Effects of Soy Protein Hydrolysates Prepared by Varying Subcritical Media on the Physicochemical Properties of Pork Patties

Two soybean species of DWK (39.4% crude protein, 19.3% crude fat, and 10.4% moisture) and SDB (48.7% crude protein, 16.3% crude fat, and 9.8% moisture) harvested in 2013 were kindly donated by the National Institute of Crop Science (Rural development Administration, Korea). The soybean was milled 10 times using a commercial miller (Duksan Machinery Co., Korea) and stored in vacuum package prior to use (within 7 d).

The Soybean powder was hydrolyzed using a lab-scale subcritical water processor as described in our previous study (Lee et al., 2013). As the reacting media, distilled water, 20% (v/v) and 50% (v/v) ethanol were prepared. The soybean powder was suspended into each media as the mass ratio of 1:15. The suspension was filled into reactor and heated from ambient temperature to 190℃ at 1.5℃/min under controlled pressure of 25 MPa. When the inside temperature of reactor was reached to 190℃, the reactor was cooled down to 40℃ at 1.7℃/min. Finally, the suspension was centrifuged at 10,000 g for 10 min and the supernatant (SPH solution) was transferred to test tube. The SPH solution was kept at 4℃ prior to use (within 6 h).

SPH was characterized according to an operation procedure as described in our previous study (Lee et al., 2013). Total crude protein of the SPH solution was determined by kjeldahl methods (%N×6.25), and solubility was calculated by percentage of solubilized nitrogen over initial crude protein. Free amino group content was measured by the method of Benjakul and Morrissey (1997) and expressed in terms of L-leucine (Nagarajan et al., 2012). Molecular weight (Mw) distribution of SPH was estimated using a YL 9100 Gel permeation chromatograph (Younglin Instrument Co., Ltd., Korea) equipped with a YL 9100 refractive index detector and an Ultrahydrogel^TM 120 column (Waters, USA) by the method of Gu et al. (2011).

Pork loin and back-fat were purchased in 24 h post-mortem from a local market (Seoul). The meat and fat were separately ground using 3 mm plate, and kept at 4℃ prior to use (within 1 h). Model pork patty was formulated based on our previous study (Lee et al., 2015). Ground pork loin and backfat was mixed by mass ratio of 8:2 and patty was formulated by 95.5% meat/fat mixture, 1.5% NaCl and 0.5% SPH (solution with 0.4% crude protein). For control, SPH was substituted to 0.5% water. Finally, the batch was adjusted to 100% by adding water (500 g batch for each treatment). The mixture and ingredient was mixed using a KMX51 food mixer (Kenwood Co., England) for 10 min. Approximately 80 g of the mixture was formed using a petri dish (90 mm in diameter and 15 mm in height) and each two patties was packed in a plastic container (3 containers of each treatment) and wrapped. The patties were stored at 4℃ for 14 d.

Quality characteristics of the chilled stored pork patties were analyzed as described in our previous study (Lee et al., 2015). From 3 containers of each treatment, 2 containers were randomly selected. Patties were carefully removed and each patty was put into a plastic bag separately. The samples were cooked in 75℃ water bath for 30 min, and cooled down to ambient for 1 h. The surface exudate was gently wiped out and weighed. Cooking loss of patty was expressed as percent weight loss over initial weight. For texture analysis, the cylindrical forms of cooked patties were sampled using a cork borer (4 cm in diameter). The sample cylinder was punctured to 50% of sample height by 1 mm/s using a CT3 texture analyzer (Brookfield Engineering Labs Inc., USA) equipped with plunge (5 cm in diameter). From the remaining containers, patty was removed from the package and color of the patty was measured 10 times from the random surface using a CR-10 color reader (Konica Minolta Sensing Inc., Japan). One gram of pork patties were taken and water holding capacity (WHC) of pork patty was determined by centrifuging method as described in Hong et al. (2008). Five grams of pork patties was mixed with 20 mL distilled water and pH was measured using a FF20-Five Easy pH meter (Mettler Toledo GmbH, Switzerland). From the remaining portion, 5 g of sample was taken to determine lipid oxidation. Thiobarbituric acid-reactive substances (TBARS) was conducted based on the method of Hoyland and Taylor (1991).

The completely randomized block design was adopted to estimate the effect of hydrolysis media and soybean species on the physicochemical properties of pork patties. Means were analyzed by one-way analysis of variance using SAS 9.1 (SAS Institute Inc., USA). When the main effects (subcritical media and soybean species) were significant (p<0.05), the means were separated by Duncan’s multiple range test.

The amount of soluble proteins and peptides was mainly affected by types of subcritical media (Table 1). Subcritical water processed SPH showed 80-81% solubility which was significantly higher than those prepared by subcritical water/ethanol mixture (p<0.05). When 20% ethanol was used as subcritical medium, solubility tended to decrease down to 73-78%, and the lowest solubility of SPH (67-69%) was obtained by usage of 50% ethanol (p<0.05). For species, SDB showed slightly lower solubility than that of DWK, excluding 20% ethanol subcritical process where the solubility of SDB was higher than that of DWK (p<0.05). The similar result was also found in free amino group content. The free amino group content of SPH prepared by subcritical water was higher than that prepared by ethanol (p<0.05). DWK prepared by subcritical water showed higher free amino group content than SDB (p<0.05). Neither species nor ethanol concentration affected the free amino group content of SPH, however, increasing ethanol concentration in subcritical media tended to decrease the free amino group content of SPH.

