Food Chemistry 158 (2014) 438–444
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Changes in fatty acid composition and lipid profile during koji fermentation and their relationships with soy sauce flavour Yunzi Feng, Zhiyao Chen, Ning Liu, Haifeng Zhao ⇑, Chun Cui, Mouming Zhao ⇑ College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 13 February 2014 Accepted 25 February 2014 Available online 6 March 2014
Evolution of lipids during koji fermentation and the effect of lipase supplementation on the sensory properties of soy sauce were investigated. Results showed that total lipids of the koji samples were in the range of 16–21%. The extracted lipid of initial koji consisted mainly of triacylglycerols (TAGs, >98%), followed by phospholipids (PLs), diglycerides (DAGs), monoacylglycerols (MAGs) and free fatty acids (FFAs). As the fermentation proceeded, peroxide value of the lipids decreased while carbonyl value increased (p < 0.05). Linoleic acid was utilised fastest according to the fatty acid composition of total lipids, and preferential degradation of PLs to liberate FFAs was also observed. Moreover, phospholipase supplementation had significant influence on the sensory characteristics of soy sauce, especially enhanced (p < 0.05) scores for the umami and kokumi taste attributes. All these results indicated that the control of PLs utilisation during fermentation was a potential method to improve soy sauce’s characteristic taste. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Soy sauce Phospholipase Lipids Free fatty acid Oxidative degradation Flavour
1. Introduction Soy sauce is used as a condiment or seasoning sauce around the world due to its characteristic aroma accompanied by the intense umami taste. Chinese traditional soy sauce involves two important fermentation stages, koji fermentation and moromi fermentation (Gao et al., 2009). Previous studies showed that the evolution of volatile components largely depended on lipid oxidation in the koji fermentation stage (Feng et al., 2013). In addition, soy sauce aroma was significantly influenced by the content of lipids in the raw materials (Gao et al., 2009). Thus, influence of 20% fat in soybean, the main material, on the flavour of traditional Chinese-type soy sauce is worthy of investigation. Lipolysis, release of fatty acids, and secondary reactions of fatty acids resulted in the development of some volatile substances, such as aldehydes, ketones and alcohols, which are responsible for the characteristic aroma of fermented foods (Gambacorta et al., 2009; Visessanguan, Benjakul, Riebroy, Yarchai, & Tapingkae, 2004). It has been reported that lipolysis and lipid oxidation make significant contributions to the thickness, mouthfulness and continuity taste in migaki-nishin, a dried fish product (Azad Shah, Tokunaga, Kurihara, & Takahashi, 2009). Moreover, evolution of
⇑ Corresponding authors. Tel./fax: +86 20 22236089 (H. Zhao). Tel./fax: +86 20 87113914 (M. Zhao). E-mail addresses: (M. Zhao).
[email protected]
(H.
Zhao),
http://dx.doi.org/10.1016/j.foodchem.2014.02.147 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
[email protected]
the lipid in some fermented soya products has been proved to be the main factor in forming aroma, such as natto, Japanese miso and Indonesian tempe (De Reu, Ramdaras, Rombouts, & Nout, 1994; Kiuchi, Ohta, Itoh, Takahayahsi, & Ebine, 1976; Sarkar, Jones, Gore, Craven, & Somerset, 1996; Shieh, Beuchat, Worthington, & Phillips, 1982). Much literature has concentrated on degradation of proteins (Chou & Ling, 1998; Gao et al., 2011; Lertsiri, Maungma, Assavanig, & Bhumiratana, 2001), and umami peptides (Lioe et al., 2004) of soy sauce. However, little attention has been paid to the changes of lipid and its relationship with volatile compounds during the koji fermentation of soy sauce. Therefore, the aim of this study was to investigate the changes in lipid compositions through the koji fermentation, and to evaluate their potential effects on the characteristic flavour of soy sauce. Furthermore, the effects of adding exogenous lipase on the sensory quality of soy sauce were described, in order to explore the relationship between lipolytic and sensory parameters of soy sauce. Results from this study would make us better understand the relationships between lipid compositions in soybeans and the flavour of soy sauce.
2. Materials and methods 2.1. Materials and chemicals Soybean (Guanghui Agricultural Products Co., Ltd., Heilongjiang, China), wheat flour (Runfon Flour Co., Ltd., Guangdong, China) and
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
edible salt (Zhongshan Salt Industrial Co., Ltd., Zhongshan, Guangdong, China) were purchased from a local market. Aspergillus oryzae HN3.042 spores were obtained from Jiaming Fermentation Food Co., Ltd. (Shanghai, China). Phospholipase (LecitaseÒ ultra) and Lipozyme IM 4350 were purchased from Novozymes (Copenhagen, Denmark). Heptadecanoic acid, methyl ester was purchased from Sigma Co., Ltd. (St. Louis, MO). Chloroform, n-hexane and isopropanol were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of samples Two kilograms raw soybeans were soaked in 4000 mL water for 8 h at room temperature, and steamed at 125 °C for 15 min. Wheat flour and steamed soybean were mixed at a ratio of 1:4 (w/w) and cooled to 40 °C. The mixture was then inoculated with 0.05% (w/w) of A. oryzae HN 3.042 spores, and incubated at 30 °C for 16 h and 28 °C for another 32 h. The relative humidity was about 95% during koji fermentation. The preparation of koji was completed when the colour of the culture began turning a greenish yellow. Five soy sauce koji samples were periodically taken at 0, 12, 24, 36 and 44 h during the koji fermentation. All the samples were ground and held at 18 °C until analysis. A primary supplementation test was conducted to further clarify the effect of metabolism of lipids on the flavour of soy sauce. Lipase (IM4350, Novozymes, 0.05% koji weight) or phospholipase (LecitaseÒ ultra, 0.03% koji weight, maintaining the same enzymatic activity) was mixed with koji to yield moromi, and the main processes of soy sauce were made according to the method of Gao et al. (2011). The fermented soy sauce samples were taken on the 6th month. All samples were filtered through filter papers (18-cm diameter, Shuangquan Co. Ltd., Hangzhou, China), then stored in a refrigerator at 18 °C until analysis. 2.3. Chemical analysis Moisture and lipid contents of the koji were determined according to AOAC (2000). To determine pH value, 5 g of each sample were homogenised with 10-fold volume of distilled water and the pH was measured directly by using PHS-3E pH meter (TecFront Electronics Co. Ltd., Shanghai, China). 2.4. Lipid extraction Total lipids were extracted from the koji samples with a solvent mixture of chloroform:methanol (2:1, v/v) according to the method of Azad Shah et al. (2009). The extracts were dried under vacuum on a rotary evaporator and finished with a nitrogen flow. The lipid samples were stored under nitrogen gas in the dark at 18 °C until further analysis. 2.5. Measurement of lipid oxidation Acid value and peroxide value of the extracted lipid were determined according to AOAC (2000). Acid value was analysed by titration of approximately 0.5 g of lipid, dissolved in a mixture of 100 mL of ethanol and diethyl ether (1:1, v/v), with 0.01 N potassium hydroxide. Phenolphthalein was used as the indicator. The results of acid value and peroxide value were expressed as mg KOH/g lipid and meq/kg of lipid, respectively. The peroxide value was defined as the oxidised potassium iodide content, expressed as meq of hyperoxide per kg of lipid. According to the method of Gambotti and Gandemer (1999), carbonyl compounds were evaluated by the ratio of the absorbance at 275 nm to the absorbance at 215 nm from the lipid (125 lg/mL in cyclohexane solution). An increase
439
of the ratio A275/A215 is related to an increase in carbonyl compounds. 2.6. Fractionation of total lipids Neutral lipids (NLs), free fatty acids (FFAs) and phospholipids (PLs), were separated from the total lipids by using StrataÒNH2 cartridges containing 500 mg of amine-propylic resin (Phenomenex, Torrance, CA) as described by Regueiro, Gibert, and Diaz (1994). The cartridge was activated with 6 mL of chloroform before use. The extracted lipids (10–20 mg of total lipid) were redissolved in chloroform and then loaded on the top of the cartridges. The NLs, FFAs and PLs were eluted with 2.5 mL chloroform:isopropanol (2:1, w/w), 3 mL 2% (w/w) of acetic acid/ether, and 3 mL methanol in sequential order. 2.7. Fatty acid composition analysis Fatty acid methyl esters (FAME) were prepared from total lipids and the isolated fractions (NLs, FFAs and PLs) of soy sauce koji lipids according to the method of Morrison and Smith (1964). The contents of the fatty acid methyl ester in each fraction were quantified using heptadecanoic acid, methyl ester as internal standard. The GC–MS system consisted of a Trace Ultra GC, a Trisplus autosampler and a quadropole DSQ II MS (Thermo Finnigan, San Jose, CA). Separation was performed with a TR-5MS capillary column (30 m 0.2 mm, 0.25 lm, J&W Scientific, Folsom, CA). Helium was used as carrier gas with a flow rate of 1.0 mL/min. Sample volume of 1.0 lL was injected with a split ratio of 100:1. The analytical conditions were as follows: the temperature of the column was maintained at 40 °C for 2 min, ramped to 150 °C at 10 °C/ min, holding for 2 min, and then rose to 280 °C at a rate of 10 °C/ min and held at 280 °C for 5 min. The mass spectrometer was operated in electron-impact (EI) mode. The ionisation energy, detector voltage, scan range and scan rate applied for the analysis were 70 eV, 350 V, m/z 35–350 and 3.00 scans/s, respectively. Both injector and ion source temperature were 250 °C. FAME were identified by matching the retention times and mass spectra with those of reference standards in the standard NIST 08 library and standard FAME analysed under the same experimental conditions. 2.8. Analysis of lipids composition The compositions of triacylglycerols (TAGs) and lipids were determined by a reversed-phase high-performance liquid chromatography. The chromatographic apparatus consisted of a Waters P600 pump with a quaternary gradient system (Waters, Milford, MA), and a 3300 evaporative light-scattering detector (Alltech Associates Inc., Deerfield, IL) with an atmosphere compression pump (Tianjin, China). A PurospherÒ STAR RP-18e column (250 mm 4.6 mm i.d., particle size 5 lm, Merck, Darmstadt, Germany) was used. The temperature was 40 °C, and flow rate of gas and liquid phases were 1.5 L/min and 0.6 mL/min, respectively. The injection volume was 10 lL. The gradient elution was achieved by mobile phases A (acetonitrile:acetic acid = 99.95:0.05, v/v) and B (dichloromethane). The gradient was operated as follows: 0–4 min 100% A; 4–12 min 90% A; 12–15 min 70% A; 15–19 min 20% A; 19–31 min 80% A; 31–36 min 90% A; 36–39 min 100% A; 39–42 min 100% A. The compounds in the samples were identified by HPLC/MS (Bruker Daltonics Inc., Billerica, MA), and the conditions were the same as described by Liu et al. (2012). 2.9. Identification of volatile compounds in the soy sauce The sample preparation and SPME technique were used according to the methods of Feng et al. (2013). The SPME Trisplus auto-
