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

Abstract

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.

Introduction

Common edible plant seeds are among the world's most important foods. The soybean (Glycine max (L.) Merrill) has been used as a food for more than 5,000 years. It is extensively consumed in the Asian diet as a variety of products, such as soymilk, tofu and various soy-based foods. Adzuki beans (Vigna angularis) and chestnuts (Castanea crenata) are also good nutrient sources (Tjahjadi et al., 1988; Miguelez et al., 2004; Hoshikawa, 1985), and are popular ingredients in many Asian desserts, especially in Japan.

The first step in seed processing involves the processing of seeds in large amounts of water, generating water extracts that are usually discarded. However, these extracts contain many different functional compounds (Moure et al., 2001). Plant-derived polyphenolic compounds perform various biological functions including exerting anti-oxidative effects. Polyphenols are known as principal antioxidants in the human diet (Amarowicz et al., 2008; Havsteen, 2002; Pietta, 2000). Therefore, it is proposed that water extracts, as a byproduct of food processing, have value as an underutilized natural resource.

However, in order to use the residual processing water for food or medical supplies, the polyphenols must be condensed from a large volume, which represents a costly process.

Polyphenols are known to non-specifically bind various proteins. Some researchers have reported improvement of flavor, texture and anti-oxidant activity with the addition of plant and fruit extracts to dairy products such as cheese (Aly, 1997; Bandyopadhyay et al., 2008). These dairy products are considered to be a form of polyphenol-milk protein complexes (Arts, 2002; Jöbstl, et al., 2004; Papadopoulou et al., 2005).

Recently, Han et al. (2011) investigated the development of functional cheese and demonstrated that added phenolic compounds are highly recuperative in cheese curd, i.e., there is a minimal loss of phenolic compounds during the cheese-making process.

The traditional cheese-making process using enzymatic coagulants does not require excessive heating or chemical substances, representing an economical method of condensing milk protein (primarily casein).

Furthermore, if “affinity of casein and polyphenols” and “efficient protein concentration by coagulant” are applied, it is proposed that as a casein-polyphenol complex, the polyphenols contained in large amounts of residual cooking water can be effectively utilized upon recovery by concentration.

This study investigated the polyphenol residual ratio and antioxidant activity of cheese curd in order to evaluate the novel usage of cooking water extracts as an under-utilized natural resource.

Materials and Methods

Preparation of sample extract    Yellow and black soybeans (Glycine max), adzuki beans (Vigna angularis) and Japanese chestnuts (Castanea crenata) were obtained from a local market. All plants were harvested in Japan.

Whole beans or chestnut inner shell (25 g dry weight per sample) were packed into electrolytic tinplate cans (100 × 100 mm). The cans were filled with reverse osmosis (RO) water (250 mL) and heated to an internal temperature of 90°C. The cans were sealed immediately as a retort for thermal treatment (115°C for 5 min) and then cooled to room temperature. Subsequently, the supernatants were decanted from the can and clarified by centrifugation at 1300 g for 10 min at room temperature. The resulting supernatants were used as the plant extracts.

Determination of total phenolics    Total phenolic content of plant extracts was estimated by the Folin-Ciocalteu colorimetric method (Singleton and Rossi, 1965). Extracts were diluted (1:10) with RO water. A 1 mL volume of the standard or sample solution was added to 1 mL of the Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO.) and 1 mL of 10% (w/v) sodium carbonate. The reaction mixture was kept in the dark for 15 min at room temperature, and absorbance was then measured at 700 nm. A standard curve was obtained with gallic acid solution (GA) (mg/mL) as a standard. Results were expressed as µg of gallic acid per milliliter of plant extracts.

