Note Research on the Application of Soybean, Adzuki Bean

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Research on the Application of Soybean, Adzuki Bean and Chestnut Inner Shell Polyphenols Contained in Cooking Water to Cheese Manufacturing

* Takayuki Miura , Kaori Tokumaru and Ryozo Akuzawa

Department of Food Science, Nippon Veterinary and Life Science University, 1-7-1, Musashino, Tokyo 180-8602, Japan

Received July 13, 2014 ; Accepted February 20, 2015

The application of cooking water abundant in phenolic compounds, obtained from cooking three types of beans (black soybean, yellow soybean and adzuki bean) and chestnut inner shell, to functional cheese product manufacturing was evaluated. Total phenolics in the cooking water were estimated by the Folin-Ciocalteu colorimetric method and showed a wide range, from 24.4 to 761 µg/mL. The antioxidant activity of extracts was determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and oxygen radical antioxidant activity (ORAC) assays. Results of both antioxidant assays showed similar trends in total phenolic content; DPPH values ranged from 24.6 to 309.3 µmol TE/mL, while ORAC values ranged from 30.6 to 325 µmol TE/mL. High estimated residual phenolic values were observed for cheese curd made with chestnut inner shell, yellow soybean, adzuki bean and black soybean extracts; 96, 87.2, 84.9 and 82.3%, respectively.

Keywords: cheese, soybean, adzuki, chestnuts, polyphenol

Introduction utilized natural resource. Common edible plant seeds are among the world’s most However, in order to use the residual processing water for food important foods. The soybean (Glycine max (L.) Merrill) has been or medical supplies, the polyphenols must be condensed from a used as a food for more than 5,000 years. It is extensively consumed large volume, which represents a costly process. in the Asian diet as a variety of products, such as soymilk, tofu and Polyphenols are known to non-specifically bind various proteins. various soy-based foods. Adzuki beans (Vigna angularis) and Some researchers have reported improvement of flavor, texture and chestnuts (Castanea crenata) are also good nutrient sources (Tjahjadi anti-oxidant activity with the addition of plant and fruit extracts to et al., 1988; Miguelez et al., 2004; Hoshikawa, 1985), and are dairy products such as cheese (Aly, 1997; Bandyopadhyay et al., popular ingredients in many Asian desserts, especially in Japan. 2008). These dairy products are considered to be a form of The first step in seed processing involves the processing of polyphenol-milk protein complexes (Arts, 2002; Jöbstl, et al., 2004; seeds in large amounts of water, generating water extracts that are Papadopoulou et al., 2005). usually discarded. However, these extracts contain many different Recently, Han et al. (2011) investigated the development of functional compounds (Moure et al., 2001). Plant-derived functional cheese and demonstrated that added phenolic compounds polyphenolic compounds perform various biological functions are highly recuperative in cheese curd, i.e., there is a minimal loss of including exerting anti-oxidative effects. Polyphenols are known as phenolic compounds during the cheese-making process. principal antioxidants in the human diet (Amarowicz et al., 2008; The traditional cheese-making process using enzymatic coagulants Havsteen, 2002; Pietta, 2000). Therefore, it is proposed that water does not require excessive heating or chemical substances, extracts, as a byproduct of food processing, have value as an under- representing an economical method of condensing milk protein

*To whom correspondence should be addressed. E-mail: [email protected] 354 T. Miura et al.