440
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
mated sampler equipped with a 75 lm carboxen/polydimethylsiloxane fibre (CAR/PDMS, Supelco, Inc., Bellefonte, PA) was employed for the extraction of volatile compounds in soy sauces. Eight millilitres of soy sauce were transferred into 20-mL gas-tight glass vessels (Supelco) and saturated with 1 g NaCl. Prior to analysis, 20 lL of 2-methyl-3-heptanone (1.724 mg/L in methanol), as an internal standard, were added and mixed. After extraction for 40 min at 45 °C with continued heating and agitation extraction, the fibre was then inserted into the GC injector for 3 min to desorb analytes. Each soy sauce sample was extracted in triplicate. In all cases, the fibres were conditioned for 10 min at 260 °C between injections to prevent any contamination. Separation was performed with a TR-5MS column (30 m 0.25 mm 0.25 lm, J&W Scientific) and TR-Wax column (30 m 0.32 mm 0.25 lm, J&W Scientific). Helium was used as carrier gas with a flow rate of 1.0 mL/min. The split ratio was 20:1. The analytical conditions were as follows: the temperature of the column was maintained at 40 °C (2 min), ramped to 120 °C (2 min) at 5 °C/min, and then raised to 220 °C (5 min) at a rate of 7 °C/min. Injection temperature was 250 °C and the ion source temperature was set at 230 °C. The mass spectrometer was operated in electron-impact (EI) mode. The ionisation energy, detector voltage, scan range and scan rate applied for the analysis were 70 eV, 350 V, m/z 35–350 and 3.00 scans/s, respectively. Identification was based on the retention indices of reference standards and through matching the mass spectra provided in the standard NIST 08 library. 2.10. Sensory evaluation Sensory analysis was performed on the soy sauces supplemented with lipase or phospholipase. Quantitative descriptive analysis (QDA) was carried out with a trained analytical panel of 7 members, using a nine-point scale according to the modified methods of Gao et al. (2011) and Lioe et al. (2004). All samples were coded with random three-digit numbers, and served to the panellists in a randomized complete block design. The sensory evaluation was divided into two parts: taste and aroma analysis. The attributes that best expressed the soy sauce taste and aroma characteristics were selected by the panel. Taste sensory attributes were salty (12 mM NaCl), umami (4 mM monosodium glutamate), sweet (40 mM sucrose), sour (10 mM lactic acid), bitter (1.5 mM caffeine) and kokumi (thickness, mouthfulness and continuity) (Gao et al., 2011). For the training of kokumi intensity, the panel was asked to compare the model chicken broth and savoury matrix solutions containing sodium chloride and monosodium glutamate. The aroma sensory evaluation was based on the procedure suggested by Steinhaus and Schieberle (2007). Aroma attributes alcohols (ethanol), acids (acetic acid), malty (3-methylbutanal), caramel-like (HEMF) and beany (boiled soybean) were used to characterise the sensory properties of soy sauce samples. The training sessions were conducted according to our previous paper (Feng et al., 2014). Fifty millilitres of sample were put in a plastic cup marked with a three-digit number (100 mL) and covered with lids prior to serving for evaluation. Significant difference for each taste attribute was analysed by analysis of variance, and the results were plotted in a spider diagram. 2.11. Statistical analysis All tests were conducted in triplicate and the results were reported as means ± standard deviations. Analysis of variance and significant differences among means were tested by one-way ANOVA using SPSS 16.0 (SPSS Inc., Chicago, IL). Student–Newman–Keuls test was used for comparison of mean values among samples, and to identify significant differences (p < 0.05).
3. Results and discussion 3.1. Chemical composition The changes of chemical compositions of soy sauce koji during fermentation are shown in Table 1. The value of pH gradually decreased from 6.5 to 6.0 during 24 h of fermentation (p < 0.05), then increased to 6.3 at 44 h of koji fermentation. As a major constituent in the koji mixture, protein accounted for approximately 41.48%, followed by lipid (16.55% dry weight) and trace amounts of sugar and ash. As shown in Table 1, a continuous increase in crude lipids was observed during the whole fermentation. The percentage of crude lipids increased significantly (p < 0.05) from 16.55% to 20.22% after the koji fermentation. Comparable changes occurred during A. oryzae fermentation of a combination of peanuts and soya beans to produce miso-like products (Shieh et al., 1982), and Bacillus subtilis fermentation to produce kinema, a fermented soya bean food in Nepal (Sarkar et al., 1996). However, these increases were probably due to decreases in non-lipid materials, such as active assimilation of carbohydrates, rather than an actual increase in lipid during fermentation (Shieh et al., 1982). 3.2. Lipid oxidation Acid value, representing the content of FFAs, was used to assess the level of lipid rancidity in the koji samples (Fig. 1A). Acid value increased rapidly from 7.25 to 46.49 mg KOH/g oil during the whole koji fermentation, indicating lipolysis of soybean lipids into FFAs in this stage. A similar result was found in dry-cured sausages (Gambacorta et al., 2009), in which the production of FFAs was the major reason for the increase of acid value. However, the pH of koji samples showed an increase at the later stage of fermentation (as in Table 1) despite the relatively large amount of acid being liberated. Peroxide value measures the primary products formed from lipids in the initial stage of oxidation, which further decompose to volatile and non-volatile fatty acids, aldehydes, ketones, etc. (Jerkovic´, Mastelic´, & Tartaglia, 2007). As shown in Fig. 1B, peroxide value decreased significantly (p < 0.05) from 12.72 to 3.79 meq/kg lipid during 0–36 h of fermentation, then increased slightly to 4.59 meq/kg lipid till 44 h of fermentation. Reduction of peroxide value in the fermented chickpea was also noted by Paredes-López, González-Castañeda, and Cárabez-Trejo (1991) during solid substrate fermentation using Phyzopus olgosporus. It demonstrated that antioxidant could be used to prevent the formation of free radicals, resulting in reduced oxidation rates of soybean oil. Thus, the decrease of peroxide value in this study might be attributed to the generation of strong antioxidant activity during the koji making period, such as increased total phenolic contents (Lin, Wei, & Chou, 2006). The accumulation of secondary oxidation products was measured by determining the carbonyl compounds. The data from A275/A215 in koji lipids are shown in Fig. 1C. Significant (p < 0.05) increase in the value of A275/A215 was observed during 36–44 h of fermentation, indicating that the level of carbonyls in the koji lipids increased during koji fermentation. This finding was in agreement with our previous report on the changes of soy sauce koji flavour, in which we observed rapid increases of aldehydes and ketones compounds during the koji fermentation (Feng et al., 2013). 3.3. Changes in lipid composition during the koji fermentation Fig. 1E shows the profiles of different lipid classes in the koji samples. Predominant components were triacylglycerols (TAGs, area ratio 85.24–98.28%), followed by diacylglycerols (DAGs, area
441
Y. Feng et al. / Food Chemistry 158 (2014) 438–444 Table 1 pH and proximate composition of 5 koji samples at different fermentation times. Fermentation time (h)
pH
Moisture (g/100 g wet matter)
Lipid (g/100 g dry matter)
Protein (g/100 g dry matter)
0 12 24 36 44
6.5 ± 0.12a 6.3 ± 0.11b 6.0 ± 0.10c 6.1 ± 0.08bc 6.3 ± 0.14b
52.71 ± 1.25a 51.87 ± 1.86a 47.72 ± 1.91b 41.60 ± 1.70c 38.65 ± 1.66d
16.55 ± 0.86a 16.78 ± 0.47a 18.60 ± 1.02b 20.80 ± 0.52c 20.22 ± 0.30c
41.48 ± 0.47a 42.22 ± 0.31a 47.03 ± 1.34b 47.36 ± 0.75b 47.16 ± 0.14b
Values are means ± standard deviations of triplicate determinations and different letters (a, b, c) in the same column indicate significant differences (p < 0.05).
Changes in TAGs composition (expressed by percentage) during koji fermentation are shown in Table 2. Fifteen different molecular species were detected in the lipid extracted from koji samples. The three-letter designation does not suggest fatty acyl positional isomers in the TAG: M, myristic; P, palmitic; S, stearic; O, oleic; L, linoleic; and Ln, linolenic fatty acid moieties. The proportion of LLL + OLLn was the richest (28.63–30.61%), followed by OLL + LLM (21.29–23.37%) and LLP (15.40–16.80%), which was in agreement with the results reported by Fasciotti and Pereira Netto (2010). In lipids from koji samples, unsaturated fatty acid such as linoleic, linolenic and oleic acids were predominantly located in the 2-position of the TAG molecules, whilst saturated fatty acids, such as palmitic and stearic acids, primarily occupied the 1-position or 3-position. Preferential losses of LLnLn and LLP were observed during koji fermentation. Wagenknecht, Mattick, Lewin, Hand, and Steinkraus (1961) pointed out that the lipases from Aspergillus always selectively target unsaturated fatty acid at exterior glycerol carbons 1,3 in triacylglycerol. 3.4. Changes in fatty acid composition of total lipid
Fig. 1. Changes in acid value (A), peroxide value (B), and A275/A215 (C) of lipids during koji fermentation.
ratio 1.52–4.58%), monoacylglycerols (MAGs, area ratio 0.00–1.37%) and free fatty acids (FFAs, area ratio 0.20–8.80%). There were no significant (p > 0.05) changes in the four lipid classes until 24 h, which might be explained by the low level of lipolysis enzymes activity at this period. According to the study of Yong and Wood (1977), the maximum level of lipase activity occurred at about 30 h of fermentation. The FFAs, MAGs and DAGs increased after the koji fermentation, which should be caused by the hydrolysis of TAGs and phospholipids (Gambacorta et al., 2009).