Oxygen radical absorbance capacity (ORAC) assay    The ORAC assay was performed using the OxiSelectTM Assay Kit (STA-345; Cell Biolabs Inc., San Diego, CA) according to the manufacturer's instructions. In this assay, AAPH acts as a peroxyl radical inhibitor that quenches fluorescein over time. The plant extracts present in the assay system blocked peroxyl radical-mediated fluorescein oxidation until the antioxidants in the sample were depleted. The area under curve (AUC) of each Trolox concentration (0 – 50 µM) was used to plot the standard curve for ORAC activity, and each extract was calculated and expressed as µmol Trolox equivalents (TE) per mL of sample. This experiment was conducted in triplicate.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay    Antioxidant activity was determined using DPPH as a free radical (Peschel et al., 2006). A 25 µL aliquot of sample or 100 mM Tris HCl buffer (pH 6.8, control sample) was added to 150 µL of 400 µM DPPH in 50% ethanol / Tris-HCl buffer (v/v) solution. The decrease in absorbance was determined at 520 nm after the reaction plateaued (after a 30 min reaction). The DPPH radical scavenging capacity is expressed as µmol Trolox equivalents (TE) per mL of sample. This experiment was conducted in triplicate.

Milk coagulation    One mL of the plant extracts was individually added to 9 mL of milk. Rennet solution (0.5 mL, 1% w/v; Chy-Max Extra, Chr. Hansen, Milwaukee, WI) was added to 10 mL of the plant extract / milk solution. Then, the samples were heated in a water bath at 35°C for 30 min to induce coagulation. Whey was separated from the cheese curd by centrifugation at 1300 g for 15 min at 21°C. Separated whey (9 mL) was stored in a refrigerator for subsequent residual polyphenol determinations. Curds were stored in a refrigerator until used in experiments.

Estimation of residual percentages of phenolics (RPP) and antioxidant activity (RPA) in cheese curd.

The RPP and RPA in cheese curd were estimated by subtracting the amount of RPP and RPA in the whey from the total phenolic content and ORAC value in the extracts.

The whey polyphenol content and ORAC value were calculated by subtracting the whey values obtained from milk without added extracts from the whey values obtained from milk with added extracts. All data are the mean ± standard deviation (SD) of triplicate determinations, and were evaluated by one-way ANOVA and Tukey's honestly significant difference test. Differences were considered statistically significant at P < 0.05. Statistical analysis was performed using Kaleida Graph software version 4.5 (Synergy Software, Reading, PA).

Finally, the RPP or RPA in cheese curd was calculated as follows:

RPP or RPA = 100 − (Phenolic content or ORAC activity in whey/Phenolic content or ORAC activity in milk×90).

Results and Discussions

Phenolic content and antioxidant activity of plant extracts    To determine the antioxidant activity of plant extracts (residual cooking water) of the 3 beans and chestnut inner shell, total phenolic content and antioxidant activity of samples were determined (Table 1).

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

Plant extracts	Total phenolics (µg/mL)	DPPH (µmol TE/mL)	ORAC (µmol TE/mL)	pH	Extract color
Black soybean	250.0±1.12	181.1±1.3	187.6±1.1	6.05	black
Yellow soybean	24.4±0.96	24.6±1.2	30.6±1.2	6.18	clear
Adzuki bean	183.5±18.8	135.6±1.3	144.4±1.3	6.29	reddish brown
Chestnut inner shell	761.0±12.9	309.3±1.3	325.0±1.5	5.03	dark brown

Results are the mean ± SD of triplicate determinations.

The total phenols of extracts 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 and ORAC assays. Both antioxidant assay results 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. The results of antioxidant activity showed a high correlation (r² of 0.993) between the assay methods used.

The chestnut extract showed higher antioxidant activity than the three bean extracts; the reason for this was that the chestnut sample was comprised of only the inner shell, which is high in polyphenol content (Hwang et al., 2001). Barreira et al. (2008) also showed that the skins of chestnuts are rich in phenolics and high in antioxidant activity.

In the bean samples, the black soybean extract showed higher antioxidant activity than adzuki beans and yellow soybean extracts, possibly due to its higher amounts of polyphenols, 250 µg/mL, compared to 183.5 and 24.4 µg/mL, for adzuki beans and yellow soybeans, respectively.

It is generally known that light-colored beans contain a lower amount of phenolic compounds than dark-colored beans (Barampama and Simard, 1994).

De Mejía et al. (2003) demonstrated that most phenolics are located in the seed coat. Smaller seeds usually have a greater seed coat area by weight than larger seeds; therefore, smaller seeds are likely to have higher phenolic content.