(primarily casein). (pH 6.8, control sample) was added to 150 μL of 400 µM DPPH in Furthermore, if “affinity of casein and polyphenols” and 50% ethanol / Tris-HCl buffer (v/v) solution. The decrease in “efficient protein concentration by coagulant” are applied, it is absorbance was determined at 520 nm after the reaction plateaued proposed that as a casein-polyphenol complex, the polyphenols (after a 30 min reaction). The DPPH radical scavenging capacity is contained in large amounts of residual cooking water can be expressed as µmol Trolox equivalents (TE) per mL of sample. This effectively utilized upon recovery by concentration. experiment was conducted in triplicate. This study investigated the polyphenol residual ratio and Milk coagulation One mL of the plant extracts was antioxidant activity of cheese curd in order to evaluate the novel individually added to 9 mL of milk. Rennet solution (0.5 mL, 1% usage of cooking water extracts as an under-utilized natural w/v; Chy-Max Extra, Chr. Hansen, Milwaukee, WI) was added to resource. 10 mL of the plant extract / milk solution. Then, the samples were heated in a water bath at 35℃ for 30 min to induce coagulation. Materials and Methods Whey was separated from the cheese curd by centrifugation at Preparation of sample extract Yellow and black soybeans 1300 g for 15 min at 21℃. Separated whey (9 mL) was stored in a (Glycine max), adzuki beans (Vigna angularis) and Japanese refrigerator for subsequent residual polyphenol determinations. chestnuts (Castanea crenata) were obtained from a local market. Curds were stored in a refrigerator until used in experiments. All plants were harvested in Japan. Estimation of residual percentages of phenolics (RPP) and Whole beans or chestnut inner shell (25 g dry weight per sample) antioxidant activity (RPA) in cheese curd. were packed into electrolytic tinplate cans (100 × 100 mm). The cans The RPP and RPA in cheese curd were estimated by subtracting were filled with reverse osmosis (RO) water (250 mL) and heated to the amount of RPP and RPA in the whey from the total phenolic an internal temperature of 90℃. The cans were sealed immediately content and ORAC value in the extracts. as a retort for thermal treatment (115℃ for 5 min) and then cooled to The whey polyphenol content and ORAC value were calculated room temperature. Subsequently, the supernatants were decanted by subtracting the whey values obtained from milk without added from the can and clarified by centrifugation at 1300 g for 10 min at extracts from the whey values obtained from milk with added room temperature. The resulting supernatants were used as the plant extracts. All data are the mean ± standard deviation (SD) of extracts. triplicate determinations, and were evaluated by one-way ANOVA Determination of total phenolics Total phenolic content of and Tukey’s honestly significant difference test. Differences were plant extracts was estimated by the Folin-Ciocalteu colorimetric considered statistically significant at P < 0.05. Statistical analysis method (Singleton and Rossi, 1965). Extracts were diluted (1:10) was performed using Kaleida Graph software version 4.5 (Synergy with RO water. A 1 mL volume of the standard or sample solution Software, Reading, PA). was added to 1 mL of the Folin-Ciocalteu reagent (Sigma-Aldrich, Finally, the RPP or RPA in cheese curd was calculated as St. Louis, MO.) and 1 mL of 10% (w/v) sodium carbonate. The follows: reaction mixture was kept in the dark for 15 min at room RPP or RPA = 100 _ (Phenolic content or ORAC activity in temperature, and absorbance was then measured at 700 nm. A whey/Phenolic content or ORAC activity in milk×90). standard curve was obtained with gallic acid solution (GA) (mg/ mL) as a standard. Results were expressed as µg of gallic acid per Results and Discussions milliliter of plant extracts. Phenolic content and antioxidant activity of plant extracts To Oxygen radical absorbance capacity (ORAC) assay The determine the antioxidant activity of plant extracts (residual ORAC assay was performed using the OxiSelectTM Assay Kit cooking water) of the 3 beans and chestnut inner shell, total (STA-345; Cell Biolabs Inc., San Diego, CA) according to the phenolic content and antioxidant activity of samples were manufacturer’s instructions. In this assay, AAPH acts as a peroxyl determined (Table 1). radical inhibitor that quenches fluorescein over time. The plant The total phenols of extracts were estimated by the Folin- extracts present in the assay system blocked peroxyl radical- Ciocalteu colorimetric method and showed a wide range, from 24.4 mediated fluorescein oxidation until the antioxidants in the sample to 761 µg/mL. were depleted. The area under curve (AUC) of each Trolox The antioxidant activity of extracts was determined by DPPH concentration (0 _ 50 µM) was used to plot the standard curve for and ORAC assays. Both antioxidant assay results showed similar ORAC activity, and each extract was calculated and expressed as trends in total phenolic content; DPPH values ranged from 24.6 to µmol Trolox equivalents (TE) per mL of sample. This experiment 309.3 µmol TE/mL, while ORAC values ranged from 30.6 to was conducted in triplicate. 325 µmol TE/mL. The results of antioxidant activity showed a high 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay Antioxidant correlation (r2 of 0.993) between the assay methods used. activity was determined using DPPH as a free radical (Peschel et The chestnut extract showed higher antioxidant activity than al., 2006). A 25 µL aliquot of sample or 100 mM Tris HCl buffer the three bean extracts; the reason for this was that the chestnut Application of Cooking Water to Cheese 355

Table 1. Phenolic content and antioxidant activity (DPPH and ORAC) of plant extracts.