Table 3 shows the fatty acid compositions of the total lipids (TLs) in the soy sauce koji lipids. The major fatty acids in the koji lipids were palmitic acid (C16:0), linoleic acid (C18:2), oleic acid (C18:1), linolenic acid (C18:3) and stearic acid (C18:0). The percentage of these fatty acids mainly reflected the characteristic fatty acid composition of soybean oil, which was similar to the results reported previously by Shibata et al. (2008). Linoleic acid (C18:2) accounted for the largest proportion (over 50%), while it showed an obvious decrease in its percentage in the koji lipid, from 54.64% to 51.81% of the total fatty acids in the koji samples. Moreover, it was stated in our previous report that most of C8 aliphatic compounds, which might be formed by the oxidation of linoleic or linolenic acids, increased rapidly in the later period of koji fermentation (Feng et al., 2013). These results differed from a previous report by Wagenknecht et al. (1961), in which the fatty acids liberated by the hydrolysis of soybean oil were not subsequently utilised. Interestingly, the contents of all fatty acids in total lipid showed increasing trends during koji fermentation. This might be due to the existence of lipoprotein complexes, which could be extracted simultaneously at the early stage of koji fermentation (Wang, Swain, Wallen, & Hesseltine, 1975). Indeed, Wang et al. (1975) studied the trypsin-inhibitory activity observed in cooked soybeans fermented by Rhizopus oligosporus, revealing that the lipoprotein could be hydrolysed to release fatty acid after fermentation. 3.5. Changes in fatty acid compositions of FFAs, NLs and PLs fractions Fatty acid compositions of the neutral lipids (NLs), phospholipids (PLs) and free fatty acids (FFAs) in the soy sauce koji lipids are demonstrated in Table 4. The contents of fatty acids in FFAs and NLs showed increases during koji fermentation. The fatty acids in NLs fraction showed a constant order (polyunsaturated FAs > monounsatured FAs > saturated FAs) during the different stages
442
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
Table 2 Changes in the composition of lipids and the major molecular species of triacylglycerol (TAG) during the koji fermentation. Compounds
FFA MAG DAG TAG LLnLn LLLn LLL + OLLn OLL + LLM LLP OLO LOP + SLL PLP SLO + OOO SLP SOM
Fermentation time 0h
12 h
24 h
36 h
44 h
0.20 ± 0.24a 0.00 ± 0.00a 1.52 ± 0.88a 98.28 ± 1.12a 1.21 ± 0.41a 8.8 ± 0.55 28.63 ± 0.82 21.29 ± 0.04b 16.59 ± 0.44a 8.63 ± 1.12 7.47 ± 0.15 4.62 ± 0.62 1.81 ± 0.22 0.75 ± 0.03 0.21 ± 0.01
0.38 ± 0.37a 0.01 ± 0.00a 1.62 ± 1.31a 97.99 ± 1.68b 0.77 ± 0.17ab 8.52 ± 0.69 29.06 ± 0.79 21.33 ± 0.1b 16.8 ± 0.39a 8.6 ± 1.11 7.43 ± 0.33 4.57 ± 0.5 1.93 ± 0.09 0.77 ± 0.04 0.22 ± 0.03
0.80 ± 0.68a 0.20 ± 0.17a 1.60 ± 1.27a 97.39 ± 2.12b 0.89 ± 0.03ab 8.06 ± 0.73 29.45 ± 0.51 21.28 ± 0.68b 16.56 ± 0.13a 8.51 ± 0.6 7.34 ± 0.33 4.64 ± 0.62 1.94 ± 0.38 0.96 ± 0.33 0.37 ± 0.17
1.78 ± 0.88a 0.40 ± 0.16a 3.28 ± 0.84a 94.54 ± 1.88b 0.53 ± 0.07b 7.37 ± 0.53 30.46 ± 0.83 21.81 ± 0.59b 16.35 ± 0.08a 8.58 ± 0.85 7.51 ± 0.17 4.56 ± 0.61 1.81 ± 0.24 0.77 ± 0.21 0.25 ± 0.07
8.80 ± 0.73b 1.37 ± 0.56b 4.58 ± 0.72ab 85.24 ± 0.89b 0.48 ± 0.05b 7.31 ± 0.34 30.61 ± 0.99 23.37 ± 0.8a 15.4 ± 0.21b 8.52 ± 0.98 7.5 ± 0.16 4.32 ± 0.49 1.6 ± 0.27 0.71 ± 0.18 0.19 ± 0.11
Each value is expressed as relative wt% contents of lipids or triacylglycerol. Values with different letters (a, b) in the same row are significantly different (p < 0.05). M, myristic (C14:0); P, palmitic (C16:0); S, stearic (C18:0); O, oleic (C18:1); L, linoleic (C18:2) and Ln, linolenic (C18:3).
Table 3 Changes in fatty acid composition of total lipids (TLs) during koji fermentation. Fermentation time
C16:0 % C18:2 % C18:1 % C18:3 % C18:0 %
(mg/g dry matter) (mg/g dry matter) (mg/g dry matter) (mg/g dry matter) (mg/g dry matter)
0h
12 h
24 h
36 h
44 h
2.95 ± 0.29a 15.22 10.6 ± 1.12a 54.64 3.3 ± 0.49a 16.96 1.82 ± 0.04a 9.41 0.73 ± 0.09a 3.77
3.52 ± 0.45a 14.08 13.2 ± 1.67ab 52.85 4.39 ± 0.71ab 17.51 2.94 ± 0.41b 11.76 0.95 ± 0.14a 3.79
4.23 ± 0.43a 14.66 15.14 ± 1.67b 52.5 5.31 ± 0.37b 18.45 2.98 ± 0.21b 10.34 1.17 ± 0.16a 4.04
5.74 ± 0.25b 14.26 20.89 ± 0.56c 51.9 7.6 ± 0.54c 18.86 4.31 ± 0.36c 10.7 1.72 ± 0.12b 4.28
6.3 ± 0.88b 14.15 22.98 ± 1.76c 51.81 8.26 ± 1.14c 18.56 4.89 ± 0.61c 10.99 2 ± 0.31b 4.49
Values with different letters (a, b, c) in the same row are significantly different (p < 0.05).
Table 4 Changes in lipid composition (mg/g dry matter) of free fatty acids (FFAs), neutral lipids (NLs) and phospholipids (PLs) fractions during koji fermentation. Fermentation time 0h
12 h
24 h
36 h
44 h
FFAs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
0.49 ± 0.04a 0.62 ± 0.05a 0.22 ± 0.02a 0.08 ± 0.01a 0.5 ± 0.04a 0.94
1.51 ± 0.13b 3.79 ± 0.34b 1.21 ± 0.14b 0.57 ± 0.06b 0.81 ± 0.08b 2.40
2.66 ± 0.2c 7.52 ± 0.59c 2.49 ± 0.14c 1.27 ± 0.07c 1.05 ± 0.09b 3.04
3.75 ± 0.19d 11.06 ± 0.61d 3.77 ± 0.26d 2.36 ± 0.18d 1.62 ± 0.12c 3.20
4.17 ± 0.41d 12.85 ± 0.7e 4.58 ± 0.45e 2.62 ± 0.22d 1.71 ± 0.19c 3.41
NLs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
1.21 ± 0.08a 4.95 ± 0.37a 1.85 ± 0.2a 0.98 ± 0.03a 0.39 ± 0.02a 4.87
1.31 ± 0.13a 5.58 ± 0.51a 2.26 ± 0.26a 1.04 ± 0.1a 0.46 ± 0.05a 5.01
1.35 ± 0.11a 6.01 ± 0.47a 2.28 ± 0.14a 1.44 ± 0.09b 0.47 ± 0.06a 5.33
1.88 ± 0.12b 8.63 ± 0.52b 3.55 ± 0.18b 1.63 ± 0.14b 0.63 ± 0.04b 5.52
2.41 ± 0.24c 10.71 ± 0.78c 4.01 ± 0.39b 2.39 ± 0.21c 0.81 ± 0.09c 5.32
PLs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
0.59 ± 0.04d 1.86 ± 0.14d 0.35 ± 0.04b 0.29 ± 0.03b 0.17 ± 0.01a 3.30
0.24 ± 0.02a 0.52 ± 0.05a 0.14 ± 0.02a 0.1 ± 0.01a 0.14 ± 0.01a 2.00
0.2 ± 0.01a 0.43 ± 0.03a 0.15 ± 0.02a 0.08 ± 0.02a 0.15 ± 0.01a 1.90
0.37 ± 0.02b 1.11 ± 0.08b 0.34 ± 0.02b 0.24 ± 0.02b 0.18 ± 0.01a 3.09
0.45 ± 0.05c 1.44 ± 0.09c 0.46 ± 0.05c 0.27 ± 0.03b 0.23 ± 0.03b 3.19
Each value is expressed as mg/g dry matter of koji. Values in parentheses are relative wt% contents of individual lipids in total lipids. Values with different superscript letters in the same row are significantly different (p < 0.05). FFAs, free fatty acids; NLs, neutral lipids; PLs, phospholipids; UNSAT, unsaturated fatty acid; SFA, saturated fatty acid.
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
of culture time and the content of FFAs was positively related to the acid value (r = 0.97). Similar changes of FFAs were reported in kinema (Sarkar et al., 1996), tempeh (Wagenknecht et al., 1961) and natto (Kiuchi et al., 1976) production. While as to the PLs, the fatty acids decreased significantly (p < 0.05) first and then increased to a small extent. In the PLs, linoleic acid (C18:2) was the main fatty acid, followed by oleic acid (C18:1) and palmitic acid (C16:0). In the FFAs fraction, the ratio of unsaturated fatty acid to saturated fatty acid (UFA/SFA) was found notably enhanced from 0.94 to 3.41, and this trend mainly resulted from the increase of polyunsaturated fatty acids (PUFAs), especially linoleic acid (C18:2). Moreover, the decreased and increased tendencies of the linoleic acid (C18:2) proportion were observed in the PLs and NLs, respectively. This suggested that the FFAs might be mainly hydrolysed from PLs, caused by microbial lipases or phospholipases (Yong & Wood, 1977). In addition, at the late stage of the koji fermentation (36–44 h), the increasing rate of fatty acid contents in FFAs reduced, which implied a delicate equilibrium might exist between the formation and oxidation of the FFAs (Andres, Cava, Martin, Ventanas, & Ruiz, 2005). These results are supported by the report that a series of volatile compounds from lipid oxidation, containing aliphatic aldehydes, certain ketones and alcohols, increased quickly in the later part of koji fermentation (Feng et al., 2013). In this sense, the PLs fraction might play an important role in the development of the characteristic taste and flavour precursors in soy sauce fermentation (Gao, Zhao, Zhao, Cui, & Ren, 2009).
