Alternative of Phosphate by Freeze- or Oven-Dried Winter Mushroom Powder in Beef Patty

Winter mushrooms (Flammulina velutipes) were purchased from a local market (Daejeon, Korea). The mushrooms were frozen at −70°C for 24 h, and lyophilized using a freeze dryer (Bondiro, Ilshin, Seoul, Korea) at room temperature and the 5 mTorr of chamber pressure, and then pulverized using a food processor (FPM250, Kenwood, Havant, UK), yielding the freeze-dried powder (FDP). The ODP was prepared using a drying oven (JSOF-150, JS Research, Gongju, Korea) at 170°C for 15 min and pulverized using a food processor (FPM250, Kenwood). The moisture contents of FDP and ODP were 5.72% and 4.67%, respectively. The powders were stored at −20°C until their use.

The methanolic extracts of winter mushroom powder were prepared by the extraction of 0.5 g mushroom powder in 49.5 mL of 70% (v/v) methanol while shaking for 60 min.

The total phenolic content in the methanolic extracts of FDP and ODP was measured using the Folin-Ciocalteu method (Subramanian et al., 1965) and is expressed as gallic acid equivalents (g/kg).

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the methanolic extract was estimated using the method described by Jung et al. (2017), and the half-maximal effective concentration (EC₅₀) for scavenging DPPH radicals was calculated.

Reducing power was determined based on the reduction rate of ferric iron to ferrous iron using the method described by Oyaizu (1986). The sample solutions of FDP and ODP (15, 12.5, 10, 7.5, and 5 mg/100 mL in 70% (v/v) methanol and 7, 5.75, 4.5, 3.25, and 2 mg/100 mL in 70% (v/v) methanol, respectively) were used to measure the reducing power. The half-maximal effective concentration (EC₅₀) for 0.5 absorbance at 700 nm was calculated using a spectrophotometer (DU^®530, Beckman Instruments, Fullerton, CA, USA).

The 1 g of FDP and ODP were homogenized with 9 mL distilled water using a homogenizer (T25 basic, IKA^®-Werke GmbH & Co. KG, Staufen, Germany) and centrifuged at 2,090×g for 10 min using a centrifuge machine (ScanSpeed 1580R, Labogene ApS, Lillerød, Denmark). The supernatant was filtered using filter paper (No. 4 filter paper, Whatman, Maidstone, UK) and the pH value was measured using a pH meter (SevenEasy, Mettler-Toledo Intl., Schwerzenbach, Switzerland).

The instrumental color (CIE L*, a*, and b*) of FDP and ODP was measured using a spectrophotometer (CM-3500d, Konica Minolta, Tokyo, Japan). The measurements were taken perpendicular to the measuring mini petri dish (CM-A128, Konica Minolta) containing powder with an illumination area of 30 mm, illuminant D65, and 10° standard observer. The results were analyzed using SpectraMagic Software (SpectraMagic^TM NX, Konica Minolta).

Three fresh bottom rounds from three heifer carcasses were prepared, and the bottom round from each carcass was allocated to each batch. The bottom round of beef (390 g) was mixed with pork back fat (60 g), bread powder (30 g), chopped onion (30 g), chopped garlic (12 g), isolated soybean powder (12 g), salt (6 g), and water (60 g) in a mixer for 5 min. The formula for each of the four treatments are as follows: 1) control: patty manufactured without sodium pyrophosphate, 2) Positive control (BP): patty manufactured with sodium pyrophosphate (0.3%); 3) BFW: patty manufactured with FDP (1%); and 4) BOW: patty manufactured with ODP (1%). The meat batter was prepared thrice for each treatment, and the patty (100 g meat batter) was molded with a stainless container (80 mm×45 mm). Five patties were made from each meat batter and cooked in an electric oven (COR-055KE, SK magic, Seoul, Korea) at 170°C for 15 min. Total nine beef patties (three patties /each batch) were used for the analysis of physicochemical properties.

Meat batter (1 g) was homogenized with distilled water (9 mL) using a homogenizer (T25 basic, IKA^®-Werke GmbH & Co. KG). The homogenates were centrifuged at 2,090×g for 10 min (1580R, Labogene ApS) and filtered using filter paper (No. 4 filter paper, Whatman). The pH value of the filtrate was measured using a pH meter (SevenEasy, Mettler-Toledo Intl.).

