François Cormier

Glycosylation of encapsulated crocetin by a Crocus sativus L. cell culture

Crocus sativus cell suspension culture converted crocetin into several glycosyl esters when the culture was fed with the encapsulated substrate. Glycosylated pigments were stored in vacuoles. Crocetin did not affect the cell growth at concentrations up to 30 mg 100 ml−1. The major pigment has been identified as the crocetin di-neapolitanosyl ester, a new pigment not yet reported in Crocus sativus plant. The other pigments were mixed forms of neapolitanosyl, gentiobiosyl, and glucosyl esters. Glucosyltransferase activity was measured during the culture cycle and indicated that the bioconversion capacity was higher at the end of the exponential phase. The formation of glycosyl esters was highest during the first 24 h of incubation and the maximal concentration of products was achieved after 4 days. The culture had the capacity to form up to 9 mg g−1 dry wt. glycosyl esters with 30 mg substrate per 100 ml culture. Glucose and 2,4-dichlorophenoxyacetic acid added to a 12-day-old culture enhanced the glucosyltransferase activity but did not affect the yield. The formation of glycosyl esters was higher when the substrate was added by steps each day rather than supplied all at once at the beginning of the incubation period, indicating the occurrence of impediment to crocetin uptake.

Isolation and characterization of a UDP-glucose: cyanidin 3-O-glucosyltransferase from grape cell suspension cultures (Vitis vinifera L.)

Abstract An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been isolated from a Vitis vinifera cell suspension culture. The enzyme was purified 75-fold by ion-exchange, chromatofocusing and gel filtration liquid chromatography with a recovery of 3.8%. The enzyme had a pH optimum at 8.0. The glucosyltransferase showed the highest activity with cyanidin as acceptor but also utilized delphinidin to a significant degree. Pelargonidin, peonidin and malvidin were glucosylated but at a lower rate. An apparent K m value of 1.2 mM for UDP-glucose was determined. At an equal concentration of UDP-glucose the apparent K m values of the enzyme for the acceptors were 18 μM for cyanidin (3′, 4′ OH) and 28 μM for delphinidin (3′, 4′, 5′ OH). The purified enzyme had an isoelectric point of 4.55 and a molecular weight of 56 kDa. Substrate affinity of the enzyme indicates that it is the first step of two anthocyanin branches which begin with cyanidin 3-glucoside and delphinidin 3-glucoside. Also, the lower activity of the enzyme with 3′- and 5′- O -methylated derivatives of cyanidin and delphinidin would explain in part why methylation occurs after the glucosylation step.

Characterization and activities of S-adenosyl-l-methionine:cyanidin 3-glucoside 3′-O-methyltransferase in relation to anthocyanin accumulation in Vitis vinifera cell suspension cultures

Abstract A cell suspension of Vitis vinifera cv Gamay Freaux was grown in a maintenance and an anthocyanin-promoting medium (APM). Time-course changes in anthocyanin accumulation and S -adenosyl- l -methionine:cyanidin 3-glucoside 3′- O -methyltransferase (CGMT) activity were examined throughout the growth cycle. An increase in anthocyanin accumulation mainly due to peonidin 3-glucoside and peonidin 3- p -coumaroylglucoside i.e. 3′-methylated derivatives of cyanidin 3-glucoside (Cy3G), of cells grown in APM was observed. The higher activity of CGMT in cells grown in the APM might be responsible for the enhanced formation of 3′-methylated anthocyanins. CGMT isolated from cell suspension cultures has been purified approx. 35-fold with 3.4% enzyme activity recovery using anion exchange, chromatofocusing and gel filtration liquid chromatography. Mg 2+ ions strongly enhanced the reaction rate at a concentration of 5 mM. Optimal pH of CGMT ranged from 7.75 to 9.75. Michaelis-Menten kinetics were observed at pH 8.75 with respect to the substrates Cy3G and S -adenosyl- l -methionine with K m values of 199 and 18 μM respectively. S -adenosyl- l -homocysteine showed a non-competitive inhibition with an approx. K i value of 50 μM. Relative to Cy3G, the activity of CGMT was 13% with cyanidin. No activity was observed with delphinidin or cyanidin 3- p -coumaroylglucoside.

PHYSICAL FACTORS INFLUENCING THE PRODUCTION OF STRAWBERRY AROMA BY PSEUDOMONAS FRAGI GROWN IN SKIM MILK

SummaryThe influence of temperature, agitation speed, pH and biomass on the synthesis of 19 metabolites contributing to a strawberry aroma was followed over a 72 h fermentation of skim milk byPseudomonasfragi. Amongst the major odor-active metabolites were ethyl butyrate, ethyl hexanoate, ethyl 2-hexenoate, ethyl crotonate, ethyl isovalerate and ethyl 2-methyl hexanoate. Up to 17 ppm of some of these metabolites were detected at 60 h of fermentation, approximately 36 h after the beginning of the stationary growth phase. The production of most odor-active metabolites was higher at 11°C and 150 RPM than at 15, 20 or 25°C and 200 or 250 RPM. The development of off-aromas was observed at high temperatures and at high agitation speeds.

