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And Β-Glucans in Natural Products and Ingredients

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And Β-Glucans in Natural Products and Ingredients Challenges in the specific measurement of a- and b-glucans in natural products and ingredients Barry V. McCleary, Ciara McLoughlin and Lucie M.J. Charmier Measurement of a-Glucans and b-Glucans a-Glucans - Starch (amylose/amylopectin) (+ maltodextrins) - Glycogen / phytoglycogen Starch Measurement - Total starch - Digestible / Resistant Starch - Damaged / Gelatinized Starch - Amylose / Amylopectin - Dietary fibre component - Structure of Starch b-Glucan Cereal b-glucan (1,3:1,4-b-glucan; mixed linkage b-glucan) Yeast and Fungal (mushroom) b-glucan (b-1,3- / b-1,6-linkage glucan) Megazyme’s contribution to Official Methods Analyte CODEX AOAC official method AACC official method method β-D-Glucan Type II 995.16 32-22.01, 32-23.01 Total Starch 996.11 76-13.01 Damaged 76-31.01 Starch Total Fructan Type III 999.03 32-32.01 α-Amylase 2002.01 22-02.01 (Ceralpha) Activity 22-05.01 (Amylazyme) Resistant Type II 2002.02 32-40.01 Starch Dietary fiber Type I 2009.01, 2011.25, 2017.16 32-45.01, 32-50.01 Total Fructan 2018.07 (First action 2017) Ethanol (First action 2019) Lactose (First action 2019) Why is measurement important? Starch - Supplies ~ 60% of the body’s energy - Readily digested starch – obesity / Type II diabetes - Total digestible starch - major source of available CHO - Resistant starch – a component of dietary fiber. Cereal b-Glucan – soluble dietary fiber (health claims) Mushroom b-glucan (health claims) - stimulates the immune system - sales value as a nutraceutical (US$18 billion in 2017). Yeast b-Glucan - stimulates the immune system - reduces use of antibiotics in animal industries Glucan polymers Cereal b-Glucan Fungal b-Glucan Measurement of Barley and Oat b-Glucan AOAC method 995.16 Hydrolysis of b-glucans by lichenase and b-glucosidase Hydrolysis of b-glucan in barley flour sample SDS gel-electrophoresis Lichenase b-Glucosidase 10 U/assay 0.2 U/assay Measurement of Yeast b-Glucan Principle: Selective enzymatic degradation followed by glucose quantification Partially degraded yeast cell walls; b-glucan and phytoglycogen 2 M KOH, 30 min + Acetate buffer ~ pH 4.5 Hydrated b-Glucan endo-1,6-b-glucanase Devoid of starch degrading endo-1,3-b-glucanase Enzymes: Amyloglucosidase exo-1,3-b-glucanase a-glucosidase b-glucosidase a-amylase Glucose Glucose determination with GOPOD reagent Measurement of Mushroom b-Glucan Principle: Selective enzymatic degradation followed by glucose quantification endo-1,6-b-Glucanase endo-/exo-1,3-b-Glucanase Incomplete hydrolysis Assay procedure: Mushroom b-Glucan Mill dried mushroom sample to pass a 1.0 mm screen Total glucan determination a-Glucan determination Pre-incubation: 12 M H2SO4. 2 h at 0ºC. Starch/glycogen solvation: 2 M NaOH. 20 min at 0ºC. Acid Hydrolysis: 2 M H2SO4. 2 h at 100ºC. Starch/glycogen/sucrose hydrolysis: Neutralise and add AMG/invertase mixture. 30 min at 40ºC. Enzyme Hydrolysis: Neutralise and add exo-1,3- b-glucanase/b-glucosidase mixture. 1 h at 40ºC. Glucose determination: Measure using GOPOD. Glucose determination: Measure using GOPOD. b-Glucan = Total glucan - a-glucan Assay optimization Total glucan (g/100 g) Total glucan (g/100 g) 12 M H SO 2 4 12 M H SO 12 M HCl 12 M HCl 12 M H SO Without exo-1,3- 2 4 12 M HCl 2 4 With exo-1,3-b- Without exo-1,3- With exo-1,3-b- Sample Pre-incubation b-glucanase and Pre-incubation glucanase and b-glucanase and glucanase and time (min) b-glucosidase time (min) b-glucosidase b-glucosidase b-glucosidase 30 48.2 52.4 45 25.1 26.7 Ganoderma 60 52.2 53.8 90 27.6 29.9 lucidum 120 56.2 56.3 180 29.7 31.4 30 56.8 67.3 45 61.5 69.7 Poria cocus 60 64.5 71.2 90 69.9 73.3 120 74.4 75.9 180 73.1 74.7 30 58.7 72.3 45 48.4 58.5 Curdlan 60 65.8 76.1 90 53.7 65.0 120 77.7 81.5 180 66.8 75.2 30 14.9 16.0 45 15.5 16.3 Alpha-cellulose 60 14.1 15.1 90 21.8 22.5 120 14.6 15.4 180 24.5 25.0 Commercial products Sample Type Sample Details Total Glucan (g/100 g) a-Glucan (g/100 g) b-Glucan (g/100 g) Polyporus umbellatus 51.1 0.6 50.5 Trametes versicolor 47.3 0.2 47.1 Inonotus obliqus 8.1 0.2 7.9 Ganoderma lucidum (B) 54.2 0.2 54.0 Agaricus blazei 16.5 3.4 13.1 Grifola frondosa 36.4 3.6 32.8 Ganoderma lucidum (A) 55.4 1.6 53.8 Poria cocus 74.7 0.8 73.9 Lentinula edoses 39.4 3.8 35.6 Mushroom Cordyceps militaris 36.5 2.2 34.3 Hericium erinaceus 37.1 3.2 33.9 Agaricus bisporus 7.3 1.3 6.0 Pleurotus ostreatus 32.7 0.4 32.3 Tremella fuciformis 16.1 1.2 14.9 Grifola frondosa 33.3 1.8 31.5 Lentinula edodes 24.4 0.9 23.5 Pleurotus eryngii 37.5 0.4 37.1 Flammulina velutipes 20.7 0.7 20.0 Agaricus bisporus 9.8 4.1 5.7 Ganderma lucidum 74.3 29.2 45.1 16 Basidomycete species blend 69.5 66.4 3.2 7 Basidomycete species blend 73.7 72.5 1.3 Ganderma lucidum A 44.6 22.6 22.0 Ganderma lucidum B 87.7 83.2 4.3 Encapsulated mushroom Ganderma lucidum/ 59.9 41.9 18.0 and mycelium-based Lentinula edodes products Cordyceps sp. A 64.8 53.9 10.9 Cordyceps sp. B 65.5 64.0 1.5 Ganderma lucidum C 52.5 45.2 7.3 Cordyceps sinensis A 13.9 3.0 10.9 Cordyceps sinensis B 29.3 24.1 5.2 Inonotus obliquus 69.8 70.0 ~ 0.0 Industrial Mushroom Production Measurement of Starch Measurement of Starch - Digestible / Resistant Starch - Damaged / Gelatinized Starch - Amylose / Amylopectin - Total starch - Dietary fibre component - Structure of Starch (isoamylase, b-amylase, a-amylase and AMG) Tools of the trade High purity, well defined enzymes: a-Amylases with different action patterns and stabilities - bacterial (purified) - stable at 100oC - fungal (chromatographically purified) - pancreatic (purified)- active on starch granules Amyloglucosidase (chromatographically purified) b-Amylase (re-crystallised) – ultra-pure Isoamylase (chromatographically purified) – ultra-pure Pullulanase (affinity purified) – ultra-pure a-Glucosidase (recombinant) – ultra-pure Digestible Starch and Resistant Starch PAA 4 KU/assay AMG 1.7 KU/assay pH 6, 37oC, 4 h. Gently stirring Pancreatic a-amylase + amyloglucosidase Rapidly Digested Starch (20 min) (major component of glycemic carbohydrates) Total Digestible Starch (4 h) (major component of available carbohydrates) Resistant Starch (left after 4h) (part of dietary fiber) Measurement of Damaged Starch (effect of concentration of A. niger a-amylase ) Wheat flour Sample A A. niger a-amylase followed by Damaged starch: amyloglucosidase ▪ Flour water absorption ▪ Sugars for yeast fermentation ▪ Bread crust colour Wheat flour Sample B Undamaged Starch granules Amylose/Amylopectin Content of Starch ▪ Solubilization of starch ▪ Con A precipitation and removal of branched amylopectin ▪ Determination of amylose (AM) Hydrolysis of amylose (in solution) with AMG + AA Glucose determination. ▪ Determination of Total Starch (TS) by hydrolysis with AMG + AA Glucose determination. ▪ Determination of amylose content (% w/w) Amylose % w/w = AM (mg/mL) / TS (mg/mL) x 100 Measurement of Total Starch (AOAC Method 996.11) • High purity starch degrading enzymes • Heat stable a-amylase • Simple, rapid, quantitative high throughput glucose determination (K-TSTA) Interlaboratory evaluation of Starch Methods - A comparison of study results - Thermostable a-amylase (stable at 100oC at pH 5.0) Comparison of starch determination methods for the measurement of total starch in a range of animal feeds and pet foods Measurement of Glucose Using the GOPOD Reagent GOPOD Linearity Reaction end-point and GOPOD stability Measurement of Starch; Challenging Samples ➢ Heterogenous samples e.g. corn silage. Solution: grinding in a Nutri-bullet homogenizer ➢ Samples containing low levels of starch (< 2%) e.g. distillers dry grains; pet foods; Solution: increased sample size (~ 500 mg). ➢ Chemically modified starches e.g. chemically cross-linked starches. Solution: pre-incubation in alkaline solutions; extended hydrolysis of sample with a-amylase at 100oC Preparation of samples for starch analysis Measurement of Total Starch Content of Phosphate cross-linked starch (Fibersym® & Hylon VII® Sample pre-treatment and a-amylase incubation Total Starch conditions (g/100 g) dwb. Hylon VII® Fibersym® AOAC Method 2002.01 (KOH Format) 94.5 41.7 RTS Method / a-amylase 30 min at 100oC (no KOH) 87.6 80.5 NaOH (4oC, 20 min) / a-amylase 30 min at 100oC 95.1 81.6 NaOH (50oC, 20 min) / a-amylase 30 min at 100oC 95.6 82.8.
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