Cooking loss of beef patties was calculated by comparing the weight difference between the cooked beef patties and raw beef patties.

Lipid oxidation in beef patties was detected by measuring the MDA content. MDA was extracted from beef patties and then analyzed using a high-performance liquid chromatography (HPLC) system (1200 series, Agilent Technologies, Santa Clara, CA, USA) with an Atlantis T3 C18 RP column (Waters, Milford, MA, USA) (4.6×250 mm, 5 μm-particles). The analysis procedures were performed in accordance with the method described by Jung et al. (2016). 1,1,3,3-tetraethoxypropane was used as the standard compound, and the MDA content in beef patties was expressed as mg MDA/kg beef patties.

Protein oxidation in beef patties was measured by analyzing the total carbonyl content using the derivatization method of 2,4-dinitrophenylhydrazine (DNPH), as described by Jo et al. (2020b). The carbonyl content was expressed as nmol/mg of protein using an absorption coefficient of 21.0 nM⁻¹ cm⁻¹ at 370 nm for protein hydrazones.

A colorimeter (CM-3500d, Konica Minolta) was used to measure the color (CIE L*, a*, and b*) of each beef patty. The measurements were taken perpendicular to the inner surface of the patty at two different locations per sample with an illumination area of 30 mm, illuminant D65, and 10° standard observer. The results were then analyzed using SpectraMagic Software (SpectraMagic^TM NX, Konica Minolta).

The texture of beef patties was measured with a two-bite system using a texture analyzer (Model A-XT2, Stable Micro Systems, Godalming, UK) with a compression probe that had a diameter of 70 mm. The patties were cut to regular size (2×2×1.5 cm³) and subjected to two cycles of 70% compression at a test speed of 2 mm/s. The texture characteristics of beef patties were expressed as hardness, springiness, cohesiveness, gumminess, and chewiness.

Winter mushrooms are fungi that exhibit a high antioxidant activity (Shah et al., 2018; Zhang et al., 2013). Phenolic compounds are anti-oxidizing substances that remove reactive oxygen species existing in plants and play a role in reducing oxidative stress. The total phenolic content of the ODP was 5.45 g GAE/kg that was significantly higher than that of the freeze-dried winter mushroom powder (FDP) at 3.50 g GAE/ kg (Table 1, p<0.05). The EC₅₀ values of FDP and ODP for DPPH radical scavenging were 16.89 g/kg and 11.52 g/kg, respectively, which were significantly different between treatments (p<0.05). In addition, ODP had a significantly lower EC₅₀ value of reducing power than that of FDP (p<0.05), indicating that ODP has a higher antioxidant potential than FDP. This is analogous to the phenomenon that previous studies report of an increase in phenolic compound content and antioxidant activity of natural products subjected to heat treatment (Li and Shah, 2013; Saad et al., 2014). Another study suggests that heat treatment of plants could improve the antioxidant activity by releasing phenolic compounds from the collapsed cell wall and the rapid inactivation of polyphenol oxidase (PPOs) such as tyrosinase, catecholase, and laccase (Nguyen et al., 2018). In addition, Maillard reaction products have antioxidant activity, which might be due to the action of reductones and melanoidins formed in the reaction process (Namiki, 1988). The reductone can effectively donate hydrogen, and melanoidin can block the reaction of metal ions or residues generating radicals (Namiki, 1988).

The pH of the ingredients is an important property of meat products because the increase in pH with the addition of ingredients can improve water retention in meat products (Choe et al., 2018; Jo et al., 2020a). When the pH of fresh winter mushroom was 6.81 (data not shown), the pH of ODP and FDP was 7.00 and 6.73, respectively, and the pH of ODP was significantly higher than that of FDP (Table 1, p<0.05). Evidently, the oven-drying process resulted in the increased pH of winter mushrooms. However, these results may not be common. Brands and Van Beokel (2002) reported that formic acid and acetic acid are the main products of the Maillard reaction. Lertittikul et al. (2007) also reported a decrease in the pH value of porcine plasma protein–glucose Maillard reaction products with increasing heating time. Previous studies found that the pH of foods containing proteins increased after heat treatment, which might be attributed to the release of basic amino acids from heat-denatured proteins (Oz et al., 2017; Vasanthi et al., 2007). Thus, a similar effect may occur in winter mushrooms subjected to heat treatment.

