Improvement of Intracellular Glutathione Content. in Baker s Yeast. for Nutraceutical Application

Transcription

1 Improvement of Intracellular Glutathione Content in Baker s Yeast for Nutraceutical Application Manuela Rollini, Alida Musatti DeFENS, Section of Food Microbiology and Bioprocessing Vienna, 28 th June 2012

2 Aim of the research To obtain new food supplements formulations from baker s yeast (Saccharomyces cerevisiae) Compressed baker s yeast Dry baker s yeast Food supplement - Nutraceutical

3 State of the Art Directive 2002/46/EC Food supplements: foodstuffs the purpose of which is to supplement the normal diet and which are concentrated sources of nutrients or other substances with a nutritional or physiological effect, alone or in combination, marketed in dose form, namely forms such as capsules, pastilles, tablets, pills and other similar forms, sachets of powder, ampoules of liquids, drop dispensing bottles, and other similar forms of liquids and powders designed to be taken in measured small unit quantities. Inadequate intake in healthy population: socio-economic circumstances increased requirements increased losses

4 The supplement: GLUTATHIONE (GSH) GLYCINE (GLY) GLUTAMIC ACID (GLU) CYSTEINE (CYS)

5 Glutathione (GSH): useful for GSH Antioxidant GSH-reductase GSSG GSH-peroxidase Amino acids transport Maintenance of reduced form of ascorbic acid Detoxification Reactive Oxygen Species (ROS) Xenobiotics and heavy metals

6 mau M inu te s mau GSH is an intracellular metabolite Centrifugation Supernatant discharged Cell pellet Wash Ascorbic acid solution Heat treatments (permeabilization) 100 C x 10 min Centrifugation Supernatant Intracellular GSH cell debris HPLC determination Eluent Sample UV detector (210 nm), 30 C column: mm Purospher RP-18 endcapped 5 µm Pump Column and oven UV detector elution conditions: 25 mm NaH 2 PO 4 ph 2.8, 0.3 ml/min.

7 Scheme of the research Topic 1. GSH production during cell growth Topic 2. Post-growth GSH accumulation Topic 3. Improvement of GSH post-growth production Topic 4. GSH bioavailability experiments

8 Isolated from commercial baker s yeast Inoculum 10% v/v Culture conditions: 1L flasks 30 C, 24 h 60 spm 4 cm run Inoculum 10% v/v Culture conditions: 14 L fermenter 30 C, 24 h 300 rpm aeration rate 1 vvm Culture medium TN (g/l): Biomass GSH (NH 4 ) 2 SO 4 5 K 2 HPO 4 1 2,50 MgSO yeast extract 1 glucose 10 ph 5.8 GSH (%dcw) Biomass (g dcw/l) 2,00 1,50 1,00 0,50 GSH + 4 mm cysteine (CYS) 0,00 TN TN+CYS Biomass CYS in the growth media = biomass decrease

9 Scheme of the research Topic 1. GSH production during cell growth Topic 2. Post-growth GSH accumulation Topic 3. Improvement of GSH post-growth production Topic 4. GSH bioavailability experiments

10 Compressed and dried baker s yeast: performance Shelf life: 30 gg, 5 C Shelf life: 1 year, room T Activation solution: glucose GSH amino acids precursors (CYS GLY GLU - SER) cofactors 5% dcw 28 C 60 spm 4 cm run Similar initial GSH levels %dcw %dcw Determination of intracellular GSH For both types of yeast, the highest yields can be obtained during the first 24 h of activation, doubling the initial intracellular GSH levels

11 Evidence of GSH intracellular accumulation At the beginning of incubation and after 24 h incubation Transmission electron micrographs of ultrathin section of S. cerevisiae (Rollini et al., Process Biochem. 2006)

12 Compressed yeast form: influence of yeast shelf-life Intracellular GSH ratio between 24 hours and t0 during biotransformation trials, employing yeast in compressed form at 1, 10, 20 and 30 days of shelf life At 1 day of storage the yeast is able to double its GSH content During shelf-life, yeast attitude towards the activation procedures decreases

13 Scheme of the research Topic 1. GSH production during cell growth Topic 2. Post-growth intracellular GSH production Topic 3. Improvement of GSH post-growth production Topic 4. GSH bioavailability experiments New Biotechnology

14 Response surface central composite design (face centered) 4 variables A: CYSTEINE B: GLYCINE C: SERINE D: GLUTAMIC ACID Tested between 0 and 4 g/l

15 A: CYSTEINE B: GLYCINE C: SERINE D: GLUTAMIC ACID Trial n Block A g/l B g/l C g/l D g/l Compressed GSH (% dcw) Dried No CYS = no GSH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02

16 Sum of Squares df Mean of Squares F p (p>f) Model < A-CYS < B-GLY < C-SER D-GLU AC < BC A < D Confidence interval 95% R 2 : ; Predicted R 2 : Adjusted R 2 : Standard deviation: %Confidence variation: 7.15 Adequate precision: GSH. = A 0.15 B C D 0.14 AC BC+0.76A D 2 Sum of Squares df Mean of Squares F p (p>f) Model < A-CYS < B-GLY C-SER D-GLU A < Confidence interval 95% R 2 : ; Predicted R 2 : Adjusted R 2 : Standard deviation: %Confidence variation: 5.35 Adequate precision: /GSH = A B C D+0.39A 2

