2014 年 61 巻 4 号 p. 109-112
A cross-linked (CL) collagen peptide (CP)-potato starch (PS) compound (CL-CP-PS) was prepared by autoclaving PS and CP and subsequently cross-linking with a microbial transglutaminase (MTGase). CP-compounded PS (CP-PS) was prepared by autoclaving a mixture of PS and CP at 120°C for 120 min. After suspending in an MTGase solution, CP-PS was cross-linked with MTGase at room temperature for 24 h while shaking. The reaction product was washed three times with distilled water, and then air-dried to obtain CL-CP-PS. CL-CP-PS showed a clear polarized image almost the same as that of PS, and had a 0.7% CP content. The median diameter of CL-CP-PS was significantly larger than that of CP-PS or of PS, suggesting the formation of multiple granules through cross-linking among the compounded CP moieties. CL-CP-PS exhibited a grater thermal structural stability, lower swelling index and solubility, as well as higher heat resistance for maintaining the swollen starch granules at 120°C for 20 min than those of CP-PS and PS. Cross-linking of CP-PS with MTGase should thus be valuable for providing a starch material having high rigidity, low swelling index and solubility, and enhanced heat resistance.
Starch is applied to many processed foods to build or modify their physical properties, because it exhibits characteristic thermal behavior as represented by gelatinization. A number of studies have thus been carried out to investigate the effects of many coexisting substances on such gelatinization and swelling behavior, and to control the texture of processed foods with the chemically and physically-modified starches. Heating at a high temperature such as that involved a retort treatment still results in extensive swelling and subsequent collapse of the swollen starch granules, thus developing a sticky and pasty texture that is often disliked in starchy foods. However, it has not been possible to inhibit such degradation by conventional means. We have paid particular attention to controlling the gelatinization behavior by adding charged amino acids and charged amino acid-rich peptides,1) 2) 3) 4) 5) 6) 7) 8) and by conjugating or compounding non-starchy substances by simultaneous modification with a heat-moisture treatment and the Maillard reaction.9) 10) 11) 12) These studies have clarified that charged amino acids would be valuable for providing an improved paste with a higher gelatinization temperature and depressed swelling. It has also become apparent that conjugating or compounding non-starchy substances, in particular, conjugating with a fatty acylated saccharide through lysine,12) could substantially contribute to endowing its pasting properties with a high gelatinization temperature, low solubility, swelling and digestibility, high thermal resistance, and easy vaporization. However, controlling only by adding amino acids and peptides would be insufficient, because changes in environmental conditions during practical food processing would have varied effects and result in unstable control of starch. The fatty acylated saccharide-conjugated starch through compounding with lysine resulted in unfavorable browning due to heating at a high temperature. It has recently been demonstrated that oleylating with lipase could provide a conjugated starch without involving browning and with such improved pasting properties as markedly low swelling and viscosity.13) However, high heat resistance could not be achieved because of complete dissolution by retorting at 120°C for 20 min. A further high heat resistance thus needs to be achieved by other suitable and unconventional means. Cross-linking between starch chains by phosphorylation could be an effective solution. However, no such cross-linking method has been adopted for food use, because chemical modification involving phosphorylation is generally disliked. Instead of direct cross-linking in this way, enzymatic cross-linking between the peptide moieties of peptide-compounded starch without resulting browning, this being called indirect cross-linking, may thus be more appropriate to provide a starch material with high thermal resistance for food use. A low-molecular-weight collagen peptide (CP) and transglutaminase as a respective compounding agent and cross-linking enzyme were selected in this study: CP has shown lack of thermal coagulation and such effects as improving skin damages14) 15) and an antihypertensive action,16) while transglutaminase has been widely applied to improving physical properties of proteins containing CP by ε-(γ-glutaminyl)lysine cross-linking.17) 18) 19)
The objectives of this present study are to prepare CP-compounded and transglutaminase-cross-linked PS, and to demonstrate increased structural thermal stability, reduced swelling and solubility, and enhanced heat-resistance.
Potato starch (PS, Hokuren Research Institute, Sapporo, Japan) were used after being washed ten times with distilled water at 4°C and then air-dried (15.3% moisture). Microbial transglutaminase (MTGase) and low-molecular-weight collagen peptide (CP, M w 1,010; M w/M n = 1.46) from tilapia scale were respectively supplied by Ajinomoto Co., Inc. (Kawasaki, Japan) and Nippi Inc. (Tokyo, Japan). All other reagents used for the measurements described below were commercially available.
CP-compounded potato starch (CP-PS) was prepared according to a previously described method.9) In brief, PS (1 kg) and CP (0.3 mol/kg of PS) were well mixed in a polyethylene bag, and then 10 g of the mixture packed in a retort pouch was autoclaved at 120°C for 120 min to obtain CP-PS. This was added to an MTGase solution (10 mg/10 mL) and then reacted at room temperature for 24 h, while shaking at 125 strokes/min. The reaction product was washed three times with distilled water, and then air-dried to obtain cross-linked CP-PS (CL-CP-PS). DSC was conducted on the starch samples to determine the gelatinization temperature and enthalpy by using an SSC-5020 DSC-6100 instrument (Hitachi High-Tech Science Co., Tokyo, Japan) as previously described.20) Triplicate measurements were taken. The swelling index and solubility of starch samples heated at 70°C for 30 min while stirring at 500 rpm were measured as previously described.1) Triplicate measurements were also taken. The granular size distribution of the starch samples was evaluated by an Sald-2100 laser diffraction particle size analyzer (Shimadzu Co., Kyoto, Japan) as previously described.21) Triplicate measurements were again taken. The starch samples heated at 120°C for 20 min while stirring at 500 rpm were observed with a PM-10AD polarizing microscope (Olympus Co., Tokyo, Japan) at a magnification of 100 times, after staining with iodine, as previously described.22) The brightness of CL-CP-PS and PS was evaluated by a CM-3600D spectrophotometer (Konica Minolta Inc., Tokyo, Japan) as the L＊ value as previously described.13) The CP content was determined by measuring the nitrogen content as previously described.2) Multiple comparisons by the Tukey-Kramer method were made to compare mean values of the data for DSC, swelling index, and solubility at the 5% significance level.