443
3.6. Sensory evaluation In this study, it was suggested that hydrolysis of PLs and NLs during koji fermentation could play an important role in taste perception and flavour development of soy sauce. Thus, supplementation of phospholipase or lipase in soy sauce fermentation might enhance the characteristic taste and flavour of soy sauce. As a result, sensory evaluation and GC–MS analysis were carried out to determine the effects of exogenous phospholipase or lipase on soy sauces. As shown in Fig. 2, sensory evaluation demonstrated that phospholipase or lipase supplementation significantly affected the flavour of soy sauce. Compared to the untreated soy sauce, in particular, the aroma notes of caramel-like and alcoholic were significantly decreased, whereas the malty, acids, beany notes were markedly higher in the sample with phospholipase and lipase supplementation (Fig. 2A). Moreover, the percentage of aldehydes, especially 2/3-methylbutanal (malty aroma), in test samples were significantly increased (p < 0.05) by 2-folds compared to the controls, whereas the alcohols and pyrazines (roasty and nutty) decreased remarkably (data not shown), which was in agreement with the aroma sensory evaluation. In addition, beany attribute was enhanced by lipase supplementation (Fig. 2A). Sugawara et al. (1985) identified that hexanal, 1-hexanol and 1-octen-3-ol, which might be liberated from autoxidation and enzymatic oxidation of unsaturated fatty acids, contributed to greeny and musty odours of soybeans. Thus, the phospholipase and lipase supplementation in soy sauce would have both positive and negative impacts on the aroma.
Fig. 2. Flavour and taste profile of soy sauces supplemented with lipase or phospholipase by sensory evaluation.
444
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
The values for the taste evaluation are shown in Fig. 2B, containing sour, sweet, salty, bitter, umami, kokumi and preference attributes. The effects on the taste of soy sauce were different between supplementation of phospholipase and lipase. The intensities of sour and bitter attributes were positively influenced by lipase addition in soy sauce, while phospholipase supplementation significantly enhanced (p < 0.05) the intensities of umami, kokumi and preference of soy sauce. Similar results have been found in dried seafood products by Azad Shah et al. (2009), who stated that addition of PUFAs could significantly enhance the intensities of thickness, mouthfulness and continuity. These results suggested that the metabolism of PLs had positive effects on the characteristic flavour of soy sauce. 4. Conclusions In conclusion, the present study has clearly demonstrated the evolution of fatty acid composition and lipid profile during koji fermentation. The changes in lipid composition suggested lipolysis of both TAGs and PLs during koji fermentation of soy sauce. LLnLn and LLP were lost significantly among the TAGs. The essential fatty acid linoleic acid (C18:2), which was the major fatty acid in NLs, FFAs and PLs content, was utilised fastest during fermentation. Preferential degradation of PLs to liberate FFAs was observed in this study and the sensory evaluation results of lipase supplementation also suggested that PLs could be the key source for the flavour development in soy sauce fermentation. DAGs, MAGs and FFAs released step by step during koji fermentation might become flavour precursors contributing to typical taste and aroma of soy sauce. On the basis of results obtained from this study, further work on understanding of the flavour formed from lipids metabolism is in progress to improve the flavour of soy sauce. Acknowledgements The authors gratefully acknowledge the National Science Technology Supporting Project for 12th Five-Year Plan (No. 2012 BAD34B03), the National High Technology Research and Development Program of China (863 Program) (No. 2012AA022502) and the Key Technology R&D Program of Zhongshan City (No. 2012 cxy004) for their financial support. References Andres, A. I., Cava, R., Martin, D., Ventanas, J., & Ruiz, J. (2005). Lipolysis in dry-cured ham: Influence of salt content and processing conditions. Food Chemistry, 90, 523–533. AOAC (2000). Official methods of analysis (17th ed.). Gaithersburg, Maryland: Association of Official Analytical Chemists. Azad Shah, A. K. M., Tokunaga, C., Kurihara, H., & Takahashi, K. (2009). Changes in lipids and their contribution to the taste of migaki-nishin (dried herring fillet) during drying. Food Chemistry, 115, 1011–1018. Chou, C., & Ling, M. (1998). Biochemical changes in soy sauce prepared with extruded and traditional raw materials. Food Research International, 31, 487–492. De Reu, J. C., Ramdaras, D., Rombouts, F. M., & Nout, M. (1994). Changes in soya bean lipids during tempe fermentation. Food Chemistry, 50, 171–175.
Fasciotti, M., & Pereira Netto, A. D. (2010). Optimization and application of methods of triacylglycerol evaluation for characterization of olive oil adulteration by soybean oil with HPLC–APCI-MS–MS. Talanta, 81, 1116–1125. Feng, Y., Cui, C., Zhao, H., Gao, X., Zhao, M., & Sun, W. (2013). Effect of koji fermentation on generation of volatile compounds in soy sauce production. International Journal of Food Science and Technology, 48, 609–619. Feng, Y. Z., Cai, Y., Su, G. W., Zhao, H. F., Wang, C. X., & Zhao, M. M. (2014). Evaluation of aroma differences between high-salt liquid-state fermentation and low-salt solid-state fermentation soy sauces from China. Food Chemistry, 145, 126–134. Gambacorta, G., Sinigaglia, M., Schena, A., Baiano, A., Lamacchia, C., Pati, S., et al. (2009). Changes in free fatty acid and diacylglycerol compounds in shortripening dry-cured sausage. Journal of Food Lipids, 16, 1–18. Gambotti, C. C., & Gandemer, G. (1999). Lipolysis and oxidation in subcutaneous adipose tissue during dry-cured ham processing. Food Chemistry, 64, 95–101. Gao, X., Cui, C., Ren, J., Zhao, H., Zhao, Q., & Zhao, M. (2011). Changes in the chemical composition of traditional Chinese-type soy sauce at different stages of manufacture and its relation to taste. International Journal of Food Science and Technology, 46, 243–249. Gao, X., Zhao, H., Zhao, M., Cui, C., & Ren, J. (2009). Comparative study on volatile flavor compounds of traditional Chinese-type soy sauces prepared with soybean and defatted soy meal. Food Science and Biotechnology, 18, 1447–1458. Jerkovic´, I., Mastelic´, J., & Tartaglia, S. (2007). A study of volatile flavor substances in Dalmatian traditional smoked ham: Impact of dry-curing and frying. Food Chemistry, 104, 1030–1039. Kiuchi, K., Ohta, T., Itoh, H., Takahayahsi, T., & Ebine, H. (1976). Studies on lipids of natto. Journal of Agriculture and Food Chemistry, 24, 404–407. Lertsiri, S., Maungma, R., Assavanig, A., & Bhumiratana, A. (2001). Roles of the Maillard reaction in browning during moromi process of Thai soy sauce. Journal of Food Processing Preservation, 25, 149–162. Lin, C., Wei, Y., & Chou, C. (2006). Enhanced antioxidative activity of soybean koji prepared with various filamentous fungi. Food Microbiology, 23, 628–633. Lioe, H. N., Apriyantono, A., Takara, K., Wada, K., Naoki, H., & Yasuda, M. (2004). Low molecular weight compounds responsible for savory taste of Indonesian soy sauce. Journal of Agricultural and Food Chemistry, 52, 5950–5956. Liu, N., Wang, Y., Zhao, Q., Cui, C., Fu, M., & Zhao, M. (2012). Immobilisation of lecitaseÒ ultra for production of diacylglycerols by glycerolysis of soybean oil. Food Chemistry, 134, 301–307. Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. Food Science and Technology, 5, 600–608. Paredes-López, O., González-Castañeda, J., & Cárabez-Trejo, A. (1991). Influence of solid substrate fermentation on the chemical composition of chickpea. Fermentation and Bioengineering, 71, 58–62. Regueiro, J. A., Gibert, J., & Diaz, I. (1994). Determination of neutral lipids from subcutaneous fat of cured ham by capillary gas chromatography and liquid chromatography. Journal of Chromatography A, 667, 225–233. Sarkar, P. K., Jones, L. J., Gore, W., Craven, G. S., & Somerset, S. M. (1996). Changes in soya bean lipid profiles during Kinema production. Journal of Food Science and Agriculture, 71, 321–328. Shibata, M., Takayama, K., Ujiie, A., Yamada, T., Abe, J., & Kitamura, K. (2008). Genetic relationship between lipid content and linolenic acid concentration in soybean seeds. Breeding Science, 58, 361–366. Shieh, Y. C., Beuchat, L. R., Worthington, R. E., & Phillips, R. D. (1982). Physical and chemical changes in fermented peanut and soybean pastes containing kojis prepared using Aspergillus oryzae and Phizopus oligosporus. Journal of Food Science, 47, 523–529. Steinhaus, P., & Schieberle, P. (2007). Characterization of the key aroma compounds in soy sauce using approaches of molecular sensory science. Journal of Agricultural and Food Chemistry, 55, 6262–6269. Visessanguan, W., Benjakul, S., Riebroy, S., Yarchai, M., & Tapingkae, W. (2004). Changes in lipid composition and fatty acid profile of Nham, a Thai fermented pork sausage, during fermentation. Food Chemistry, 94, 580–588. Wagenknecht, A. C., Mattick, L. R., Lewin, L. M., Hand, D. B., & Steinkraus, K. H. (1961). Changes in soybean lipids during tempeh fermentation. Journal of Food Science, 26, 373–376. Wang, H. L., Swain, E. W., Wallen, L. L., & Hesseltine, C. W. (1975). Free fatty acids identified as antitryptic factor in soybeans fermented by Rhizopus oligosporus. The Journal of Nutrition, 105, 1351–1355. Yong, F. M., & Wood, B. (1977). Biochemical changes in experimental soy sauce koji. International Journal of Food Science and Technology, 12, 163–175.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Changes in fatty acid composition and lipid profile during koji fermentation and their relationships with soy sauce flavour Yunzi Feng, Zhiyao Chen, Ning Liu, Haifeng Zhao ⇑, Chun Cui, Mouming Zhao ⇑ College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 7 November 2013 Received in revised form 13 February 2014 Accepted 25 February 2014 Available online 6 March 2014
Evolution of lipids during koji fermentation and the effect of lipase supplementation on the sensory properties of soy sauce were investigated. Results showed that total lipids of the koji samples were in the range of 16–21%. The extracted lipid of initial koji consisted mainly of triacylglycerols (TAGs, >98%), followed by phospholipids (PLs), diglycerides (DAGs), monoacylglycerols (MAGs) and free fatty acids (FFAs). As the fermentation proceeded, peroxide value of the lipids decreased while carbonyl value increased (p < 0.05). Linoleic acid was utilised fastest according to the fatty acid composition of total lipids, and preferential degradation of PLs to liberate FFAs was also observed. Moreover, phospholipase supplementation had significant influence on the sensory characteristics of soy sauce, especially enhanced (p < 0.05) scores for the umami and kokumi taste attributes. All these results indicated that the control of PLs utilisation during fermentation was a potential method to improve soy sauce’s characteristic taste. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Soy sauce Phospholipase Lipids Free fatty acid Oxidative degradation Flavour
1. Introduction Soy sauce is used as a condiment or seasoning sauce around the world due to its characteristic aroma accompanied by the intense umami taste. Chinese traditional soy sauce involves two important fermentation stages, koji fermentation and moromi fermentation (Gao et al., 2009). Previous studies showed that the evolution of volatile components largely depended on lipid oxidation in the koji fermentation stage (Feng et al., 2013). In addition, soy sauce aroma was significantly influenced by the content of lipids in the raw materials (Gao et al., 2009). Thus, influence of 20% fat in soybean, the main material, on the flavour of traditional Chinese-type soy sauce is worthy of investigation. Lipolysis, release of fatty acids, and secondary reactions of fatty acids resulted in the development of some volatile substances, such as aldehydes, ketones and alcohols, which are responsible for the characteristic aroma of fermented foods (Gambacorta et al., 2009; Visessanguan, Benjakul, Riebroy, Yarchai, & Tapingkae, 2004). It has been reported that lipolysis and lipid oxidation make significant contributions to the thickness, mouthfulness and continuity taste in migaki-nishin, a dried fish product (Azad Shah, Tokunaga, Kurihara, & Takahashi, 2009). Moreover, evolution of
⇑ Corresponding authors. Tel./fax: +86 20 22236089 (H. Zhao). Tel./fax: +86 20 87113914 (M. Zhao). E-mail addresses: (M. Zhao).