The application of oven-drying altered the color of winter mushrooms to brown by the Maillard reaction. ODP had a lower L* value and higher a* and b* values than that of FDP (Table 1, p<0.05), and this change could be an effect of the brown pigment melanoidin as the final Maillard reaction product (Namiki, 1988).

The high pH of meat products is one of the major factors determining the quality and increasing the economic value of meat products. Meat batter containing no phosphate and winter mushroom powder (control) showed the lowest pH among the treatments (Table 2, p<0.05). Although the addition of winter mushroom powders (BFW and BOW) to meat batter at a concentration of 10 g/kg increased the pH compared to the control (p<0.05), the effect of the winter mushroom powder on the pH of the meat batter was not notable. In addition, no difference in pH between BFW and BOW was found despite the higher pH of ODP than that of FDP. The pH of BFW and BOW was significantly lower than that of BP, which was manufactured with the addition of 0.3% sodium pyrophosphate (p<0.05). Choe et al. (2018) reported no difference in pH between pork batter containing 1% winter mushroom powder and 0.3% sodium pyrophosphate. However, chicken meat batter containing 1% winter mushroom powder had a lower pH than that of chicken meat batter containing 0.3% sodium pyrophosphate (Jo et al., 2018). The different effects of winter mushroom powder addition on the pH of meat batter may be caused by the differences in buffering capacity of meats such as beef, pork, and chicken.

The increase in pH of meat batter for comminuted meat products improves their water and fat retention because the increased electrical repulsion forces among proteins increases the space available for water in myofibrillar filaments and gel structure and fat droplets in the gel structure (Chen et al., 2019). The highest and lowest cooking loss of beef patties occurred in the control group and the BP, respectively (Table 2, p<0.05). BFW showed a significantly lower cooking loss than the control group (p<0.05). However, the cooking loss in BOW was similar to that in the control (p>0.05). Previous studies found that the addition of winter mushroom powder to pork and chicken sausages with the same level of concentration as that used in the present study resulted in a decrease in cooking loss, which was attributed to the increase in pH and the water holding and fat binding capacity of dietary fiber in winter mushroom powder (Choe et al., 2018; Jo et al., 2018). In the present study, there were no notable pH changes in meat batter with the addition of winter mushroom powder. The decrease in the cooking loss of BFW might be due to dietary fiber. However, the dietary fiber in ODP might lose its water holding capacity and fat binding capacity by heat treatment. A previous study reported a reduced water-holding capacity of dietary fiber with structural damage induced by heat treatment (Miranda et al., 2010; Vega-Gálvez et al., 2015).

The content of MDA, a secondary product of lipid oxidation, in beef patties was the highest in the control and the lowest in the BP (Table 3, p<0.05). This result was attributed to the antioxidant activity of phosphate in the BP. Phosphate suppresses lipid oxidation through metal-ion chelating activity in the meat system (Choe et al., 2018). Beef contains considerable myoglobin, and the iron ions released from myoglobin can generate hydroxyl radicals via the Fenton reaction (Lloyd et al., 1997). Therefore, the antioxidant activity of phosphate by chelating metal ions may be maximized in beef compared to that in chicken and pork. Previous studies found that freeze-dried winter mushroom at l.0% level of concentration in pork and chicken sausages served as an alternative to phosphate and decreased lipid oxidation (Jo et al., 2018; Jo et al., 2020b). However, there was no inhibition of lipid oxidation in BFW compared to that in the control used in the present study (p>0.05).

This result might be attributed to the different lipid oxidation potential with the major oxidants and substrates among beef, pork, and chicken (Rhee et al., 1996). Phenolic compounds in winter mushroom powder can effectively suppress lipid oxidation in pork and poultry. However, its effect as a prooxidant might be less in beef because the main factor of lipid oxidation in beef is metal ions (Min et al., 2010). Nonetheless, the MDA content of BOW was lower than that of the control and BFW (p<0.05), although it was higher than that of BP (p<0.05). These results were attributed to the increased antioxidant activity of winter mushrooms after oven-drying. The ODP showed an increase in antioxidant activity with an increase in phenolic content and the generation of Maillard reaction products (Table 1). Alfawaz et al. (1994) and Smith and Alfawaz (1995) reported the inhibitory effects of Maillard reaction products on lipid oxidation in cooked ground beef.

