers based on tandem-MS (MS2) spectra. Total folate quantification revealed differences in formyl folate contents depending on the cartridge chemistry used for purification. These are supposedly a result of interconversion reactions occurring during sample preparation due to variation in pH adjustments for the different purification protocols. The occurrence of interconversion and oxidation reactions should be taken into consideration in sample preparation procedures for metabolomics analyses with a focus on folates.

2009 ◽  
Vol 75 (9) ◽  
pp. 2765-2774 ◽  
Author(s):  
Esben H. Hansen ◽  
Birger Lindberg Møller ◽  
Gertrud R. Kock ◽  
Camilla M. Bünner ◽  
Charlotte Kristensen ◽  
...  

ABSTRACT Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.


Author(s):  
Ida N Jamal ◽  
Reiny A Tumbol ◽  
Remy E.P Mangindaan

Motile Aeromonas Septicaemia disease (MAS) attacking tilapia has increased in recent years as a consequence of intensive aquaculture activities, which led to losses in aquaculture industry. The agent causing MAS disease is Aeromonas hydrophila. The disease can be controlled with the β-glucan. As immunostimulants, β-glucans can also increase resistance in farmed tilapia. Studies on the use of β-glucan extracted from baker's yeast Saccharomyces cerevisiae was intended to evaluate the non-specific immune system of tilapia that were challenged with Aeromonas hydrophila. The method used was an experimental method with a completely randomized design consisting of four treatments with three replicats. The dose of β-glucan used as treatments were 0 mg.kg-1 fish (Control), 5 mg.kg-1 fish (B), 10 mg.kg-1 fish (C) and 20 mg.kg-1 fish (D), each treatment as injected three times at intervals of 3 days, the injection volume of 0.5 ml/fish for nine days and resistance surveillance for seven days. The results showed that the difference in the amount of β-glucan and the frequency of the injected real influence on total leukocytes, phagocytic activity and resistance. Total leukocytes, phagocytic activity and resistance to treatment was best achieved by the administration of C a dose of  10 mg.kg-1 of the fish© Penyakit Motil Aeromonas Septicaemia (MAS) yang menyerang ikan nila mengalami peningkatan selama beberapa tahun terakhir sebagai konsekuensi dari kegiatan akuakultur intensif, yang menyebabkan kerugian dalam industri budidaya. Agen utama penyebab penyakit MAS adalah Aeromonas hydrophila. Untuk mengendalikan penyakit tersebut dapat dilakukan dengan pemberian β-glukan. Sebagai imunostimulan, β-glukan juga dapat  meningkatkan resistensi pada ikan nila yang dibudidayakan. Pengkajian mengenai pemanfaatan β-glukan yang diekstrak dari ragi roti Saccharomyces cerevisiae dimaksudkan untuk menguji sistem imun non spesifik ikan nila yang diuji tantang dengan bakteri Aeromonas hydrophila. Metode yang digunakan yaitu metode eksperimen dengan rancangan acak lengkap yang terdiri dari empat perlakuan dan tiga ulangan. Dosis β-glukan  yang digunakan sebagai perlakuan sebesar 0 mg.kg-1 ikan (Kontrol), 5 mg.kg-1 ikan (B), 10 mg.kg-1 ikan (C) dan 20 mg.kg-1 ikan (D), masing-masing perlakuan diinjeksi sebanyak 3 kali dengan interval waktu 3 hari selama 9 hari, volume injeksi 0,5 mL/ekor ikan dan pengamatan resistensi selama tujuh hari. Hasil penelitian menunjukkan perbedaan jumlah β-glukan dan frekuensi pemberian yang diinjeksikan memberikan pengaruh nyata terhadap total leukosit, aktivitas fagositosis dan resistensi. Total leukosit, aktivitas fagositosis dan resistensi terbaik dicapai pada perlakuan C dengan dosis 10 mg.kg-1 ikan©


Fermentation ◽  
2019 ◽  
Vol 6 (1) ◽  
pp. 2 ◽  
Author(s):  
Antonio Alfonzo ◽  
Raimondo Gaglio ◽  
Marcella Barbera ◽  
Nicola Francesca ◽  
Giancarlo Moschetti ◽  
...  

The present work was carried out to evaluate the microbiological, physicochemical, and sensory characteristics of fortified pistachio breads. Pistachio powder (5% w/w) was added to flour or semolina and fermented by a commercial baker’s yeast (Saccharomyces cerevisiae). Pistachio powder did not influence the biological leavening of the doughs. The kinetics of pH and total titratable acidity (TTA) during dough fermentation showed that the leavening process occurred similarly for all trials. The concentration of yeasts increased during fermentation and reached levels of 108 CFU/g after 2 h. Pistachio powder decreased the height and softness of the final breads and increased cell density of the central slices. The amount of lysine after baking increased in pistachio breads and this effect was stronger for semolina rather than flour trials. Sensory evaluation indicated that fortified breads processed from semolina were those more appreciated by the judges. This work clearly indicated that the addition of pistachio powder in bread production represents a promising strategy to increase the availability of lysine in cereal-based fermented products.


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