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Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu
Food Control 30 (2013) 590e595
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Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu Xuefeng Zeng, Wenshui Xia*, Qixing Jiang, Fang Yang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2012 Received in revised form 16 July 2012 Accepted 24 July 2012
This paper reports the first study on the chemical and microbial properties of Suan yu, a sanitary Chinese traditional low-salt fermented whole fish product of high nutrition, unique flavor, and long-storage capability. Six brands of Suan yu from different locations in China were studied. Significant differences were observed in some characteristics of this traditional fermented fish, raw material and other spontaneously fermented fish. The values of Enterobacteria and Pseudomonads were under the detection limits in six different brands. In addition, the products contained higher protein and lower moisture ranging from 17.2% to 22.9% and 52.9%e58.1%, respectively. The pH values ranged between 4.27 and 5.18, and lactic acid was the major organic acid. The proteolysis of the myosin heavy chain and actin chain were observed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Moreover, low concentrations of biogenic amine (BA) and thiobarturic acid (TBARS) values were found in all samples. The characteristics of the six Suan yu brands were all influenced by raw materials, ingredients, and fermentation processes and conditions. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Suan yu Fermented fish Composition Protein Biogenic amines
1. Introduction Low-salt fermented whole fish or fish pieces products, such as Plaa-som (Riebroy, Benjakul, Visessanguan, Kijrongrojana, & Tanaka, 2004) and Enam NeeSetaakye (Asiedu & Sanni, 2002), are mainly prepared and consumed in Southeast Asia and subSaharan Africa. These products are highly nutritious and of relatively firm and springy texture, providing an ideal and cheap protein source. However, the relatively short shelf life (four to five days) under ambient conditions and the strong fishy odor of these traditional fermented fish pose major limitations with respect to marketing (Valyasevi & Rolle, 2002). The high amounts of biogenic amine (BA) (mainly histamine) and pathogenic bacteria in these foods also increase concern on food safety. Suan yu is also a Chinese low-salt fermented whole fish snack with a characteristic flavor. It is stable during storage and free of fishy odor and taste and retains all the nutritional advantages of fish compared with other traditional fermented fish products. It is manufactured using traditional technologies without the addition of starter cultures in small-scale processing units. Fish used are mainly freshwater fish mixed with cooked carbohydrate, salt, and
* Corresponding author. Tel./fax: þ86 510 85919121. E-mail address: [email protected] (W. Xia). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.07.037
spice. Long-term spontaneous fermentation is conducted to develop flavor in anaerobic conditions. Studies have shown that the spontaneous fermentation of meat is characterized by the participation of lactic acid bacteria (LAB), Gram-positive and catalase-positive cocci, yeasts, and molds (Buckenhüskes, 1993). Among them, LAB is mainly responsible for acidification, ensuring the safety of products by reducing the pH level through sugar fermentation (Lücke, 2000). Coagulasenegative staphylococci and yeast participate in the development of aroma, flavor, and color of fermented products, such as cassava fish (Anihouvi, Sakyi-Dawson, Ayernor, & Hounhouigan, 2007) and Plaa-som (Riebroy et al., 2004). Proteolysis is an important biochemical change during the ripening of fermented meats. This process influences both texture and flavor development through the formation of several low-molecular weight compounds, mainly peptides, amino acids, aldehydes, organic acids, and amines, which are known as important flavor compounds or precursors of flavor compounds (Roseiro et al., 2008). Besides, the occurrence of high amounts of BA in these products is of great concern in food safety issues. When high amounts of BA are consumed or normal pathways of amine catabolism are inhibited, various physiological effects, namely, hypotension (in the case of histamine, putrescine, cadaverine) or hypertension (in the case of tyramine), nausea, headache, rash, dizziness, cardiac palpitation, and even intracerebral hemorrhage and death, may occur in very severe cases (Rawles,
X. Zeng et al. / Food Control 30 (2013) 590e595
Flick, & Martin, 1996). Thus, these complex chemical and biochemical reactions probably contributed to the unique flavor and safety properties of Suan yu. However, the characteristics of this product have not been widely studied. Therefore, the aim of the present study was to investigate the chemical and microbial properties of Suan yu. 2. Materials and methods 2.1. Sampling Six types of high-quality commercial Suan yu produced through traditional fermentation technology were purchased from handicraft shops in four Chinese counties, namely, Fenghuang, Jishou, Huayuan, and Baojin. It is traditionally prepared from freshwater fish, thawed in running tap water and then eviscerated, scale-removed and gills-removed. Fish, salt and various spices were added according to the consumer preferences. After being mixed thoroughly, the mixtures were cured at ambient temperature for 12 h. Next, they were partially dried in the sun or oasthouse. Then they were manually mixed with ground roasted carbohydrates, such as cornmeal or glutinous rice flour (COFCO, Shanghai, China), and some of the roasted carbohydrates were stuffed into the fish bellies. The mixtures were placed in smallsized Pickle Pot with lids and then sealed tightly by surrounded water or wet mud. The products were fermented at different seasons until the desirable taste and aroma were produced. The special manufacturing conditions of the six different Suan yu are illustrated in Table 1. Prior to analyses, the bone and cornmeal or glutinous rice flour in Suan yu were removed using a sterilized knife. Samples were cut and ground in a meat grinder (MX-T2G National, Tokyo, Japan) for 3 min and kept at 4 C for further analysis.