[email protected]
(H.
Zhao),
http://dx.doi.org/10.1016/j.foodchem.2014.02.147 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
[email protected]
the lipid in some fermented soya products has been proved to be the main factor in forming aroma, such as natto, Japanese miso and Indonesian tempe (De Reu, Ramdaras, Rombouts, & Nout, 1994; Kiuchi, Ohta, Itoh, Takahayahsi, & Ebine, 1976; Sarkar, Jones, Gore, Craven, & Somerset, 1996; Shieh, Beuchat, Worthington, & Phillips, 1982). Much literature has concentrated on degradation of proteins (Chou & Ling, 1998; Gao et al., 2011; Lertsiri, Maungma, Assavanig, & Bhumiratana, 2001), and umami peptides (Lioe et al., 2004) of soy sauce. However, little attention has been paid to the changes of lipid and its relationship with volatile compounds during the koji fermentation of soy sauce. Therefore, the aim of this study was to investigate the changes in lipid compositions through the koji fermentation, and to evaluate their potential effects on the characteristic flavour of soy sauce. Furthermore, the effects of adding exogenous lipase on the sensory quality of soy sauce were described, in order to explore the relationship between lipolytic and sensory parameters of soy sauce. Results from this study would make us better understand the relationships between lipid compositions in soybeans and the flavour of soy sauce.
2. Materials and methods 2.1. Materials and chemicals Soybean (Guanghui Agricultural Products Co., Ltd., Heilongjiang, China), wheat flour (Runfon Flour Co., Ltd., Guangdong, China) and
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
edible salt (Zhongshan Salt Industrial Co., Ltd., Zhongshan, Guangdong, China) were purchased from a local market. Aspergillus oryzae HN3.042 spores were obtained from Jiaming Fermentation Food Co., Ltd. (Shanghai, China). Phospholipase (LecitaseÒ ultra) and Lipozyme IM 4350 were purchased from Novozymes (Copenhagen, Denmark). Heptadecanoic acid, methyl ester was purchased from Sigma Co., Ltd. (St. Louis, MO). Chloroform, n-hexane and isopropanol were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Preparation of samples Two kilograms raw soybeans were soaked in 4000 mL water for 8 h at room temperature, and steamed at 125 °C for 15 min. Wheat flour and steamed soybean were mixed at a ratio of 1:4 (w/w) and cooled to 40 °C. The mixture was then inoculated with 0.05% (w/w) of A. oryzae HN 3.042 spores, and incubated at 30 °C for 16 h and 28 °C for another 32 h. The relative humidity was about 95% during koji fermentation. The preparation of koji was completed when the colour of the culture began turning a greenish yellow. Five soy sauce koji samples were periodically taken at 0, 12, 24, 36 and 44 h during the koji fermentation. All the samples were ground and held at 18 °C until analysis. A primary supplementation test was conducted to further clarify the effect of metabolism of lipids on the flavour of soy sauce. Lipase (IM4350, Novozymes, 0.05% koji weight) or phospholipase (LecitaseÒ ultra, 0.03% koji weight, maintaining the same enzymatic activity) was mixed with koji to yield moromi, and the main processes of soy sauce were made according to the method of Gao et al. (2011). The fermented soy sauce samples were taken on the 6th month. All samples were filtered through filter papers (18-cm diameter, Shuangquan Co. Ltd., Hangzhou, China), then stored in a refrigerator at 18 °C until analysis. 2.3. Chemical analysis Moisture and lipid contents of the koji were determined according to AOAC (2000). To determine pH value, 5 g of each sample were homogenised with 10-fold volume of distilled water and the pH was measured directly by using PHS-3E pH meter (TecFront Electronics Co. Ltd., Shanghai, China). 2.4. Lipid extraction Total lipids were extracted from the koji samples with a solvent mixture of chloroform:methanol (2:1, v/v) according to the method of Azad Shah et al. (2009). The extracts were dried under vacuum on a rotary evaporator and finished with a nitrogen flow. The lipid samples were stored under nitrogen gas in the dark at 18 °C until further analysis. 2.5. Measurement of lipid oxidation Acid value and peroxide value of the extracted lipid were determined according to AOAC (2000). Acid value was analysed by titration of approximately 0.5 g of lipid, dissolved in a mixture of 100 mL of ethanol and diethyl ether (1:1, v/v), with 0.01 N potassium hydroxide. Phenolphthalein was used as the indicator. The results of acid value and peroxide value were expressed as mg KOH/g lipid and meq/kg of lipid, respectively. The peroxide value was defined as the oxidised potassium iodide content, expressed as meq of hyperoxide per kg of lipid. According to the method of Gambotti and Gandemer (1999), carbonyl compounds were evaluated by the ratio of the absorbance at 275 nm to the absorbance at 215 nm from the lipid (125 lg/mL in cyclohexane solution). An increase
439
of the ratio A275/A215 is related to an increase in carbonyl compounds. 2.6. Fractionation of total lipids Neutral lipids (NLs), free fatty acids (FFAs) and phospholipids (PLs), were separated from the total lipids by using StrataÒNH2 cartridges containing 500 mg of amine-propylic resin (Phenomenex, Torrance, CA) as described by Regueiro, Gibert, and Diaz (1994). The cartridge was activated with 6 mL of chloroform before use. The extracted lipids (10–20 mg of total lipid) were redissolved in chloroform and then loaded on the top of the cartridges. The NLs, FFAs and PLs were eluted with 2.5 mL chloroform:isopropanol (2:1, w/w), 3 mL 2% (w/w) of acetic acid/ether, and 3 mL methanol in sequential order. 2.7. Fatty acid composition analysis Fatty acid methyl esters (FAME) were prepared from total lipids and the isolated fractions (NLs, FFAs and PLs) of soy sauce koji lipids according to the method of Morrison and Smith (1964). The contents of the fatty acid methyl ester in each fraction were quantified using heptadecanoic acid, methyl ester as internal standard. The GC–MS system consisted of a Trace Ultra GC, a Trisplus autosampler and a quadropole DSQ II MS (Thermo Finnigan, San Jose, CA). Separation was performed with a TR-5MS capillary column (30 m 0.2 mm, 0.25 lm, J&W Scientific, Folsom, CA). Helium was used as carrier gas with a flow rate of 1.0 mL/min. Sample volume of 1.0 lL was injected with a split ratio of 100:1. The analytical conditions were as follows: the temperature of the column was maintained at 40 °C for 2 min, ramped to 150 °C at 10 °C/ min, holding for 2 min, and then rose to 280 °C at a rate of 10 °C/ min and held at 280 °C for 5 min. The mass spectrometer was operated in electron-impact (EI) mode. The ionisation energy, detector voltage, scan range and scan rate applied for the analysis were 70 eV, 350 V, m/z 35–350 and 3.00 scans/s, respectively. Both injector and ion source temperature were 250 °C. FAME were identified by matching the retention times and mass spectra with those of reference standards in the standard NIST 08 library and standard FAME analysed under the same experimental conditions. 2.8. Analysis of lipids composition The compositions of triacylglycerols (TAGs) and lipids were determined by a reversed-phase high-performance liquid chromatography. The chromatographic apparatus consisted of a Waters P600 pump with a quaternary gradient system (Waters, Milford, MA), and a 3300 evaporative light-scattering detector (Alltech Associates Inc., Deerfield, IL) with an atmosphere compression pump (Tianjin, China). A PurospherÒ STAR RP-18e column (250 mm 4.6 mm i.d., particle size 5 lm, Merck, Darmstadt, Germany) was used. The temperature was 40 °C, and flow rate of gas and liquid phases were 1.5 L/min and 0.6 mL/min, respectively. The injection volume was 10 lL. The gradient elution was achieved by mobile phases A (acetonitrile:acetic acid = 99.95:0.05, v/v) and B (dichloromethane). The gradient was operated as follows: 0–4 min 100% A; 4–12 min 90% A; 12–15 min 70% A; 15–19 min 20% A; 19–31 min 80% A; 31–36 min 90% A; 36–39 min 100% A; 39–42 min 100% A. The compounds in the samples were identified by HPLC/MS (Bruker Daltonics Inc., Billerica, MA), and the conditions were the same as described by Liu et al. (2012). 2.9. Identification of volatile compounds in the soy sauce The sample preparation and SPME technique were used according to the methods of Feng et al. (2013). The SPME Trisplus auto-
440
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
