Article pubs.acs.org/JAFC
Volatile Compounds as Markers of Tofu (Soybean Curd) Freshness during Storage Seonmi Lee, Heera Cho, and Kwang-Geun Lee* Department of Food Science and Biotechnology, Dongguk UniversitySeoul, 26, 3-Ga, Pil-dong, Chung-gu, Seoul 100-715, Republic of Korea ABSTRACT: Volatile compounds in packed and unpacked commercial tofu were extracted by solid phase microextraction fibers and analyzed by gas chromatography−mass spectrometry. The tofu samples were stored at 4 °C for 15 days to measure freshness and quality changes during storage. Totals of 41 and 35 volatile compounds were identified from packed tofu and unpacked tofu, respectively. Peak area ratio (PAR) of hexanal in packed tofu increased from 11.44 (day 0) to 496.30 (day 9) and finally decreased to 11.06 on day 15 of storage. PAR of ethanol and 1-hexanol increased from 5.41 and 0.76 (day 1) to 66.93 (day 9) and 47.46 (day 15), respectively. Changes in these volatiles in unpacked tofu were similar to those in packed tofu. The results show that hexanal, ethanol, and 1-hexanol are characteristic volatile compounds for providing quantitative and qualitative information regarding deterioration of tofu. KEYWORDS: volatile compounds, tofu, storage, freshness, HS-SPME, GC-MS
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method is a solvent-free technique and is relatively inexpensive.21 SPME coupled with gas chromatography−mass spectrometry (GC-MS) was adopted in this study. Few studies have investigated changes in volatile compounds of fresh commercial tofu during storage.18,22 The objective of our study was to investigate the changes in volatile compounds in commercial tofu using the SPME method and GC-MS during storage. Analyses of volatiles and microbial contamination were carried out to evaluated the freshness and quality change in tofu. The relationship between tofu freshness and volatile compounds was determined.
INTRODUCTION Tofu (soybean curd) is a healthy, popular, and traditional soy food in Asian diets, as it is rich in protein, calcium, B-vitamins, iron, and magnesium.1 Tofu is becoming more popular in other countries because isoflavones in soybean are very beneficial to human health.2 However, tofu is very perishable and has a short shelf life of 2−3 days.3 Tofu is very sensitive to microbial growth even under refrigeration due to its high moisture content (>80%) and neutral pH (5.8−6.2).4−6 Mass production of tofu is currently carried out in Korea through a pasteurizing process at a low temperature and packaging in sealed containers. Tofu is distributed by a cold-chain system. Sales of tofu increased rapidly from $336 million (2004) to $414 million (2008) in Korea. The cold-chain system provides easy handling of tofu and improves its storability.7 Nevertheless, the only way to check the freshness of leftover tofu is to look at the expiration date on the label or smell the tofu. A few studies have investigated the development of a freshness indicator for tofu.8 Many studies have been conducted on the volatile compounds in fermented tofu and other soybean products. However, these studies were restricted to volatile compounds of fermented soybean products such as Thai thua nao, Chinese sufu, and Korean doenjang.9−13 Among the volatiles, hexanal, ethanol, and 1-hexanol are found in all fermented tofu. In particular, hexanal contributes a disagreeable green grass-like aroma to tofu and soy milk.14,15 The content of hexanal in the nonfermented tofu is higher than that in fermented tofu. This phenomenon suggests that fermentation may remove the undesirable green grass flavor from soybean products16,17 and indicates that volatile compounds in tofu could be a durable index chemical to determine the quality of tofu during storage. The typical methods for extracting volatile compounds from tofu are simultaneous distillation extraction, supercritical fluid extraction, and solid phase microextraction (SPME).13,18,19 SPME has recently replaced older techniques for volatile analysis as it is fast and less labor intensive.20 In addition, the SPME © 2014 American Chemical Society
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MATERIALS AND METHODS
Materials. Two types of tofu, a packed tofu and an unpacked tofu, were purchased at a commercial market in Korea. In Korea the market for tofu is divided into packed and unpacked tofu. The packed tofu selected in this study is the first in market share in Korea. The unpacked tofu was also selected due to higher market share in Korean traditional market. Petri film was used for microbiological analysis (3M, St. Paul, MN, USA). The C8−C20 alkanes for calculating the retention indices (RI) were purchased from Aldrich Chemical Co. (St. Louis, MO, USA). Three kinds of SPME fibers with different coats were purchased from Supelco Inc. (Bellefonte, PA, USA); they were polydimethylsiloxane (PDMS, 100 μm film thickness), Carboxen−polydimethylsiloxane (CAR-PDMS, 75 μm film thickness), and polydimethylsiloxane− divinylbenzene (PDMS-DVB, 65 μm film thickness). Sample Preparation and Storage Condition. The tofu was cut into cubed samples (5 cm ×5 cm ×3 cm) and placed in a plastic container with 160 mL of sterile purified water. Tofu was classified according to temperature and storage day. The samples were stored at 4 °C for 15 days. Microbiological Analysis. Ten grams of sample was mixed with 90 mL of buffered peptone water (0.1%) in a sterile stomacher bag. Received: Revised: Accepted: Published: 772