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Manitol Salt Agar (MSA) plates incubated at 37 C for 48 h. The Enterobacteriaceae count was determined using violet red bile glucose agar (VBGA) was incubated at 37 C for 24 h. The Pseudomonas count was determined using a pseudomonas-aeromonasselective (GSP) agar base incubated at 26 C for 72 h. Yeast and mold were grown on PDA (nature pH) containing 0.01% (w/v) chloramphenicol (selective supplement, Oxoid) at 25 C for 3e4 days, and single colonies and ivory strains were enumerated and identified as yeast. The results are reported as colony-forming units per gram (cfu/g). 2.3. Proximate chemical analyses Protein content was analyzed using the Kjeldahl Method AOAC (1997). A factor 6.25 was used for conversion content of nitrogen to crude protein. Crude fat content was measured through an extraction technique using petroleum ether AOAC (1997). Ash content was determined from the weight of the sample burned at 550 C for 4 h. Moisture content was calculated from the weight loss of the sample (5 g) after being oven-dried at 105 C for 2 h until constant weight. 2.4. Determination of salt content Salt content was determined according to the AOAC (1997) procedure. Accurately weighed 1 g of the sample was treated with 10 ml of 0.1 M AgNO3 and 10 ml of HNO3. The mixture was heated gently on an induction-cooker for 10 min. Then the mixture was cooled with running water, to which were added distilled water (50 ml) and ferric alum indicator (5 ml). The mixture was titrated with standard 0.1 M KSCN until the solution became permanent brownish-red. The results were calculated and expressed as % NaCl.
2.2. Microbiological analysis
2.5. Determination of pH, total acidity, and Aw
From each Suan yu, samples (25 g) were taken aseptically, transferred to sterile plastic pouches, and homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) for 120 s with 225 ml of 0.1% peptone water. Appropriate decimal dilutions of the samples were made using the same diluents, and 0.1 ml of each dilution was inoculated in duplicate in different growth media to estimate microbial counts. LAB was grown anaerobically on DeMan Rogosa Sharpe (MRS) agar at 30 C for two to three days. The Micrococcaceae count was determined using
The pH levels of the samples were determined according to the procedure proposed by Wang (2000). Ten-gram samples were homogenized with 90 ml of deionized water at 12,000 rpm for 1 min. The pH levels were then measured using a digital pH meter (Mettler Toledo 320-s, Shanghai, China). Titrable acidity (TA) was determined via AOAC (1997), and the results were expressed as lactic acid percentage. Water activity (Aw) was measured using a digital water activity meter (Rotronic Hygroskop DT, Zurich, Switzerland) after equilibrium at 25 C.