Since the diameter of the bean samples used in this study was about 7 – 9 mm, the above-mentioned factor likely had minimal influence on the results.

The RPP and RPA in cheese curd was estimated by subtracting the amount of RPP and RPA in whey from the total phenolic content and ORAC value of extracts.

Residual percentage of phenolic content (RPP) and antioxidant activity (RPA) in cheese curd    Table 2 shows the RPP and RPA according to the ORAC value in cheese curd.

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

Extracts	Phenolics (µg/mL)		RPP in curd (%)	ORAC (µmol TE/mL)		RPA in curd (%)
	Milk with extracts*	whey**		Milk with extracts*	whey**
Black soybean	25.0	4.90±2.10a	82.3	18.7	9.2±0.08a	55.7
Yellow soybean	2.4	0.34±0.05b	87.2	3.06	0.9±0.09b	73.5
Adzuki bean	18.0	3.02±0.59a	84.9	14.4	8.0±0.09a	50.0
Chestnut inner shell	76.1	3.33±2.00a	96.0	32.5	18.1±0.11c	49.8

*  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).

With respect to phenolics, the chestnut inner shell, yellow soybean, adzuki bean and black soy bean extracts showed high RPPs of 96.0, 87.2, 84.9 and 82.3%, respectively.

Notably, the RPP of black soybean was the lowest (82.3%) even though its total phenolic content was the 2^nd highest among samples.

Most edible plants and common beans contain various phenolic acids and flavonoids, especially beans with a dark-colored seed coat, which are rich in anthocyanin.

Although hydrophobic associations can occur between hydroxyl groups on polyphenols with proteins, structural differences in polyphenols strongly affect this association (Arts, 2002; Jöbstl et al., 2004; Papadopoulou et al., 2005).

Miura and Akuzawa (2010) reported that gallated green tea catechins, such as ECG and EGCG, are among many molecules that interact with milk casein.

Han et al. (2011) also showed that the retention coefficient value (which in this article corresponds to the residual percent) was dependent on the kind of crude polyphenol extracted from fruits or green tea. Since these crude extracts contained a variety of hydrophobic and hydrophilic compounds of differing composition, it is likely that the RPP changed.

In contrast, the RPA showed a lower level than the RPP. The yellow bean extract was the highest at 73.5%, and the values for black soy bean, adzuki bean and chestnut were 55.7, 50 and 49.8%, respectively.

Pasteurized milk has high antioxidant activity, which can be attributed to milk protein derivatives (mainly casein), vitamin E, carotenoids, and other substances. Most of these components are transferred to the curd; however, some remain in the whey and exhibit about one-tenth to one-twentieth the antioxidant activity of whole milk (Zulueta et al., 2009). In this experiment, the ORAC of whey obtained from milk without added extracts was 1.79 µ mol TE/mL, and so was used to calculate the antioxidant value derived from extracts by subtracting from the value of whey with extract added. The RPA value of yellow soy bean is thought to be transferred efficiently to curd because of the low phenolic content in the undiluted extract solution, showing the highest value. The RPA of black soybean, adzuki bean, and chestnut inner shell were approx. equal, however, no correlations were seen with the amount of phenolics transferred to curd or the antioxidant value. Since the phenolic content and ORAC in the extracts were correlated (Table 1), detailed investigation of the residual phenolic component in curd and whey is needed.

These results suggested that cooking water with a low polyphenol concentration could also be efficiently condensed in cheese curd production.

Research investigating the use of exogenous phenolics to improve the quality of dairy products dates back to the 1970s (O'Connell and Fox, 2001). Although a large number of studies have been conducted addressing purified polyphenols or extracts of edible plants, little is known about under-utilized plant resources, such as residual cooking water and inedible fruit skins or shells.

Conclusions

This study demonstrated that phenolic compounds in the crude extracts of three bean varieties and chestnut inner shell interacted with cheese curd at a high percentage.

These results indicate that antioxidants found in the residual cooking water can be efficiently collected during the milk enzymatic coagulation process, allowing for the development of novel dairy products.

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