October 29, 2013 January 3, 2014 January 6, 2014 January 6, 2014 dx.doi.org/10.1021/jf404847g | J. Agric. Food Chem. 2014, 62, 772−779
Journal of Agricultural and Food Chemistry
Article
Figure 1. Relationship between microbial growth and peak area ratio of volatile compounds in packed tofu (a) and unpacked tofu (b) during storage at 4 °C. Bars represent, from left to right in each group, hexanal, 1-hexanol, and ethanol. The mixture was blended for 1 min using a Stomacher Lab Blender (Bag mixer model, Inter Science), serially (1:10) diluted with buffered peptone water (0.1%), and plated on Petri film (3M). The Petri film was incubated at 37 °C for 48 h, and total bacterial counts were carried out. Volatile Compound Analysis. The ground samples (2.5 g) were mixed with 2.5 mL of tofu soaking water, and the mixtures were placed into a 15 mL headspace vials (Supelco). The headspace vials were sealed with a polypropylene hole cap and PTFE/silicone septa (Supelco). Then they were pre-equilibrated for 21 min at 46 °C in a thermostatic bath. Before the volatile compound extraction, 20 μL (1 ppm) of internal standard (1,2-dichlorobenzene) was added to the sample solution. Samples were analyzed using headspace-SPME-GC-MS. Three fibers were tested and compared: PDMS (100 μm film thickness), CAR-PDMS (75 μm film thickness), and PDMS-DVB (65 μm film thickness). Each fiber was preconditioned in the injector of the GC system according to the conditions provided by the manufacturer. The selected fiber (CAR-PDMS (75 μm film thickness) fiber) was pushed through the vial septum of the prepared sample. The fiber was pushed out of the housing and exposed to the headspace (3 cm in depth) at 46 °C for 14 min. After extraction, the fiber was pulled into the housing, and the SPME device was removed from the vial and inserted into the injection port of a gas chromatograph (GC) for thermal desorption of the analysis (4 cm in depth). The fiber was desorbed for 10 min at 230 °C to eliminate any residual volatile compounds.
GC-MS analysis was performed using an Agilent 6890 gas chromatograph with a 5975 mass spectrometry detector (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a DB-WAX column (30 m length × 0.32 mm i.d × 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). The analytical conditions were as follows: inlet temperature, 220 °C; column head pressure, 14.14 psi; splitless time, 1 min; helium carrier gas flow rate, 1.3 mL/min; oven temperature, held at 40 °C for 4 min, raised at 5 °C/min to 185 °C, and held for 20 min. Mass spectra were generated in the electron impact mode (MSEI) at 70 eV and an ion source temperature of 230 °C. The total ion chromatograms acquired via GC-MS was used for peak area integration. Agilent MSD Chemstation software (G1701BA ver.D.02.00) was used for data acquisition. Compound Identification and Quantification. The volatile compounds were positively identified by comparing Kovats retention indices (KI)23 and the MS fragmentation patterns with those of reference compounds or with mass spectra in the Wiley 275 mass spectral database (Hewlett-Packard, Palo Alto, CA, USA) and previously reported KIs. The KIs of the unknown compounds were determined via sample injection with a homologous series of alkanes (C8−C20). The GC-MS conditions were the same as described above. To quantify volatiles, the samples were run in duplicate, and the integrated areas based on the total ion chromatograms were normalized to the areas of the internal standard and averaged. The relative volatile concentrations 773
dx.doi.org/10.1021/jf404847g | J. Agric. Food Chem. 2014, 62, 772−779
Journal of Agricultural and Food Chemistry
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Article
RESULTS AND DISCUSSION
Microbiological Analysis. Figure 1 shows the microbial population and volatiles of packed and unpacked tofu during the storage period at 4 °C. The initial colony-forming units (CFUs) of aerobic mesophilic microorganisms (total bacterial count) of packed tofu and unpacked tofu were different. No microorganisms were detected until storage day 9 for packed tofu. Then, their level reached 7.29 log CFU/g at day 15. The initial number of microbial flora in unpacked tofu was 3.76 log CFU/g, which was much higher than that of packed tofu. The number of microbial flora ranged from 3.76 to 7.67 log CFU/g in unpacked tofu for all storage days. The maximum log CFU/g in unpacked tofu was found after 9 days of storage. The unpacked tofu had rapidly growing microbial flora compared with that in the packed tofu because it was exposed to air without any packaging material. In Korea the microbial (aerobic mesophilic microorganisms) count exceeding the safe level is approximately 6−7 log CFU/g.8 However, coli form should be