CL-CP-PS was prepared by compounding PS with CP and then cross-linking with MTGase. The reaction conditions were selected from the results obtained by preliminary experiments, being carried out by varying the amount of CP (0.2, 0.3 and 0.5 mol/kg of PS), the autoclaving time (90 and 120 min), and the MTGase reaction conditions (10, 20 and 30 mg/10 mL of MTGase; room temperature, 30 and 40°C for the reaction; and 24 and 48 h for the reaction time) to give the highest gelatinization temperature and lowest swelling index. Cross-linking of an acid-treated gelatin, a typical CP, with MTGase has been confirmed by forming a gelatin gel with high breaking strength due to the production of ε-(γ-glutaminyl)lysine.17) 18)
A small amount of compounded CP (about 0.7%) was contained in CL-CP-PS. The CL-CP-PS showed no decrease in brightness (L＊ value of 94.5 ± 0.6, n = 10) when compared with that (94.7 ± 0.7, n = 30) for PS. Each CP-PS and CL-CP-PS sample showed a clear polarized image similar to that of PS (Fig. 1), suggesting no substantial change in the crystallinity of PS by compounding with CP and cross-linking with MTGase. Measurements of granular size distribution showed the median diameter (51.8 ± 0.5 μm) of CP-PS to be almost the same as that (52.7 ± 0.7 μm) of PS, whereas the median diameter (58.5 ± 0.6 μm) of CL-CP-PS was significantly greater than that of CP-PS or PS, presumably due to the formation of multiple granules through inter-granular cross-linking among the compounded CP moieties.
Polarized micrographs of the native and heated CL-CP-PS, CP-PS and PS samples.
The heated samples were prepared by heating at 120°C for 20 min while stirring at 500 rpm, before being stained with iodine. Polarized micrographs were taken at a direct magnification of 100 times.
The structural thermal stability levels of CL-CP-PS, CP-PS, and PS were investigated by DSC. CL-CP-PS showed a significantly higher gelatinization temperature (T o) than that of CP-PS or PS (Fig. 2). The gelatinization enthalpy (ΔH) values of CL-CP-PS and CP-PS were significantly lower than that of PS, and ΔH tended to be lower than that of CP-PS, although not significantly. The gelatinization entropy (ΔS) derived from T o and ΔH (ΔS = ΔH/T, where T o is the absolute temperature, and ΔH and ΔS are calculated on the basis of the glucose residue of starch) represents the rigidity of starch.23) 24) The ΔS values of CL-CP-PS and CP-PS were significantly lower than that of PS, and of CL-CP-PS tended to be lower than that of CP-PS, although not significantly (Fig. 2). Cross-linking of CP-PS with MTGase could thus improve the rigidity, i.e. increase the structural thermal stability.
Thermal characteristics (T o, ΔH and ΔS) of CL-CP-PS, CP-PS and PS evaluated by DSC.
T o, onset gelatinization temperature; ΔH, gelatinization enthalpy; ΔS, gelatinization entropy, being derived from T o and ΔH as previously described.15) 24) Each value is the mean ± SD (n = 3). Different letters show significant difference (P < 0.05).
The swelling index and solubility of CL-CP-PS, CP-PS and PS were evaluated at 70°C, because the conclusion temperature of these samples by DSC was in the 68‒75°C range. The swelling index value of CL-CP-PS was significantly lower than that for CP-PS or PS (Fig. 3). The solubility of CL-CP-PS was also significantly lower than the values for CP-PS and PS. The lower swelling index and solubility for CL-CP-PS were probably due to the inhibition of hydration and dissolution of the starch chains resulting from cross-linking with MTGase among the compounded CP moieties. Each CL-CP-PS, CP-PS and PS suspension was autoclaved at 120°C for 20 min while stirring at 500 rpm to observe the morphological change. The swollen starch granules were completely collapsed and dissolved in PS, while being elongated and incompletely dissolved in CP-PS, probably due to the inhibited hydration of the starch chains with compounding with CP. In contrast, the elongated, swollen granules in CL-CP-PS did not collapse (Fig. 1), matching the results of our previous microscopic observation of a fatty acylated saccharide and Lys-PS conjugate.13) This result indicates that CL-CP-PS had been endowed with enhanced heat stability. It is thus concluded that compounding with CP and subsequently cross-linking with MTGase will be valuable as an improved starch material without browning and possessing high rigidity, markedly low swelling index and solubility, and high heat resistance probably available for retort-packed food and fried coatings maintaining a dry texture.
Swelling indices (A) and solubility levels (B) of CL-CP-PS, CP-PS and PS.
The starch samples were heated at 70°C for 30 min while stirring at 500 rpm, and the resulting suspensions were then centrifuged at 31,000 × G and 20°C for 30 min. The swelling index and solubility were respectively evaluated from the dry weight of the precipitate and dissolved saccharides of the supernatant. Each value is the mean ± SD (n = 3). Different letters show significant difference (P < 0.05). Any omitted error bars were too limited to show.