mated sampler equipped with a 75 lm carboxen/polydimethylsiloxane fibre (CAR/PDMS, Supelco, Inc., Bellefonte, PA) was employed for the extraction of volatile compounds in soy sauces. Eight millilitres of soy sauce were transferred into 20-mL gas-tight glass vessels (Supelco) and saturated with 1 g NaCl. Prior to analysis, 20 lL of 2-methyl-3-heptanone (1.724 mg/L in methanol), as an internal standard, were added and mixed. After extraction for 40 min at 45 °C with continued heating and agitation extraction, the fibre was then inserted into the GC injector for 3 min to desorb analytes. Each soy sauce sample was extracted in triplicate. In all cases, the fibres were conditioned for 10 min at 260 °C between injections to prevent any contamination. Separation was performed with a TR-5MS column (30 m 0.25 mm 0.25 lm, J&W Scientific) and TR-Wax column (30 m 0.32 mm 0.25 lm, J&W Scientific). Helium was used as carrier gas with a flow rate of 1.0 mL/min. The split ratio was 20:1. The analytical conditions were as follows: the temperature of the column was maintained at 40 °C (2 min), ramped to 120 °C (2 min) at 5 °C/min, and then raised to 220 °C (5 min) at a rate of 7 °C/min. Injection temperature was 250 °C and the ion source temperature was set at 230 °C. The mass spectrometer was operated in electron-impact (EI) mode. The ionisation energy, detector voltage, scan range and scan rate applied for the analysis were 70 eV, 350 V, m/z 35–350 and 3.00 scans/s, respectively. Identification was based on the retention indices of reference standards and through matching the mass spectra provided in the standard NIST 08 library. 2.10. Sensory evaluation Sensory analysis was performed on the soy sauces supplemented with lipase or phospholipase. Quantitative descriptive analysis (QDA) was carried out with a trained analytical panel of 7 members, using a nine-point scale according to the modified methods of Gao et al. (2011) and Lioe et al. (2004). All samples were coded with random three-digit numbers, and served to the panellists in a randomized complete block design. The sensory evaluation was divided into two parts: taste and aroma analysis. The attributes that best expressed the soy sauce taste and aroma characteristics were selected by the panel. Taste sensory attributes were salty (12 mM NaCl), umami (4 mM monosodium glutamate), sweet (40 mM sucrose), sour (10 mM lactic acid), bitter (1.5 mM caffeine) and kokumi (thickness, mouthfulness and continuity) (Gao et al., 2011). For the training of kokumi intensity, the panel was asked to compare the model chicken broth and savoury matrix solutions containing sodium chloride and monosodium glutamate. The aroma sensory evaluation was based on the procedure suggested by Steinhaus and Schieberle (2007). Aroma attributes alcohols (ethanol), acids (acetic acid), malty (3-methylbutanal), caramel-like (HEMF) and beany (boiled soybean) were used to characterise the sensory properties of soy sauce samples. The training sessions were conducted according to our previous paper (Feng et al., 2014). Fifty millilitres of sample were put in a plastic cup marked with a three-digit number (100 mL) and covered with lids prior to serving for evaluation. Significant difference for each taste attribute was analysed by analysis of variance, and the results were plotted in a spider diagram. 2.11. Statistical analysis All tests were conducted in triplicate and the results were reported as means ± standard deviations. Analysis of variance and significant differences among means were tested by one-way ANOVA using SPSS 16.0 (SPSS Inc., Chicago, IL). Student–Newman–Keuls test was used for comparison of mean values among samples, and to identify significant differences (p < 0.05).
3. Results and discussion 3.1. Chemical composition The changes of chemical compositions of soy sauce koji during fermentation are shown in Table 1. The value of pH gradually decreased from 6.5 to 6.0 during 24 h of fermentation (p < 0.05), then increased to 6.3 at 44 h of koji fermentation. As a major constituent in the koji mixture, protein accounted for approximately 41.48%, followed by lipid (16.55% dry weight) and trace amounts of sugar and ash. As shown in Table 1, a continuous increase in crude lipids was observed during the whole fermentation. The percentage of crude lipids increased significantly (p < 0.05) from 16.55% to 20.22% after the koji fermentation. Comparable changes occurred during A. oryzae fermentation of a combination of peanuts and soya beans to produce miso-like products (Shieh et al., 1982), and Bacillus subtilis fermentation to produce kinema, a fermented soya bean food in Nepal (Sarkar et al., 1996). However, these increases were probably due to decreases in non-lipid materials, such as active assimilation of carbohydrates, rather than an actual increase in lipid during fermentation (Shieh et al., 1982). 3.2. Lipid oxidation Acid value, representing the content of FFAs, was used to assess the level of lipid rancidity in the koji samples (Fig. 1A). Acid value increased rapidly from 7.25 to 46.49 mg KOH/g oil during the whole koji fermentation, indicating lipolysis of soybean lipids into FFAs in this stage. A similar result was found in dry-cured sausages (Gambacorta et al., 2009), in which the production of FFAs was the major reason for the increase of acid value. However, the pH of koji samples showed an increase at the later stage of fermentation (as in Table 1) despite the relatively large amount of acid being liberated. Peroxide value measures the primary products formed from lipids in the initial stage of oxidation, which further decompose to volatile and non-volatile fatty acids, aldehydes, ketones, etc. (Jerkovic´, Mastelic´, & Tartaglia, 2007). As shown in Fig. 1B, peroxide value decreased significantly (p < 0.05) from 12.72 to 3.79 meq/kg lipid during 0–36 h of fermentation, then increased slightly to 4.59 meq/kg lipid till 44 h of fermentation. Reduction of peroxide value in the fermented chickpea was also noted by Paredes-López, González-Castañeda, and Cárabez-Trejo (1991) during solid substrate fermentation using Phyzopus olgosporus. It demonstrated that antioxidant could be used to prevent the formation of free radicals, resulting in reduced oxidation rates of soybean oil. Thus, the decrease of peroxide value in this study might be attributed to the generation of strong antioxidant activity during the koji making period, such as increased total phenolic contents (Lin, Wei, & Chou, 2006). The accumulation of secondary oxidation products was measured by determining the carbonyl compounds. The data from A275/A215 in koji lipids are shown in Fig. 1C. Significant (p < 0.05) increase in the value of A275/A215 was observed during 36–44 h of fermentation, indicating that the level of carbonyls in the koji lipids increased during koji fermentation. This finding was in agreement with our previous report on the changes of soy sauce koji flavour, in which we observed rapid increases of aldehydes and ketones compounds during the koji fermentation (Feng et al., 2013). 3.3. Changes in lipid composition during the koji fermentation Fig. 1E shows the profiles of different lipid classes in the koji samples. Predominant components were triacylglycerols (TAGs, area ratio 85.24–98.28%), followed by diacylglycerols (DAGs, area
441
Y. Feng et al. / Food Chemistry 158 (2014) 438–444 Table 1 pH and proximate composition of 5 koji samples at different fermentation times. Fermentation time (h)
pH
Moisture (g/100 g wet matter)
Lipid (g/100 g dry matter)
Protein (g/100 g dry matter)
0 12 24 36 44
6.5 ± 0.12a 6.3 ± 0.11b 6.0 ± 0.10c 6.1 ± 0.08bc 6.3 ± 0.14b
52.71 ± 1.25a 51.87 ± 1.86a 47.72 ± 1.91b 41.60 ± 1.70c 38.65 ± 1.66d
16.55 ± 0.86a 16.78 ± 0.47a 18.60 ± 1.02b 20.80 ± 0.52c 20.22 ± 0.30c
41.48 ± 0.47a 42.22 ± 0.31a 47.03 ± 1.34b 47.36 ± 0.75b 47.16 ± 0.14b
Values are means ± standard deviations of triplicate determinations and different letters (a, b, c) in the same column indicate significant differences (p < 0.05).
Changes in TAGs composition (expressed by percentage) during koji fermentation are shown in Table 2. Fifteen different molecular species were detected in the lipid extracted from koji samples. The three-letter designation does not suggest fatty acyl positional isomers in the TAG: M, myristic; P, palmitic; S, stearic; O, oleic; L, linoleic; and Ln, linolenic fatty acid moieties. The proportion of LLL + OLLn was the richest (28.63–30.61%), followed by OLL + LLM (21.29–23.37%) and LLP (15.40–16.80%), which was in agreement with the results reported by Fasciotti and Pereira Netto (2010). In lipids from koji samples, unsaturated fatty acid such as linoleic, linolenic and oleic acids were predominantly located in the 2-position of the TAG molecules, whilst saturated fatty acids, such as palmitic and stearic acids, primarily occupied the 1-position or 3-position. Preferential losses of LLnLn and LLP were observed during koji fermentation. Wagenknecht, Mattick, Lewin, Hand, and Steinkraus (1961) pointed out that the lipases from Aspergillus always selectively target unsaturated fatty acid at exterior glycerol carbons 1,3 in triacylglycerol. 3.4. Changes in fatty acid composition of total lipid
Fig. 1. Changes in acid value (A), peroxide value (B), and A275/A215 (C) of lipids during koji fermentation.
ratio 1.52–4.58%), monoacylglycerols (MAGs, area ratio 0.00–1.37%) and free fatty acids (FFAs, area ratio 0.20–8.80%). There were no significant (p > 0.05) changes in the four lipid classes until 24 h, which might be explained by the low level of lipolysis enzymes activity at this period. According to the study of Yong and Wood (1977), the maximum level of lipase activity occurred at about 30 h of fermentation. The FFAs, MAGs and DAGs increased after the koji fermentation, which should be caused by the hydrolysis of TAGs and phospholipids (Gambacorta et al., 2009).