For Mw distribution of SPH, DWK prepared by subcritical water showed two major peaks at ~200 Da and ~600 Da (Fig. 1). In addition, minor Mw peak at 1,400 Da was also shown. As indicated by Pihlanto-Leppälä, (2001), physiologically active peptides were consisted of 3-20 amino acids, hence the obtained Mw distribution was expected to possess various beneficial functionalities. The SHP prepared by subcritical water/ethanol media also showed similar Mw distribution, while the peak at ~300 Da was more predominant than that at ~600 Da. Although, there was no comparative investigation, one could expect that the difference in Mw distribution manifests different functionalities. It was reported that dielectric constant of ethanol was higher than that of pure water under high temperature, indicating that polarity of ethanol was less than water (Curren and King, 2001). Therefore, ethanol/water mixture was favorable to interact with organic substances comparing to pure water, thereby producing different types of SPH. In the case of SDB, pattern was similar to DWK, indicated that hydrolysis pattern was mostly affected by processing media rather than soybean species. The results reflected that subcritical process did not hydrolyze protein randomly, but the breakage of peptide bonds would have regularity under same processing condition. Eventually, processing media appeared to be potential factor involving in the antioxidant activity of SPH.

Fig. 1. Molecular weight distribution of soy protein hydrolysates prepared by varying subcritical media.

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Addition of SPH hydrolyzed by subcritical water did not affect the L* of pork patties comparing to control (Table 2). Meanwhile, the L* of pork patties tended to decrease by adding SPH hydrolyzed under high ethanol concentration and significantly lower L* value was obtained when the added SPH was prepared under subcritical 50% ethanol (p<0.05). The types of SPH also attributed to a* of pork patties. In particular, SPH-treated pork patty showed higher a* than control, when the SPH was hydrolyzed by 50% ethanol of subcritical media. Alternately, b* of all treatments was not significantly different with control. In comparison of soybean species, all color parameters were not differ each other excluding a* of 50% ethanol treatment which was higher in DWK than SDB (p<0.05). In visual appearance, control showed locally brown discoloration after 14 d of storage which was comparable to SPH treatments (Fig. 2). All SPH treatments showed redder and local discoloration was less noticeable than control. In particular, 50% ethanol treatment showed the uniform red color regardless of applied soybean species.

Fig. 2. Effect of subcritical media on the visual appearance of pork patties.

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Discoloration during chilled storage is an indicator of freshness of pork patty for consumers. In our previous study (Lee et al., 2015), SPH prepared by subcritical water at 190℃ showed effective antioxidant activity in pork patties, showing high color stability during chilled storage. Meanwhile, less color stabilizing effect of subcritical water treatment would be explained by different amount of SPH addition. Despite small amount of SPH addition, maintaining stable color of ethanol treatments reflected that the SPH hydrolyzed by subcritical water/ethanol had better antioxidant activity than that applied in water alone, which was also confirmed by TBARS (Table 2). In SDB treatments, although, TBARS of water treatment was not differ from control, all SPH treatments showed significantly lower TBARS than 0.73 mg/kg of control (p<0.05). For comparison of supercritical media, ethanol treatments showed better stability against lipid oxidation than water treatments with irrespective of species (p<0.05), while the TBARS between 20% and 50% ethanol treatments was not different. In TBARS, still DWK showed lower TBARS than those counterpart of SDB (p<0.05).

From the results, it was concluded that the anti-oxidative activity of SPH was manifested by subcritical media. However, it should be noted that anti-oxidative substances obtained from soybean was not limited to peptides, since raw material of this study was not isolated soy protein but soybean powder including hull. Various phytochemicals were extracted from varying plants by subcritical water process (Ibañez et al., 2003) and these chemicals probably influenced on the oxidative stability of pork patties. Consequently, the reason why subcritical water/ethanol treatment showed better antioxidant activity was not understood in this study, nevertheless, the present study demonstrated that hat DWK had a potential to be used as an antioxidant in processed meat products.

The WHC of pork patties was affected neither by SPH treatment nor by soybean species and showed 81-85% (Table 3). Although, 20% ethanol treatment in DWK had 25.4% of significantly high cooking loss comparing to control (p<0.05), normally cooking loss of pork patties was not depending on the addition of SPH and ranged to 20-23%. Alternately, all treatments exhibited significantly lower hardness than control (p<0.05) with the exception of 20% ethanol treatment in DWK. It has been reported that lipid oxidation has a relationship with protein oxidation which was characterized by free carbonyl group formation (Faustman et al., 2010; Lund et al., 2011; Xiong and Decker, 1995). The protein oxidation was therefore manifest a modification of protein functionality such as water-binding properties or high shear force of muscle foods (Jia et al., 2012; Lund et al., 2011). However, no evidence of protein oxidation was found in our previous study (Lee et al., 2015) and it would be explained by short storage period (14 d). Therefore, addition of SPH did not affect the eating quality of pork patties. Finally, the results implicated that SPH prepared by subcritical ethanol/water was efficient to prevent or delay oxidative metabolisms in meat products without altering the physical qualities, and exhibited a potential application of the ethanol/water mediated SPH as an antioxidant of various fat-containing muscle foods.