Analytical Results

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The HPLC method described in this paper is a quantitative method for available starch in foods. A variety of samples, such as grains, ready-to-eat breakfast cereals, breads, crackers, fruits, vegetables, and mixed diets can be analyzed by using this method. Complex carbohydrates are determined as the sum of available starch and dietary fiber; available starch by high performance liquid chromatogra-phy determination of glucose and maltose present in the enzymatic hydrolysates of desugared sample; and dietary fiber by enzymatic gravimetric method, official final action method 991.43.

It is important to remove simple sugars prior to enzymatic hydrolysis of starches, so that simple sugars do not carry over in the sugar analysis for available starch determination. For the efficient desugaring, we used 49% ethanol in water initially for easier dissolution of sugars. Additional 95% ethanol was added to "S

make the final concentration of 78% ethanol to precipitate dietary fiber extraction. g

This procedure would ensure that simple sugars are efficiently removed without eliminating water soluble dietary fiber components. Table 5 indicates that the extraction efficiency is generally excellent, ranging from 90% to 99%. One cereal sample which showed 90% desugaring efficiency had a low sugar content (6.2%) in the original sample to begin with. Ethanolic extract of that sample contained 5.6% sugars. We consider both 6.2% and 5.6% to be in the normal analytical variability range for sugar determinations. Thus, overall efficiency of the desugar-ing step is considered excellent.

The elution orders of standard sugars in this HPLC method, using an amino-bonded silica column packing and acetonitrile-water mobile phase, follow the expected saccharide polarity: fructose, glucose, sucrose, and maltose. The enzy-

Cho and Prosky

Table 5 Sugar Extraction Efficiency

Sugars in

Sugar extracted,

sample,

% Extraction

Source

%

%

efficiency

Standard Mix

39.8

40.0

99.0

Green Bean

15.6

16.2

96.3

Wheat Bran Cereal

15.9

17.0

92.9

Prune

53.5

56.2

95.2

Bread

5.6

6.2

90.3

Cellulose

0

0

0

Wheat Starch

0

0

0

Cracker

<1.0

<1.0

0

matic digestate of the desugared sample showed mostly glucose peaks and negligible peaks from maltose and sucrose. The sample chromatograms demonstrated that the starch digestion steps using three enzymes, heat stable alpha-amylase, protease, and amyloglucosidase, were near completion, producing mostly glucose. The glucose values in the enzyme hydrolysate were multiplied by the factor 0.9 to convert into starch values.

The Final Food Labeling Regulations in the United States (6) have recognized that HPLC methods are the most appropriate for nutrition labeling purposes among all the sugar determination techniques. The HPLC approach offers the most accurate and specific determinations of mono- and di-saccha-rides. Thus, the HPLC technique can give the most accurate figures for available starch, which is derived from glucose and maltose values of the starch hydroly-sates. However, the method requires a high capital investment and trained personnel for troubleshooting. The presence of salt, especially in the hydrolysate of processed foods, interferes with the analysis because chloride ions elute quickly after glucose. It is important to wash the column for 2 hours at 1.5 mL/min with j a solution of 0.1% tetraethylenepentamine (TEPA) (pH approximately 7 with t?

acetic acid) in 78% acetonitrile in water and finally with normal mobile phase (20).

Table 6 shows the percent recovery of several standards. Percent recoveries I

of available starch and DF for these known standards are excellent. The starch digestion in our method was done by heat stable alpha-amylase and amyloglucosi- 1

dase, which are more specific to glucose-glucose bonds. Thus, most of the sucrose ©

in the sample (99%) was recovered as sucrose in the enzyme hydrolysate.

In Table 7 are the reported available starch data generated by HPLC and

Complex Carbohydrates 141

Table 6 Percent Recovery of Standards

Source

TDF

Available starch

Cellulose

105.8

0.5

Wheat Starch

0.4

99.0

Corn Starch

0.5

100.8

Standard Mix

108.9

99.9

the difference methods. The samples tested were corn cereal, rice cereal, wheat and wheat bran cereals, crackers and bread, wheat bran and green beans.

Complex carbohydrate values derived analytically as the sum of DF and available starch were precise (Table 7). The current TDF methods do not fully recover RO. For the samples containing added RO sources such as inulin and polydextrose, the supplemental method should be used (21, 22). On the other hand, the difference method cannot be used for the samples containing significant amounts of available oligosaccharides, such as malto-triose and malto-tetrose, present in candies. The difference method would significantly overestimate complex carbohydrate content in these types of products.

Based on the results, we suggest that this HPLC method for analysis of available starches be collaboratively validated by different laboratories.

Table 7 Determination of Available Starch in Foods (by HPLC) and Complex Carbohydrates*1*2

Sample

% Available starch

% Complex CHO

Table 7 Determination of Available Starch in Foods (by HPLC) and Complex Carbohydrates*1*2

Sample

% Available starch

% Complex CHO

Corn Cereal

76.2

+

2.59

78.8

+

2.59

Rice Cereal

72.5

+

0.27

73.5

+

0.27

Wheat Cereal

22.6

+

0.86

53.5

+

0.86

Wheat Bran Cereal

49.3

+

0.50

67.0

+

1.10

Cracker

71.4

+

0.59

74.6

+

0.37

Bread

57.7

+

1.01

64.9

+

0.89

Green Bean

16.6

+

0.30

46.7

+

0.30

Wheat Bran

19.6

+

1.03

62.8

+

*2 Available starch (calculation) = 100 - %H2O - %protein - %ash -%fat - %sugar - %TDF.

Relative values comparing analytical and calculated values = (analytical values/calculated values) X 100.

*2 Available starch (calculation) = 100 - %H2O - %protein - %ash -%fat - %sugar - %TDF.

Relative values comparing analytical and calculated values = (analytical values/calculated values) X 100.

Cho and Prosky

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