Table 3 shows the fatty acid compositions of the total lipids (TLs) in the soy sauce koji lipids. The major fatty acids in the koji lipids were palmitic acid (C16:0), linoleic acid (C18:2), oleic acid (C18:1), linolenic acid (C18:3) and stearic acid (C18:0). The percentage of these fatty acids mainly reflected the characteristic fatty acid composition of soybean oil, which was similar to the results reported previously by Shibata et al. (2008). Linoleic acid (C18:2) accounted for the largest proportion (over 50%), while it showed an obvious decrease in its percentage in the koji lipid, from 54.64% to 51.81% of the total fatty acids in the koji samples. Moreover, it was stated in our previous report that most of C8 aliphatic compounds, which might be formed by the oxidation of linoleic or linolenic acids, increased rapidly in the later period of koji fermentation (Feng et al., 2013). These results differed from a previous report by Wagenknecht et al. (1961), in which the fatty acids liberated by the hydrolysis of soybean oil were not subsequently utilised. Interestingly, the contents of all fatty acids in total lipid showed increasing trends during koji fermentation. This might be due to the existence of lipoprotein complexes, which could be extracted simultaneously at the early stage of koji fermentation (Wang, Swain, Wallen, & Hesseltine, 1975). Indeed, Wang et al. (1975) studied the trypsin-inhibitory activity observed in cooked soybeans fermented by Rhizopus oligosporus, revealing that the lipoprotein could be hydrolysed to release fatty acid after fermentation. 3.5. Changes in fatty acid compositions of FFAs, NLs and PLs fractions Fatty acid compositions of the neutral lipids (NLs), phospholipids (PLs) and free fatty acids (FFAs) in the soy sauce koji lipids are demonstrated in Table 4. The contents of fatty acids in FFAs and NLs showed increases during koji fermentation. The fatty acids in NLs fraction showed a constant order (polyunsaturated FAs > monounsatured FAs > saturated FAs) during the different stages
442
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
Table 2 Changes in the composition of lipids and the major molecular species of triacylglycerol (TAG) during the koji fermentation. Compounds
FFA MAG DAG TAG LLnLn LLLn LLL + OLLn OLL + LLM LLP OLO LOP + SLL PLP SLO + OOO SLP SOM
Fermentation time 0h
12 h
24 h
36 h
44 h
0.20 ± 0.24a 0.00 ± 0.00a 1.52 ± 0.88a 98.28 ± 1.12a 1.21 ± 0.41a 8.8 ± 0.55 28.63 ± 0.82 21.29 ± 0.04b 16.59 ± 0.44a 8.63 ± 1.12 7.47 ± 0.15 4.62 ± 0.62 1.81 ± 0.22 0.75 ± 0.03 0.21 ± 0.01
0.38 ± 0.37a 0.01 ± 0.00a 1.62 ± 1.31a 97.99 ± 1.68b 0.77 ± 0.17ab 8.52 ± 0.69 29.06 ± 0.79 21.33 ± 0.1b 16.8 ± 0.39a 8.6 ± 1.11 7.43 ± 0.33 4.57 ± 0.5 1.93 ± 0.09 0.77 ± 0.04 0.22 ± 0.03
0.80 ± 0.68a 0.20 ± 0.17a 1.60 ± 1.27a 97.39 ± 2.12b 0.89 ± 0.03ab 8.06 ± 0.73 29.45 ± 0.51 21.28 ± 0.68b 16.56 ± 0.13a 8.51 ± 0.6 7.34 ± 0.33 4.64 ± 0.62 1.94 ± 0.38 0.96 ± 0.33 0.37 ± 0.17
1.78 ± 0.88a 0.40 ± 0.16a 3.28 ± 0.84a 94.54 ± 1.88b 0.53 ± 0.07b 7.37 ± 0.53 30.46 ± 0.83 21.81 ± 0.59b 16.35 ± 0.08a 8.58 ± 0.85 7.51 ± 0.17 4.56 ± 0.61 1.81 ± 0.24 0.77 ± 0.21 0.25 ± 0.07
8.80 ± 0.73b 1.37 ± 0.56b 4.58 ± 0.72ab 85.24 ± 0.89b 0.48 ± 0.05b 7.31 ± 0.34 30.61 ± 0.99 23.37 ± 0.8a 15.4 ± 0.21b 8.52 ± 0.98 7.5 ± 0.16 4.32 ± 0.49 1.6 ± 0.27 0.71 ± 0.18 0.19 ± 0.11
Each value is expressed as relative wt% contents of lipids or triacylglycerol. Values with different letters (a, b) in the same row are significantly different (p < 0.05). M, myristic (C14:0); P, palmitic (C16:0); S, stearic (C18:0); O, oleic (C18:1); L, linoleic (C18:2) and Ln, linolenic (C18:3).
Table 3 Changes in fatty acid composition of total lipids (TLs) during koji fermentation. Fermentation time
C16:0 % C18:2 % C18:1 % C18:3 % C18:0 %
(mg/g dry matter) (mg/g dry matter) (mg/g dry matter) (mg/g dry matter) (mg/g dry matter)
0h
12 h
24 h
36 h
44 h
2.95 ± 0.29a 15.22 10.6 ± 1.12a 54.64 3.3 ± 0.49a 16.96 1.82 ± 0.04a 9.41 0.73 ± 0.09a 3.77
3.52 ± 0.45a 14.08 13.2 ± 1.67ab 52.85 4.39 ± 0.71ab 17.51 2.94 ± 0.41b 11.76 0.95 ± 0.14a 3.79
4.23 ± 0.43a 14.66 15.14 ± 1.67b 52.5 5.31 ± 0.37b 18.45 2.98 ± 0.21b 10.34 1.17 ± 0.16a 4.04
5.74 ± 0.25b 14.26 20.89 ± 0.56c 51.9 7.6 ± 0.54c 18.86 4.31 ± 0.36c 10.7 1.72 ± 0.12b 4.28
6.3 ± 0.88b 14.15 22.98 ± 1.76c 51.81 8.26 ± 1.14c 18.56 4.89 ± 0.61c 10.99 2 ± 0.31b 4.49
Values with different letters (a, b, c) in the same row are significantly different (p < 0.05).
Table 4 Changes in lipid composition (mg/g dry matter) of free fatty acids (FFAs), neutral lipids (NLs) and phospholipids (PLs) fractions during koji fermentation. Fermentation time 0h
12 h
24 h
36 h
44 h
FFAs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
0.49 ± 0.04a 0.62 ± 0.05a 0.22 ± 0.02a 0.08 ± 0.01a 0.5 ± 0.04a 0.94
1.51 ± 0.13b 3.79 ± 0.34b 1.21 ± 0.14b 0.57 ± 0.06b 0.81 ± 0.08b 2.40
2.66 ± 0.2c 7.52 ± 0.59c 2.49 ± 0.14c 1.27 ± 0.07c 1.05 ± 0.09b 3.04
3.75 ± 0.19d 11.06 ± 0.61d 3.77 ± 0.26d 2.36 ± 0.18d 1.62 ± 0.12c 3.20
4.17 ± 0.41d 12.85 ± 0.7e 4.58 ± 0.45e 2.62 ± 0.22d 1.71 ± 0.19c 3.41
NLs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
1.21 ± 0.08a 4.95 ± 0.37a 1.85 ± 0.2a 0.98 ± 0.03a 0.39 ± 0.02a 4.87
1.31 ± 0.13a 5.58 ± 0.51a 2.26 ± 0.26a 1.04 ± 0.1a 0.46 ± 0.05a 5.01
1.35 ± 0.11a 6.01 ± 0.47a 2.28 ± 0.14a 1.44 ± 0.09b 0.47 ± 0.06a 5.33
1.88 ± 0.12b 8.63 ± 0.52b 3.55 ± 0.18b 1.63 ± 0.14b 0.63 ± 0.04b 5.52
2.41 ± 0.24c 10.71 ± 0.78c 4.01 ± 0.39b 2.39 ± 0.21c 0.81 ± 0.09c 5.32
PLs C16:0 C18:2 C18:1 C18:3 C18:0 P P UNSAT/ SFA
0.59 ± 0.04d 1.86 ± 0.14d 0.35 ± 0.04b 0.29 ± 0.03b 0.17 ± 0.01a 3.30
0.24 ± 0.02a 0.52 ± 0.05a 0.14 ± 0.02a 0.1 ± 0.01a 0.14 ± 0.01a 2.00
0.2 ± 0.01a 0.43 ± 0.03a 0.15 ± 0.02a 0.08 ± 0.02a 0.15 ± 0.01a 1.90
0.37 ± 0.02b 1.11 ± 0.08b 0.34 ± 0.02b 0.24 ± 0.02b 0.18 ± 0.01a 3.09
0.45 ± 0.05c 1.44 ± 0.09c 0.46 ± 0.05c 0.27 ± 0.03b 0.23 ± 0.03b 3.19
Each value is expressed as mg/g dry matter of koji. Values in parentheses are relative wt% contents of individual lipids in total lipids. Values with different superscript letters in the same row are significantly different (p < 0.05). FFAs, free fatty acids; NLs, neutral lipids; PLs, phospholipids; UNSAT, unsaturated fatty acid; SFA, saturated fatty acid.
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
of culture time and the content of FFAs was positively related to the acid value (r = 0.97). Similar changes of FFAs were reported in kinema (Sarkar et al., 1996), tempeh (Wagenknecht et al., 1961) and natto (Kiuchi et al., 1976) production. While as to the PLs, the fatty acids decreased significantly (p < 0.05) first and then increased to a small extent. In the PLs, linoleic acid (C18:2) was the main fatty acid, followed by oleic acid (C18:1) and palmitic acid (C16:0). In the FFAs fraction, the ratio of unsaturated fatty acid to saturated fatty acid (UFA/SFA) was found notably enhanced from 0.94 to 3.41, and this trend mainly resulted from the increase of polyunsaturated fatty acids (PUFAs), especially linoleic acid (C18:2). Moreover, the decreased and increased tendencies of the linoleic acid (C18:2) proportion were observed in the PLs and NLs, respectively. This suggested that the FFAs might be mainly hydrolysed from PLs, caused by microbial lipases or phospholipases (Yong & Wood, 1977). In addition, at the late stage of the koji fermentation (36–44 h), the increasing rate of fatty acid contents in FFAs reduced, which implied a delicate equilibrium might exist between the formation and oxidation of the FFAs (Andres, Cava, Martin, Ventanas, & Ruiz, 2005). These results are supported by the report that a series of volatile compounds from lipid oxidation, containing aliphatic aldehydes, certain ketones and alcohols, increased quickly in the later part of koji fermentation (Feng et al., 2013). In this sense, the PLs fraction might play an important role in the development of the characteristic taste and flavour precursors in soy sauce fermentation (Gao, Zhao, Zhao, Cui, & Ren, 2009).
