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And Β-Glucans in Natural Products and Ingredients

And Β-Glucans in Natural Products and Ingredients

Challenges in the specific measurement of a- and b- in natural products and ingredients

Barry V. McCleary, Ciara McLoughlin and Lucie M.J. Charmier Measurement of a-Glucans and b-Glucans a-Glucans - (/) (+ ) - Glycogen / phytoglycogen Starch Measurement - Total starch - Digestible / Resistant Starch - Damaged / Gelatinized Starch - Amylose / Amylopectin - Dietary fibre component - Structure of Starch b- Cereal b-glucan (1,3:1,4-b-glucan; mixed linkage b-glucan) 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 Type III 999.03 32-32.01 α- 2002.01 22-02.01 (Ceralpha) Activity 22-05.01 (Amylazyme) Resistant Type II 2002.02 32-40.01 Starch 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 - Readily digested starch – obesity / Type II - 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 industries Glucan

Cereal b-Glucan

Fungal b-Glucan Measurement of Barley and Oat b-Glucan

AOAC method 995.16 of b-glucans by and b-glucosidase

Hydrolysis of b-glucan in barley 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 quantification

Partially degraded yeast 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 : 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/ hydrolysis: Neutralise and add AMG/ mixture. 30 min at 40ºC. 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- 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 (, b-amylase, a-amylase and AMG) Tools of the trade High purity, well defined enzymes: a- 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 )

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 ▪ for yeast ▪ 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 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 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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