Table 1 Product manufacture conditions of the six different types of Suan yu. Manufacture condition
Sample A
B
C
D
E
F
Origin of product Ingredientsa Fish Roasted cornmeal Glutinous rice flour NaCl Sucrose Glucose Spiceb Fermentation container Processing time Ripening temperature Ripening time (days)
Fenghuang
Fenghuang
Jishou
Baojin
Jishou
Huayuan
70% Common Carp 22% 0 3% 2% 0 3% Spices A Ceramic pots Summer 30 C 55
75% Grass Carp 18% 0 3% 2% 0 2% Spices A Glass pot Autumn 20 C 46
72% Common Carp 22% 0 3% 0 1% 2% Spices A Ceramic pots Autumn 20 C 54
75% Grass Carp 15% 0 6% 2% 0 2% Spices A Glass pot Autumn 20 C 62
70% Common Carp 23% 0 3% 2% 0 2% Spices B Ceramic pots Autumn 20 C 44
75%Common Carp 0 18% 3% 2% 0 2% Spices A Ceramic pots Winter 10 C 394
a
The weight percentages of ingredients for fermented fish was expressed as w % w (wet weight). 2% or 3% Spices A: 1e2% chili powder, 0.1e0.3% cumin, 0.1e0.3% cinnamon, 0.1e0.3% pepper; 2% Spices B: 1%e2% chili powder, 0.1e0.3% cumin, 0.1e0.3% cinnamon, 0.1e0.3% pepper, 0.1e0.3% bay leaves. The content of species A or B for fermented fish was controlled by the local acceptance, without any strict regulation. b
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2.6. Determination of organic acids
Shanghai, China). The results were expressed as mg malonaldehyde/kg sample.
Organic acids were extracted using the method proposed by Riebroy et al. (2004). The samples (15 g) were homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) with 35 ml of distilled water at 8000 rpm for 1 min and centrifuged (Sigma Laborzentrifugen, Model 4K15, Osterode, Germany) at 4500 g for 15 min. The supernatant (0.9 ml) was mixed with 0.1 ml of 5% (w/v) butyric acid as an internal standard. The mixture was added with 0.5 M of perchloric acid and allowed to stand for 5 min. Then, it was centrifuged at 12,000 g for 30 min at room temperature to remove precipitated proteins. The supernatant was filtered through a 0.45 mm membrane (Minisart RC-15, Satorious, Goettingen, Germany) prior to high-performance liquid chromatography (HPLC) analysis (Agilent1100, water, USA) with an Ecomsil C18 (4.6 250 mm). A gradient elution program was employed, and a mixture of methanol:water:phosphoric acid (5:95:0.05 v/v) was used as the mobile phase. Column temperature and flow rate were set at 30 C and 0.8 ml/min, respectively. Twenty microliters of the mobile phase was injected into the HPLC, and a photo diode array (Model Waters 996) set at 210 nm wavelength was used as the detector. The data were processed and analyzed using Agilent Chemstations. The different organic acids were identified by comparing their retention times with those of standard solutions (Sigma). Organic acid concentration was expressed as the percentage of each organic acid in the samples (w/w). 2.7. Determination of TCA-soluble peptides TCA-soluble peptides were determined according to the methods described by Visessanguan, Benjakul, Riebroy, and Thepkasikul (2004). Samples (3 g) were homogenized with 27 ml of 5% TCA (w/v) and kept at 4 C for 1 h and then centrifuged at 12,000 g for 5 min at 4 C (Sigma Laborzentrifugen, Model 4K15, Osterode, Germany). TCA-soluble peptides in the supernatant were determined via the method proposed by Lowry, Rosebrough, Farr, and Randall (1951). Bovine serum albumin was used as standard. The TCA-soluble peptides in the samples were expressed as mg tyrosine/kg sample. 2.8. Determination of thiobarbituric acid reactive substances Thiobarbituric acid (TBARS) was determined according to the method described by Buege and Aust (1978). Samples (5 g) were homogenized in 25 ml of TBARS solution (0.375% TBA, 15% TCA, and 0.25 M HCl), and the mixture was heated for 10 min in boiling water (95e100 C) to obtain a pink color. After being cooled with running water, the mixture was centrifuged at 5500 g for 25 min. Finally, the absorbance of the supernatant was determined at 532 nm using a spectrophotometer (Model 135 WFZ-UV-2100, UNICOTM,