443
3.6. Sensory evaluation In this study, it was suggested that hydrolysis of PLs and NLs during koji fermentation could play an important role in taste perception and flavour development of soy sauce. Thus, supplementation of phospholipase or lipase in soy sauce fermentation might enhance the characteristic taste and flavour of soy sauce. As a result, sensory evaluation and GC–MS analysis were carried out to determine the effects of exogenous phospholipase or lipase on soy sauces. As shown in Fig. 2, sensory evaluation demonstrated that phospholipase or lipase supplementation significantly affected the flavour of soy sauce. Compared to the untreated soy sauce, in particular, the aroma notes of caramel-like and alcoholic were significantly decreased, whereas the malty, acids, beany notes were markedly higher in the sample with phospholipase and lipase supplementation (Fig. 2A). Moreover, the percentage of aldehydes, especially 2/3-methylbutanal (malty aroma), in test samples were significantly increased (p < 0.05) by 2-folds compared to the controls, whereas the alcohols and pyrazines (roasty and nutty) decreased remarkably (data not shown), which was in agreement with the aroma sensory evaluation. In addition, beany attribute was enhanced by lipase supplementation (Fig. 2A). Sugawara et al. (1985) identified that hexanal, 1-hexanol and 1-octen-3-ol, which might be liberated from autoxidation and enzymatic oxidation of unsaturated fatty acids, contributed to greeny and musty odours of soybeans. Thus, the phospholipase and lipase supplementation in soy sauce would have both positive and negative impacts on the aroma.
Fig. 2. Flavour and taste profile of soy sauces supplemented with lipase or phospholipase by sensory evaluation.
444
Y. Feng et al. / Food Chemistry 158 (2014) 438–444
The values for the taste evaluation are shown in Fig. 2B, containing sour, sweet, salty, bitter, umami, kokumi and preference attributes. The effects on the taste of soy sauce were different between supplementation of phospholipase and lipase. The intensities of sour and bitter attributes were positively influenced by lipase addition in soy sauce, while phospholipase supplementation significantly enhanced (p < 0.05) the intensities of umami, kokumi and preference of soy sauce. Similar results have been found in dried seafood products by Azad Shah et al. (2009), who stated that addition of PUFAs could significantly enhance the intensities of thickness, mouthfulness and continuity. These results suggested that the metabolism of PLs had positive effects on the characteristic flavour of soy sauce. 4. Conclusions In conclusion, the present study has clearly demonstrated the evolution of fatty acid composition and lipid profile during koji fermentation. The changes in lipid composition suggested lipolysis of both TAGs and PLs during koji fermentation of soy sauce. LLnLn and LLP were lost significantly among the TAGs. The essential fatty acid linoleic acid (C18:2), which was the major fatty acid in NLs, FFAs and PLs content, was utilised fastest during fermentation. Preferential degradation of PLs to liberate FFAs was observed in this study and the sensory evaluation results of lipase supplementation also suggested that PLs could be the key source for the flavour development in soy sauce fermentation. DAGs, MAGs and FFAs released step by step during koji fermentation might become flavour precursors contributing to typical taste and aroma of soy sauce. On the basis of results obtained from this study, further work on understanding of the flavour formed from lipids metabolism is in progress to improve the flavour of soy sauce. Acknowledgements The authors gratefully acknowledge the National Science Technology Supporting Project for 12th Five-Year Plan (No. 2012 BAD34B03), the National High Technology Research and Development Program of China (863 Program) (No. 2012AA022502) and the Key Technology R&D Program of Zhongshan City (No. 2012 cxy004) for their financial support. References Andres, A. I., Cava, R., Martin, D., Ventanas, J., & Ruiz, J. (2005). Lipolysis in dry-cured ham: Influence of salt content and processing conditions. Food Chemistry, 90, 523–533. AOAC (2000). Official methods of analysis (17th ed.). Gaithersburg, Maryland: Association of Official Analytical Chemists. Azad Shah, A. K. M., Tokunaga, C., Kurihara, H., & Takahashi, K. (2009). Changes in lipids and their contribution to the taste of migaki-nishin (dried herring fillet) during drying. Food Chemistry, 115, 1011–1018. Chou, C., & Ling, M. (1998). Biochemical changes in soy sauce prepared with extruded and traditional raw materials. Food Research International, 31, 487–492. De Reu, J. C., Ramdaras, D., Rombouts, F. M., & Nout, M. (1994). Changes in soya bean lipids during tempe fermentation. Food Chemistry, 50, 171–175.
Fasciotti, M., & Pereira Netto, A. D. (2010). Optimization and application of methods of triacylglycerol evaluation for characterization of olive oil adulteration by soybean oil with HPLC–APCI-MS–MS. Talanta, 81, 1116–1125. Feng, Y., Cui, C., Zhao, H., Gao, X., Zhao, M., & Sun, W. (2013). Effect of koji fermentation on generation of volatile compounds in soy sauce production. International Journal of Food Science and Technology, 48, 609–619. Feng, Y. Z., Cai, Y., Su, G. W., Zhao, H. F., Wang, C. X., & Zhao, M. M. (2014). Evaluation of aroma differences between high-salt liquid-state fermentation and low-salt solid-state fermentation soy sauces from China. Food Chemistry, 145, 126–134. Gambacorta, G., Sinigaglia, M., Schena, A., Baiano, A., Lamacchia, C., Pati, S., et al. (2009). Changes in free fatty acid and diacylglycerol compounds in shortripening dry-cured sausage. Journal of Food Lipids, 16, 1–18. Gambotti, C. C., & Gandemer, G. (1999). Lipolysis and oxidation in subcutaneous adipose tissue during dry-cured ham processing. Food Chemistry, 64, 95–101. Gao, X., Cui, C., Ren, J., Zhao, H., Zhao, Q., & Zhao, M. (2011). Changes in the chemical composition of traditional Chinese-type soy sauce at different stages of manufacture and its relation to taste. International Journal of Food Science and Technology, 46, 243–249. Gao, X., Zhao, H., Zhao, M., Cui, C., & Ren, J. (2009). Comparative study on volatile flavor compounds of traditional Chinese-type soy sauces prepared with soybean and defatted soy meal. Food Science and Biotechnology, 18, 1447–1458. Jerkovic´, I., Mastelic´, J., & Tartaglia, S. (2007). A study of volatile flavor substances in Dalmatian traditional smoked ham: Impact of dry-curing and frying. Food Chemistry, 104, 1030–1039. Kiuchi, K., Ohta, T., Itoh, H., Takahayahsi, T., & Ebine, H. (1976). Studies on lipids of natto. Journal of Agriculture and Food Chemistry, 24, 404–407. Lertsiri, S., Maungma, R., Assavanig, A., & Bhumiratana, A. (2001). Roles of the Maillard reaction in browning during moromi process of Thai soy sauce. Journal of Food Processing Preservation, 25, 149–162. Lin, C., Wei, Y., & Chou, C. (2006). Enhanced antioxidative activity of soybean koji prepared with various filamentous fungi. Food Microbiology, 23, 628–633. Lioe, H. N., Apriyantono, A., Takara, K., Wada, K., Naoki, H., & Yasuda, M. (2004). Low molecular weight compounds responsible for savory taste of Indonesian soy sauce. Journal of Agricultural and Food Chemistry, 52, 5950–5956. Liu, N., Wang, Y., Zhao, Q., Cui, C., Fu, M., & Zhao, M. (2012). Immobilisation of lecitaseÒ ultra for production of diacylglycerols by glycerolysis of soybean oil. Food Chemistry, 134, 301–307. Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. Food Science and Technology, 5, 600–608. Paredes-López, O., González-Castañeda, J., & Cárabez-Trejo, A. (1991). Influence of solid substrate fermentation on the chemical composition of chickpea. Fermentation and Bioengineering, 71, 58–62. Regueiro, J. A., Gibert, J., & Diaz, I. (1994). Determination of neutral lipids from subcutaneous fat of cured ham by capillary gas chromatography and liquid chromatography. Journal of Chromatography A, 667, 225–233. Sarkar, P. K., Jones, L. J., Gore, W., Craven, G. S., & Somerset, S. M. (1996). Changes in soya bean lipid profiles during Kinema production. Journal of Food Science and Agriculture, 71, 321–328. Shibata, M., Takayama, K., Ujiie, A., Yamada, T., Abe, J., & Kitamura, K. (2008). Genetic relationship between lipid content and linolenic acid concentration in soybean seeds. Breeding Science, 58, 361–366. Shieh, Y. C., Beuchat, L. R., Worthington, R. E., & Phillips, R. D. (1982). Physical and chemical changes in fermented peanut and soybean pastes containing kojis prepared using Aspergillus oryzae and Phizopus oligosporus. Journal of Food Science, 47, 523–529. Steinhaus, P., & Schieberle, P. (2007). Characterization of the key aroma compounds in soy sauce using approaches of molecular sensory science. Journal of Agricultural and Food Chemistry, 55, 6262–6269. Visessanguan, W., Benjakul, S., Riebroy, S., Yarchai, M., & Tapingkae, W. (2004). Changes in lipid composition and fatty acid profile of Nham, a Thai fermented pork sausage, during fermentation. Food Chemistry, 94, 580–588. Wagenknecht, A. C., Mattick, L. R., Lewin, L. M., Hand, D. B., & Steinkraus, K. H. (1961). Changes in soybean lipids during tempeh fermentation. Journal of Food Science, 26, 373–376. Wang, H. L., Swain, E. W., Wallen, L. L., & Hesseltine, C. W. (1975). Free fatty acids identified as antitryptic factor in soybeans fermented by Rhizopus oligosporus. The Journal of Nutrition, 105, 1351–1355. Yong, F. M., & Wood, B. (1977). Biochemical changes in experimental soy sauce koji. International Journal of Food Science and Technology, 12, 163–175.
