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Evaluation using DSC and reverseD-phase HPLC of starch and protein extracted from two Australian lentil cultivars, Matilda and Digger

H.C. Lee1, A.K. Htoon2, S. Uthayakumaran2 and J.L. Paterson1

1Food Science and Technology, School of Chemical Engineering and Industrial Chemistry, The University of New South Wales, Sydney NSW 2052, Australia
Food Science Australia (A Joint Venture of CSIRO and The Victorian Government), North Ryde, NSW 1670, Australia


Starch extraction in the food industry is done by several methods, with each method yielding different extraction efficiency and resultant functional properties. Alkaline extraction gives high yield and purity (Han and Hamaker, 2002) but alkali-extracted starch has lower pasting temperature and higher pasting viscosity when compared with commercially wet-milled starch. Different alkalis such as detergents and sodium hydroxide and the number of extraction stages have been studied (Lim et al., 1999; Matsunaga et al., 2003). Ideal extraction conditions cause little or no structural changes to the extracted components. In the case of starch, no damage to its crystalline phase nor depolymerisation is desired (Han and Hamaker, 2002). Legume protein isolates are commonly extracted using wet processes. Alkali solubilises the protein. Protein isolation and nitrogen dispersibility have been investigated in legume flours. Nitrogen dispersibility of most legumes is least when extracting at pH 4 and its solubility increases tremendously after pH 6.0 (Fan and Sosulski, 1974). This paper describes the effect of alkaline extraction on the starch and protein qualities.

Materials and methods

Samples - The extraction of starch and protein from flour was carried out using a modified in-house extraction method adopted in Food Science Australia, CSIRO. The procedure involves many extraction and washing steps. Starches and proteins were obtained from extraction trials involving four temperatures (ambient 22oC, 30oC, 35oC and 40oC) and five extraction pHs (distilled water and pH adjusted with NaOH to 8, 8.5, 9.0 and 9.5).

Differential Scanning Calorimetry (DSC)-A Perkin-Elmer Pyris-1 DSC with internal coolant (Intracooler IP) and nitrogen purge gas was used. When determining the enthalpy and temperature for lentil proteins, 4% sodium chloride (NaCl) solution was used to dissolve the protein isolate. Pure distilled water was used when preparing the sample for starch isolate. The samples were hermetically sealed in the aluminium pan with an O-ring.

Agilent Bioanalyzer 2100-The Agilent 2100 bioanalyzer lab-on-a-chip was used to analyse the extracted protein from both Digger and Matilda. The Protein 200 Plus chips and LabChip kits used were purchased from Agilent Technologies, USA.

Reversed-phase HPLC-Reversed-phase HPLC was a rapid identification method used to separate the proteins accordingly to their hydrophobicity (Pollard et al., 1997).

Protein Functionality- The emulsifying (Yasumatsu et al., 1972), foaming ability (Patel et al., 1988) and water holding capacity (Regenstein and Regenstein, 1984; Pinnavaia and Pizzirani, 1998) of protein isolates were investigated.

Results and discussion

Extraction at high pH (pH 9.5) resulted in >1.0 % starch damage. This was not desirable as it causes structural change to the starch granules, resulting in altered rheological and functional properties. The DSC ΔH of extracted lentil starch from both Digger and Matilda increased with increases in pH and temperature. Extraction at higher pH resulted in a smoother and symmetrical peak, denoting the absence of adhered protein on the starch surface. The ΔH values and peak temperatures achieved for extracted starches increased slightly with increased severity of extraction conditions. DSC temperature corresponding to the peak was 65.7C to 66.4C for Digger and 63.6C to 65.6C for Matilda, while the corresponding enthalpy values, ΔH, were 16.12 0.36 J/g to 17.55 0.28 J/g for Digger starch and 14.94 0.99 J/g to 16.05 0.54 J/g for Matilda starch. Full fat soy protein was used as a reference to detect for the presence of 7S and 11S protein. At about 92C and 110C, 7S and 11S proteins respectively were detected. The 11S protein peak at around 110C was absent in all 3 samples of extracted Digger and Matilda protein. The extraction pH and temperatures affected protein quality and functional properties. The calculated ΔH value decreased as pH increased. Extraction pH affected the quality of extracted protein as the H values decreased significantly (P<0.05) with an increase in pH.