There were no significant differences in the carbonyl content among the treatments (Table 3, p>0.05). Phosphate and winter mushroom powders had no inhibitory effect on protein oxidation in beef patties. This result is in line with that of a previous study. Jo et al. (2018) reported that phosphate and winter mushroom powder had no effect on the protein oxidation in chicken sausages. However, the effect of winter mushrooms on protein oxidation in meat products should be further studied with other protein oxidation parameters, such as free sulfhydryl content and tryptophan fluorescence intensity. The carbonyl content might not be a suitable indicator of protein oxidation in beef patties. In this study, the carbonyl content of beef patties that included winter mushroom powders, tended to be higher than that of control and BP. This might be due to the action of quinones generated by the oxidation of phenolic compounds (Pourcel et al., 2007). Quinones can accelerate the oxidative deamination of responsive amino acids and consequently produce applicable carbonyl compounds (Estévez and Heinonen, 2010). Cando et al. (2014) reported an increase in the carbonyl content and inhibition of lipid oxidation in beef patties with the addition of the phenolic-rich extract of Willowherb (Epilobium hirsutum L.).

The intense color of natural ingredients incorporated in meat products results in color changes in meat products (Jeong et al., 2020; Lee et al., 2019). There were no significant differences in L* and a* values, except for b* value among control, BP and BFW (Table 4, p>0.05). Previous study reported that the lack of an effect of winter mushroom based on its white color on the color of meat products could be a suitable property as a natural ingredient for meat products (Choe et al., 2018; Choi et al., 2020; Jo et al., 2018; Jo et al., 2020b). BP had a higher b* value than control and BFW (p<0.05). This result might be attributed to the increase in Maillard reaction by the increase of pH with the phosphate addition. Maillard reaction rate is faster in high pH environments, and some studies reported that phosphate accelerates Mallard reaction (Bell, 1997). The ODP showed lower L* and higher a* values with the Maillard reaction compared to FDP (Table 1). From the results, BOW showed the lower L* and the higher a* values compared to those of control, BP and BFW (p<0.05). However, the difference in color among the treatments was small.

The texture properties of beef patties were presented as hardness, springiness, cohesiveness, gumminess, and chewiness (Table 5). BP showed the highest values for all the texture properties (p<0.05). The addition of phosphates in meat products increases the solubility of myofibrillar proteins by the dissociation of actomyosin and an increase in ion strength (Chen et al., 2019). The increase in solubilized myofibrillar proteins results in the strong and elastic gel structure of comminuted meat products (Choe et al., 2018). In addition, the phosphorylation of myofibrillar proteins leads to an increase in the interaction between insolubilized and solubilized myofibrillar proteins, thereby increasing the gel strength (Chen et al., 2019). There were no differences in cohesiveness, gumminess, and chewiness among the control, BFW, and BOW groups (p>0.05). The hardness of BOW and springiness of BFW and BOW were higher than those of the control (p<0.05). The effect of winter mushroom powder addition on the texture of meat products differed between beef patties and sausages. Choe et al. (2018) and Jo et al. (2018) reported a decrease in hardness and springiness of pork and chicken sausages with the addition of winter mushroom powder and reported that these results were caused by the hindrance of gelling by dietary fiber in winter mushroom. The difference in texture changes in meat products with winter mushroom addition might be different in the organization of structure among meat products. The structure of sausages, especially emulsion sausages, is densely organized by the interaction of solubilized proteins, insoluble proteins, and small particles of myofibrillar filaments (Chen et al., 2019). However, the structure of beef patties is relatively less dense compared to that of sausage because it is organized by the interaction of partly solubilized protein and meat particles. Therefore, the interactions among structural components in beef patties might not be affected by dietary fiber from winter mushrooms. In the present study, the pH of the meat batter increased for BFW and BOW (Table 2). The increase in pH of meat batter results in a decrease of the electrical attraction force among myofibrillar proteins, resulting in increased solubility of myofibrillar proteins (Thangavelu et al., 2019). Therefore, the myofibrillar proteins in BFW and BOW might be more solubilized and form a more intense structure than the control.