2.9. SDS-PAGE Samples (3 g) were homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) with 27 ml of solubilizing agent (TriseHCl 8.0 buffer, 2% SDS, 8 M urea, and 2% b-mercaptoethanol). The homogenate was heated at 85 C for 60 min and centrifuged at 10,000 g for 15 min at room temperature. Protein concentration was determined using the method proposed by Lowry et al. (1951). The supernatant was mixed (1:1, v/v) with the SDS-PAGE sample buffer (0.125 M TriseHCl, pH 6.8, 4% SDS, 10% glycerol, and 0.005% bromophenol blue). According to the method of Laemmli (1970), SDS-PAGE was performed in a vertical gel electrophoresis unit (Mini-Protean-3 Cell, Bio-Rad, Richmond, CA, USA). A 10% separating gel with 4% stacking gel was used, and aliquots of 10 ml were injected in each well. Electrophoresis was performed at 100e120 V. After electrophoresis, the gels were stained with Comassie Brilliant Blue R-250 (0.125%) in 25% methanol and 10% acetic acid. The gels were destained using 40% methanol and 10% acetic acid. 2.10. Determination of BA BA was extracted according to a slightly modified version of the procedure proposed by Ben-Gigirey, de Sousa, Villa, and BarrosVelazquez (1999). Two-gram samples with 10 ml of 0.4 M perchloric acid were homogenized at 3000 rpm using an Ultra Turrax homogenizer (IKA Labortechnik, Selangor, Malaysia) for 10 min. The homogenate was centrifuged at 12,000 g for 10 min at 4 C using a Sorvall Model RC-5B Plus centrifuge (Newtown, CT, USA). Extraction was performed twice. The supernatants were combined with 0.4 M perchloric acid to reach 25 ml. BA was derived according to the method proposed by Ben-Giglrey, Vieites Baptista de Sousa, and Villa (1998). A 1-ml extract or a standard solution was mixed with 200 ml of 2 M sodium hydroxide and 300 ml of saturated sodium bicarbonate. Two milliliters of dansyl chloride (10 mg/ml) was mixed with each sample. The mixture was incubated for 45 min at 40 C. Residual dansyl chloride was removed by adding 100 ml of 25% ammonium hydroxide. After 30 min at room temperature, the mixture was complemented to 5 ml with acetonitrile. Finally, the supernatant was filtered (0.45 mm Millipore membrane), and 5 ml of filtrate was injected into the HPLC (Agilentll00, USA). Liquid chromatographic separations were performed using Zorbax XDB C18 (4.6 250 mm). A gradient-elution program was used with the mobile phase containing 50% acetonitrile as solvent A and 90% acetonitrile as solvent B at a flow rate of 1.0 ml/min and column temperature of 30 C. Fluorescence was detected at an emission wavelength of 515 nm using an excitation wavelength of 340 nm.
Table 2 Microbiological properties in Suan yu. Sample
Lactic acid bacteria (log cfu/g)
Common Carp Grass Carp A B C D E F
3.51 3.73 7.36 7.85 7.62 5.53 7.81 7.13
0.16a 0.18a 0.26cd 0.38d 0.42cd 0.27b 0.38d 0.22c
Micrococcaceae (log cfu/g) 4.34 4.23 4.13 4.53 4.17 5.25 4.11 4.57
0.22a 0.19a 0.11a 0.23a 0.21a 0.53b 0.24a 0.32a
Yeast (log cfu/g) 2.47 2.68 6.36 6.72 6.47 5.13 6.14 6.48
0.11a 0.12a 0.31c 0.43c 0.47c 0.36b 0.35c 0.52c
Enterobacteria (log cfu/g)
Pseudomonads (log cfu/g)
3.37 0.15b 3.29 0.16b <1a <1a <1a <1a <1a <1a
3.62 0.18b 3.51 0.21b <1a <1a <1a <1a <1a <1a
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test. <1: Under the detection limit.