Enhanced crocetin glucosylation by means of maltosyl-β-cyclodextrin encapsulation

The water-soluble solvent dimethylsulfoxide proved to be inhibitory to the enzymatic glucosylation of crocetin catalyzed by cell-free extracts of saffron (Crocus sativus L.) callus cultures. This problem was circumvented by encapsulating crocetin into maltosyl-β-cyclodextrin at a 1:3 and a 1:6 ratio which made it possible to carry out the reaction with up to 1,750 and 2,500 μM crocetin respectively.

A highly specific glucosyltransferase is involved in the synthesis of crocetin glucosylesters in Crocus sativus cultured cells

Summary Saffron ( Crocus sativus ) stigmata contain rare water-soluble carotenoids and the major one is crocin, the crocetin digentiobiosyl-ester. Previous studies indicated that two glucosyltransferases might be involved in the formation of crocetin glucosyl- and gentiobiosyl-esters (Dufresne et al. 1997). A UDP-Glc: crocetin 8,8′-glucosyltransferase involved in the biosynthesis of crocetin monoglucosyl- and diglucosyl-esters was extracted from saffron cell cultures and purified 300-fold by gel filtration chromatography and preparative IEF electrophoresis, with a recovery of 13 percnt;. The purified enzyme preparation was highly specific for crocetin and formed ester bonds between the glucose moiety of UDP-Glc and the free carboxyl functions of crocetin. The enzyme did not add other glucose units to the glucosyl-esters to form crocetin gentiobiosyl-esters. A crude desalted extract of the same material was less specific and formed glucosyl-esters with several other compounds, including abscisic and retinoic acids. The purified preparation was active between pH 4.4 and 4.6. SDS-PAGE revealed a major band at 26 kDa while the native molecular mass determined by gel filtration was in the range of 49 to 55 kDa. The study provides concrete evidence for the hypothesis that more than one glucosyltransferase is involved in the biosynthesis of crocetin glycosyl-esters in saffron.

Anthocyanin release from grape (Vitisvinifera L.) cell suspension

SummaryIn order to develop a protocol for the release of intravacuolarly-stored anthocyanins from grapevine (Vitis vinifera L.) cell suspension with preserved viability, the effects of extracellular pH modifications, of solid adsorbents in the culture medium (two-phase system) and of chemical permeabilization were investigated. The solid adsorbent Amberlite IR-120 and chemical permeabilization were efficient in enabling the release of up to 70–80% of vacuolar anthocyanins. However, the release was always at the expense of a proportionate loss in cell viability. Results demonstrate the irreversible nature of anthocyanin sequestration.

ENHANCEMENT OF FRUITY AROMA PRODUCTION OF PSEUDOMONAS FRAGI GROWN ON SKIM MILK, WHEY AND WHEY PERMEATE SUPPLEMENTED WITH C3-C7 FATTY ACIDS

SummaryPseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C3-C7 fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C3-C7 fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.

Partial purification and properties of proteases from fig (Ficuscarica) callus cultures

SummaryCell culture of fig (Ficuscarica) was evaluated as a source of thermostable cysteine proteases. Extracts of 28 day old callus contained a benzoyl-arginine-p-nitroanilide (BAPNA) hydrolase activity of 2200 U/g dry wt (49.5 U/mg protein) at 25°C. Thermal stability and thermal denaturation (at 70°C) properties showed some similarities to those of commercial ficin (Sigma). High performance gel filtration revealed the presence of a peak with BAPNA hydrolase and caseinolytic activity which matched commercial ficin. Covalent chromatography using Thiopropyl Sepharose 6B (Pharmacia) demonstrated that approx 50% of the total BAPNA hydrolase activity could be attributed to thiol-proteins.

Properties of a glucosyltransferase involved in crocin synthesis.

Abstract The properties of a partially purified uridine-5′-diphosphoglucose (UDP-glucose)–crocetin 8,8′-glucosyltransferase (EC 2.4.1.) involved in crocin synthesis are described. The enzyme participates in the biosynthesis of crocin by forming ester bonds between the carboxyl groups of crocetin and the glucose moiety of UDP-glucose. The enzyme has been purified 125-fold trough a multi-step process of acetone fractionation, anion-exchange, and gel filtration chromatography. The native molecular weight is in the range of 47 kDa, as estimated by analytical gel filtration. The most purified preparation showed isoforms between pH 4.4 and 4.8 when resolved by IEF. The enzyme had maximal activity at 40°C, apparent Km values for UDP-glucose and crocetin were 0.72 and 0.17 mM, respectively, and Vmax, 30 pkatal/mg.