The molecular weight for the proteins extracted for both Digger and Matilda generally fell within the range of 10 kDa to 140 kDa. When comparing the gel patterns and elution profiles achieved for Matilda protein extracted at same pH condition (distilled water and pH 9.5) but at different temperatures, no distinct difference in pattern was observed. However, extraction pH conditions had an effect on the molecular weight of extracted proteins. From the protein gel patterns obtained for Matilda protein extracted at 22C, the intensity of the protein bands at 85 kDa and 90 kDa were darker (more intensified) for the proteins extracted at pH 9.0 and 9.5 when compared to those extracted at distilled water, pH 8.0 and 8.5 (Figure 1). Protein extracted using higher extraction pH had more higher molecular weight protein and less low molecular weight protein. Qualitatively, the molecular weight profile did not change significantly with extraction conditions although the intensity of the molecular weight of the extracted proteins changed with increased in extraction pH. Extraction temperatures have little effect on the molecular weight of extracted protein.



Figure 1: Matilda proteins extracted at same temperature with varying extraction pHs obtained from Agilent 2100 bioanalyzer; (A) gel patterns and (B) elution profiles

Extraction pH and temperature affected the quality of protein obtained. Chromatograms from reversed-phase HPLC showed the loss of hydrophilic proteins. Protein peaks eluted only after 32 minutes, for both Digger and Matilda, denoting the presence of more hydrophobic proteins in all alkaline extracted proteins as compared to lentil flour, where the initial protein peaks eluted out at about 10.4 mins for Digger and 11.1 mins for Matilda.

Figure 2. Chromatograms of reversed-phase HPLC at two extreme extraction conditions for red lentil protein (Digger): (A) lentil flour; (B) distilled water [22οC] and (C) pH 9.5, [40οC]

Figure 2B and 2C shows the reversed-phase HPLC chromatogram for Digger proteins extracted at the two extreme conditions. Out of the 20 peaks found, 10 peaks were significantly different (P<0.05). In the case of Matilda protein, 11 out of 16 detected peaks were significantly different. Values were achieved from four analyses SD. The difference in peaks observed may be due to alteration of the protein structures during extraction.

Table 1. Emulsifying, foaming and water holding capacity properties of extracted Digger and Matilda proteins at three different extraction conditions

Lentil Protein

Emulsion Activity (%)

Emulsion Stability (%)

Water-Holding Capacity (%)

Foam Expansion (%)

Foam Stability (%)

Red Lentil (Digger)


Distilled Water, 22C

46.7 1.0

89.9 0.3

2.7 0.2

43.3 11.0

20.9 6.1

pH 8.5, 35C

45.9 0.8

83.2 0.5

2.8 0.4

31.3 4.2

22.8 5.4

pH 9.5, 40C

41.1 0.5

82.0 0.9

3.1 0.1

24.0 2.0

31.8 6.7


Green Lentil (Matilda)


Distilled Water, 22C

46.3 0.6

89.4 4.3

2.1 0.3

68.0 11.1

13.3 5.1

pH 8.5, 35C

44.9 0.6

82.3 2.0

3.3 0.1

54.0 3.5

28.4 2.1

pH 9.5, 40C

42.9 0.9

79.1 0.6

3.8 0.1

42.0 7.2

62.4 7.1

Values are triplicates means SD

Extraction conditions affected the emulsifying properties of lentil proteins. Both the emulsifying activity and emulsifying stability of extracted lentil proteins (Digger and Matilda) decreased with increase in extraction pH and temperature. Digger proteins extracted using distilled water at 22oC had an emulsifying activity of about 46.7%. This emulsifying property was decreased to about 41.1% when the Digger protein was extracted by the most severe extraction condition (pH 9.5, 40οC). Although the emulsifying activity in neither Digger nor Matilda decrease significantly as extraction conditions became more severe, the decrease in emulsifying stability was notable. It decreased from about 90% to 82% for Digger and about 89% to 79% for Matilda. The water-holding capability of both Digger and Matilda proteins (adjusted to pH 7.0) increased slightly with increasing extraction pH. The foaming capacity of both proteins, Digger and Matilda, decreased with higher extraction pH, while foam stability increased with higher extraction pH. Foaming capacity of both proteins decreased with higher extraction pH, while foam stability increases with higher extraction pH. Matilda proteins showed greater foam forming capacity than Digger.


Severe extraction conditions (high pH and high temperature) generally affected the quality of protein and starch isolates obtained. The results from the DSC, bioanalyzer and reversed-phase HPLC chromatograms showed the changes in protein profiles with extraction conditions. These changes affect the overall functional behaviour of the proteins, in terms of their emulsifying properties, water-holding capacity as well as its foaming properties.


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