Results are the mean ± SD of triplicate determinations. sample was comprised of only the inner shell, which is high in catechins, such as ECG and EGCG, are among many molecules polyphenol content (Hwang et al., 2001). Barreira et al. (2008) also that interact with milk casein. showed that the skins of chestnuts are rich in phenolics and high in Han et al. (2011) also showed that the retention coefficient antioxidant activity. value (which in this article corresponds to the residual percent) was In the bean samples, the black soybean extract showed higher dependent on the kind of crude polyphenol extracted from fruits or antioxidant activity than adzuki beans and yellow soybean extracts, green tea. Since these crude extracts contained a variety of possibly due to its higher amounts of polyphenols, 250 µg/mL, hydrophobic and hydrophilic compounds of differing composition, compared to 183.5 and 24.4 µg/mL, for adzuki beans and yellow it is likely that the RPP changed. soybeans, respectively. In contrast, the RPA showed a lower level than the RPP. The It is generally known that light-colored beans contain a lower yellow bean extract was the highest at 73.5%, and the values for amount of phenolic compounds than dark-colored beans black soy bean, adzuki bean and chestnut were 55.7, 50 and 49.8%, (Barampama and Simard, 1994). respectively. De Mejía et al. (2003) demonstrated that most phenolics are Pasteurized milk has high antioxidant activity, which can be located in the seed coat. Smaller seeds usually have a greater seed attributed to milk protein derivatives (mainly casein), vitamin E, coat area by weight than larger seeds; therefore, smaller seeds are carotenoids, and other substances. Most of these components are likely to have higher phenolic content. transferred to the curd; however, some remain in the whey and Since the diameter of the bean samples used in this study was exhibit about one-tenth to one-twentieth the antioxidant activity of about 7 _ 9 mm, the above-mentioned factor likely had minimal whole milk (Zulueta et al., 2009). In this experiment, the ORAC of influence on the results. whey obtained from milk without added extracts was 1.79 µ mol The RPP and RPA in cheese curd was estimated by subtracting TE/mL, and so was used to calculate the antioxidant value derived the amount of RPP and RPA in whey from the total phenolic from extracts by subtracting from the value of whey with extract content and ORAC value of extracts. added. The RPA value of yellow soy bean is thought to be Residual percentage of phenolic content (RPP) and antioxidant transferred efficiently to curd because of the low phenolic content activity (RPA) in cheese curd Table 2 shows the RPP and RPA in the undiluted extract solution, showing the highest value. The according to the ORAC value in cheese curd. RPA of black soybean, adzuki bean, and chestnut inner shell were With respect to phenolics, the chestnut inner shell, yellow approx. equal, however, no correlations were seen with the amount soybean, adzuki bean and black soy bean extracts showed high of phenolics transferred to curd or the antioxidant value. Since the RPPs of 96.0, 87.2, 84.9 and 82.3%, respectively. phenolic content and ORAC in the extracts were correlated (Table Notably, the RPP of black soybean was the lowest (82.3%) 1), detailed investigation of the residual phenolic component in even though its total phenolic content was the 2nd highest among curd and whey is needed. samples. These results suggested that cooking water with a low Most edible plants and common beans contain various phenolic polyphenol concentration could also be efficiently condensed in acids and flavonoids, especially beans with a dark-colored seed cheese curd production. coat, which are rich in anthocyanin. Research investigating the use of exogenous phenolics to Although hydrophobic associations can occur between hydroxyl improve the quality of dairy products dates back to the 1970s groups on polyphenols with proteins, structural differences in (O’Connell and Fox, 2001). Although a large number of studies polyphenols strongly affect this association (Arts, 2002; Jöbstl et al., have been conducted addressing purified polyphenols or extracts of 2004; Papadopoulou et al., 2005). edible plants, little is known about under-utilized plant resources, Miura and Akuzawa (2010) reported that gallated green tea such as residual cooking water and inedible fruit skins or shells. 356 T. Miura et al.

Table 2. Residual percentage of phenolics (RPP) and antioxidant activity (RPA) of cheese curd.

*A 1-mL aliquot of plant extract was added to 9 mL of milk. Phenolic content and ORAC values in milk are shown in Table 1. **Phenolic content and ORAC were calculated by subtracting the whey value obtained from milk without added extract. Results are the mean ± SD of triplicate determinations. Different letters in the same column indicate significant differences (Tukey’s honestly significant test, P < 0.05).

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