17 Compressed yeast GLY 2 g/l GLY 4 g/l GSH (%dcw) GSH (%dcw) GSH (%dcw) SER (g/l) CYS (g/l) SER (g/l) CYS (g/l) No GSH increase without cysteine Serine to be set at 4 g/l when glycine is 2 g/l Only when glycine is 4 g/l, serine settable < 4 g/l

18 Dried yeast Performance of yeast in dried form was not found satisfactory, as the maximum GSH level was 1.18 %dcw Dried GSH (%dcw) To produce dry yeast, moisture reduction involves removal of the portion of water more closely bound to yeast cell (cell water). GLY (g/l) B: GLY CYS (g/l) A: CYS 4.00 Several researchers reported this step to cause a considerable increase of cytoplasmic membrane permeability Increased membrane permeability can lead to a faster amino acids transport, with the result of cysteine being accumulated intracellularly Reduced de novo GSH synthesis

19 Only compressed yeast was investigated on influence of other activation mixture components: X 5 glucose, X 6 Na citrate, X 7 MgSO 4 and X 8 ammonium sulphate Glucose: between 60 and 140 g/l Na citrate: g/l MgSO 4 : 0 2 g/l Ammonium sulphate: 6 10 g/l high GSH content: about 2% dcw The model was found significant, even if no strong influences had been evidenced Sum of Squares df Mean of Squares F p (p>f) Model < X 5 -GLUCOSE X 6 -SODIUM CITRATE X < X R 2 : ; Predicted R 2 : Adjusted R 2 : Confidence interval 95%

20 Scheme of the research Topic 1. GSH production during cell growth Topic 2. Post-growth GSH accumulation Topic 3. Improvement of GSH post-growth production Topic 4. GSH bioavailability experiments

21 Samples for GSH bioavailability study Standard GSH solution GSH enriched compressed yeast Lyophilised to powder to simulate supplement formulation Ingestion In vitro gastro-intestinal digestion Absorption Gastric Digestion Intestinal Digestion 1. Gastric digestion 2. Intestinal digestion GSH standard solutions Lyophilised yeasts ph 2 Pepsin 37 C 2 h agitation ph 6 Pancreatin Bile extract 37 C 2 h agitation Soluble fraction

22 In vitro gastro-intestinal digestion GSH standard solution GSH-enriched yeast 250 GSH GSSG 250 GSH GSSG GSH-GSSG (mg/l) GSH-GSSG (mg/l) t0 Gastr. Intest. 0 t0 Gastr. Intest. Gastrointestinal steps do not affect GSH levels A very limited amount of GSH (< 10%) was found to oxidize to GSSG

23 Evaluation of GSH intestinal transport Cellular models used Absorptive cells : Caco-2 Globet cells: HT29-MTX (mucus secreting cells) GC GSH (3 mm) GSH-enriched yeast cells AC Apical compartment Cell monolayer 50-70% absorptive cells (AC) % globet cells (GC) Basal compartment Permeable support with microporous membrane Calculating the apparent permeability coefficients (Papp) = (C -1 A -1 )

24 GSH transported at 2 h (%) 3 mm (%) 10 mm (%) 30 mm (%) Yeast-A (%) Yeast-B (%) Caco ± ± ± ± ±0.05 Co-culture 70: ± ± ± ± ±0.36 Co-culture 50: ± ± ± ± ±0.84 GSH transported at 2 h is constant in each cell lines: 2.2 % in Caco % in co-culture Caco-2:HT-29 (ratio 70:30) 7.0 % in co-culture Caco-2:HT-29 (ratio 50:50) GSH transport at 2 h in co-culture models, more similar to in vivo systems, is higher than in the Caco-2 cell line The intestinal transport is low in all tested cell lines

25 Cell viability to oxidative stress 125 control GSH 3 mm yeast Cell viability (% of control) no no HH2O2 2 O 2 HH2O2 2 O 2 2 mm HH2O2 2 O mm H2O2 H 2 O mm mm Cells were treated with GSH both as standard and GSH-enriched yeast at the same level (3 mm) They show different behavior to H 2 O 2 additions The GSH-enriched yeasts show to be able to prevent cell viability reduction caused by H 2 O 2

26 Conclusions S. cerevisiae yeast cells may be enriched in GSH applying a post-growth procedure Among the tested yeasts, the compressed form provides higher yields than dried one In order to obtain high GSH yields, DoE let us understand that precursors amino acids and glucose source are important, as well as yeast shelf-life The gastro-intestinal steps do not affect GSH levels Even if GSH intestinal transport is low in model systems, GSH-enriched biomass may protect intestinal cells from oxidative stress

27 Aknowledgments Lab group inside the Section of Food Microbiology and Bioprocessing (DeFENS- University of Milan) Departamento de Conservación y calidad de los alimentos (IATA-CSIC, Valencia)