Alpha-amylase inhibitory activity of collagen hydrolysate from Asian bullfrog skin and its application in dark chocolate

Figures & data

Figure 1. Inhibitory effect of collagen hydrolysate on α-amylase. (a) α-amylase inhibitory activities of collagen hydrolysate. (b) Lineweaver–Burk plot for determining the kinetic constants for α-amylase.

Table 1. Characteristics of dark chocolate bars prepared with collagen hydrolysate from Asian bullfrog skin at different addition levels.

Characteristics	Control	C-0.5	C-1.0	C-2.0
Moisture content (g/100 g)	1.27 ± 0.06^b	1.36 ± 0.04^a	1.38 ± 0.02^a	1.41 ± 0.04^a
Water activity, aw	0.478 ± 0.009^a	0.470 ± 0.002^ab	0.439 ± 0.002^c	0.462 ± 0.002^b
Colour
L*	27.45 ± 0.24^b	30.62 ± 0.12^a	30.82 ± 0.09^a	30.65 ± 0.10^a
a*	8.34 ± 0.17^a	7.53 ± 0.18^c	7.66 ± 0.08^bc	7.89 ± 0.16^b
b*	6.03 ± 0.12^a	4.41 ± 0.13^b	4.33 ± 0.12^b	4.28 ± 0.30^b
Rheological behaviour
Plastic viscosity (Pa.s)	5.30 ± 0.12^d	7.26 ± 0.26^c	7.85 ± 0.10^b	14.46 ± 0.05^a
Yield stress (Pa)	13.30 ± 0.20^d	15.13 ± 0.06^c	17.10 ± 0.40^b	22.10 ± 0.40^a
Flow behaviour index	0.86 ± 0.00^d	0.87 ± 0.00^c	0.88 ± 0.00^b	0.89 ± 0.00^a
Apparent viscosity (Pa)	2.79 ± 0.05^d	3.91 ± 0.05^c	4.34 ± 0.06^b	7.87 ± 0.10^a
Thixotropy (Pa.s^-1)	4.11 ± 0.26^d	13.42 ± 0.49^c	16.78 ± 0.10^b	29.86 ± 0.38^a
α-amylase inhibitory activity (%)	nd.	14.74 ± 0.15^c	22.97 ± 0.20^b	36.44 ± 0.79^a

nd.: not detected; Data are expressed as mean ± standard deviation (n = 3); Control: Dark chocolate bar prepared without collagen hydrolysate addition; C-0.5, -1.0, -2.0: Dark chocolate prepared with collagen hydrolysate addition at 0.5, 1.0, and 2.0% (w/w), respectively; Different lowercase superscripts in the same row indicate significant difference (p ≤ 0.05).

Figure 2. Appearance of dark chocolate bars prepared with collagen hydrolysate from Asian bullfrog skin at different addition levels. Control: Dark chocolate bar prepared without collagen hydrolysate addition. C-0.5, -1.0, -2.0: Dark chocolate prepared with collagen hydrolysate addition at 0.5, 1.0, and 2.0% (w/w), respectively.

Figure 3. Breaking strength of dark chocolate bars prepared with collagen hydrolysate from Asian bullfrog skin at different addition levels. Control: Dark chocolate bar prepared without collagen hydrolysate addition. C-0.5, -1.0, -2.0: Dark chocolate prepared with collagen hydrolysate addition at 0.5, 1.0, and 2.0% (w/w), respectively. Different letters on the bars indicate significant differences between the samples (p ≤ 0.05).

Figure 4. Thermal behaviour of dark chocolate bars prepared with collagen hydrolysate from Asian bullfrog skin at different addition levels. Control: Dark chocolate bar prepared without collagen hydrolysate addition. C-0.5, -1.0, -2.0: Dark chocolate prepared with collagen hydrolysate addition at 0.5, 1.0, and 2.0% (w/w), respectively. (a) Melting process after crystallization for 24 h at 22 °C. (b) Dynamic crystallization from 90 °C to – 50 °C. (c) Re-melting of crystals formed in segment (b). Arrow indicates the S-L-S (Solid–Liquid–Solid) transition.

Table 2. Nutritional compositions, energy value, and sensory attributes of selected dark chocolate bars prepared with collagen hydrolysate from Asian bullfrog skin at different addition levels.

Parameters	Control	C-2.0
Nutritional compositions
Protein (g/100 g)*	8.31 ± 0.10^b	9.59 ± 0.21^a
Fat (g/100 g)	37.53 ± 0.97^a	34.37 ± 0.90^b
Ash (g/100 g)	2.05 ± 0.13^a	2.16 ± 0.02^a
Total carbohydrate (g/100 g)	50.44 ± 0.77^a	51.12 ± 1.15^a
Energy value (kcal/100 g)	572.75 ± 5.35^a	552.24 ± 4.38^b
Sensory attributes (liking scores)
Appearance	4.43 ± 0.50^a	4.40 ± 0.62^a
Color	4.53 ± 0.51^a	4.47 ± 0.57^a
Taste	4.00 ± 0.64^a	3.90 ± 0.61^a
Sweetness	4.10 ± 0.61^a	4.10 ± 0.55^a
Mouthfeel	4.47 ± 0.51^a	3.67 ± 0.88^b
Hardness	4.23 ± 0.45^a	3.87 ± 0.68^b
Aftertaste**	4.00 ± 0.64^a	3.93 ± 0.64^a
Overall	4.20 ± 0.48^a	4.07 ± 0.58^a

Data are expressed as mean ± standard deviation (n = 3); *The conversion factor used is 6.25; **Aftertaste: any remaining sensations perceived after the sample has been completely swallowed; Control: Dark chocolate bar prepared without collagen hydrolysate addition; C-2.0: Dark chocolate prepared with collagen hydrolysate addition at 2.0% (w/w); Different lowercase superscripts in the same row indicate significant difference (p ≤ 0.05).