AOAC RI ERP E-Book - DS DF

ash. Perform protein analysis on residue using Kjeldahl or combustion methods. Use 6.25 factor for all cases to calculate g of protein. For ash analysis, incinerate the second residue for 5 hr at 525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite weight to determine ash.

Proceed to step [ I(a )] for calculations ) to calculate HMWDF .

(h)

H. Determination of SDFS (by HPLC) Proper deionization of the filtrate is an essential part of obtaining quality chromatographic data on SDFS. Refer to ( Figure 2017.xxE ) to see patterns of glycerol and D-glucose in the presence and absence of buffer salts. To ensure that the resins being used are of adequate deionizing capacity, add 0.1 mL of protease suspension [ C(f )] ) to 40 mL of either maleate buffer [ C(j )] ) or MES buffer [ C(k )] ) along with 3.0 mL of 0.75 M Tris base solution [ , C(l )] , ), 4.0 mL of 2M acetic acid [ , C(m )] , ), 1 mL of glycerol internal standard (100 mg/mL ) [ ), C(g )] ) and 1 mL of D-glucose solution (100 mg/mL). Concentrate this solution to dryness on a rotary evaporator and re-dissolve the residue in 32 mL of deionised water. To 5 mL of this solution in a 13 mL polypropylene tube [ B( s)] sii) , add 1.5 g of Amberlite ® FPA53 (OH − ) resin , C(s) and 1.5 g of Ambersep ® 200 (H + ) resin, C(s) and swirl the contents regularly over 5 min. Allow the resin to settle and remove the supernatant (1.5-2.0 mL) with a syringe [ B(cc )] ) and filter through a polyvinylidene fluoride filter, pore size 0.45 μm [ B(z )] . ). Inject an aliquot (50 L) of this solution onto the TSKTSKgel ® G2500PW XL columns ([ Bio-Rad ® de-ashing pre-cartridges , B(v) in place ).]. No salt peaks should be seen on HPLC. ( a) Filtrate recovery, desalting, and LC analysis.— (Set aside the filtrate from one of the sample duplicates [ , G(c )] ) to use in case of spills or if duplicate LMWDFSDFS data is desired. Transfer the filtrate [ , G(c )] ) into a 500 mL measuring cylinder. Adjust the volume to 300 mL with 78 % v/v aqueous ethanol [ , C(b )], ) , transfer to a 1 L beaker and mix thoroughly. Transfer ~ 75 mL (~ 25 %) of this solution to a 500 mL evaporator flask, and concentrate with a rotary evaporator to dryness at 50°C. [( Note: it is not essential to quantitatively transfer AOAC Research Institute ERP Use Only

Method updated 2008--08-10 This copy printed 22 August 2017

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