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Table 3 Chemical composition (%) of Suan yu. Sample
Parameter Protein (%)
Common Carp Grass Carp A B C D E F
17.3 16.3 22.8 20.5 22.9 20.6 20.3 17.2
0.43b 0.47a 0.58d 0.40c 0.62d 0.67c 0.45c 0.41b
Lipid (%) 1.58 4.46 1.13 0.97 1.08 1.48 0.91 0.88
0.07d 0.21e 0.03c 0.01ab 0.03bc 0.04d 0.02a 0.03a
Moisture (%) 79.4 78.6 52.9 56.8 55.8 53.9 58.1 57.4
2.04d 1.79d 0.82a 0.76bc 0.85b 0.75a 0.92c 0.81bc
Ash (%) 3.07 1.06 4.71 6.83 5.22 6.78 4.18 8.18
0.11b 0.04a 0.22d 0.33f 0.20e 0.33f 0.22c 0.3d
Salt (%) 0.14 0.13 3.03 2.85 3.17 6.86 3.24 2.94
pH
0.01a 0.01a 0.13bc 0.15b 0.16c 0.31d 0.18c 0.12bc
6.58 6.79 4.51 4.41 4.28 5.18 4.27 4.40
TA (%)
0.27c 0.31c 0.17a 0.12a 0.13a 0.29b 0.13a 0.22a
0.41 0.38 2.24 1.4 2.48 1.29 2.48 2.40
0.02a 0.02a 0.13c 0.19b 0.16c 0.11b 0.22c 0.14c
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test.
2.11. Statistical analysis The data obtained were subjected to the analysis of variance (ANOVA) and mean differences were evaluated by the least square difference (LSD) test (P < 0.05). Statistical analysis was performed using the SPSS statistic program (Version 13.0) for Window (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Microbiological analysis LAB, CNS, and yeast are actively involved in the development of safety, texture, color, and flavor (Ostergaard & Ben, 1998). Microbial properties of the raw materials (Common Carp and Grass Carp) and Suan yu are presented in Table 2. A large number of LAB, staphylococcus, and yeast were found in the spontaneously fermented fish products compared to the raw material (P < 0.05). Besides, the LAB cell numbers in brand D were lower (P < 0.05) than the other fermented samples, which contained similar counts. According to Achinewhu and Oboh (2002), salt concentration is likely to have a pronounced influence on the microbial growth and rate of fermentation. Therefore, in the present work, the growth of LAB was inhibited by the high salt concentrations in brand D. The result of this study is similar to those reported on other fermented fish products by Anihouvi et al. (2007) and Ostergaard and Ben (1998). Enterobacteria and Pseudomonads were under the detection limit in the fermented samples. This test met the guidelines for the microbiological quality of fermented meats published by Gilbert et al. (2000), indicating that these products are of good hygienic quality. The growth inhibition effects of Enterobacteriaceae and Pseudomonas on Suan yu might be attributed to the actions of bacteriocins and adequate acidification. 3.2. Chemical composition of Suan yu The chemical composition of the Suan yu and the raw material is shown in Table 3. Significant differences were found between Suan yu and raw material (P < 0.05). In addition, samples have typically higher protein concentrations than Thai Som-fug (11.4e16.2%) and Ghanaian Enam NeeSetaakye (15.6e15.8%), indicating that Suan yu is nutrient-rich because of the high protein and low fat content. The high protein content in the samples was most likely ascribed to the exclusion of carbohydrate and bones during Suan yu preparation. Furthermore, higher protein content probably affected by the different species, sizes, originations, and removal extent of water prior to processing (Casiraghi, Pompei, Dellaglio, Parolari, & Virgili, 1996; Comi et al., 2005). Moisture content is one of the main preservation factors in fermented fish products. High moisture content can greatly reduce shelf life. In this study, lower moisture
content was detected in Suan yu compared with raw material, Thai Som-fug and Ghanaian Enam NeeSetaakye, which contained 79.4e78.6%, 69.66e77.08% and 72.2e77.8% moisture, respectively. The low levels of the moisture in the samples may have resulted from the drying and curing processes prior to fermentation. The salt content of Suan yu is similar to Plaa-som, a traditional fermented fish product with 2.5e5.5% NaCl (Phithakpol, 1995). The different salt concentrations in different fermented fish have a pronounced influence on the microbial growth and the rate of fermentation and, thereby, on the sensory quality and safety of the product (Kose & Hall, 2010). Low levels of ash content was found in the raw material, which indicated that the variation in ash content possibly resulted from the different fermentation times of the samples and the salt and other additives Visessanguan et al. (2004). pH value,TA, Aw, and organic acid. Significantly different pH and TA values were observed between Suan yu and the raw material (P < 0.05). The pH values were between 4.27 and 5.18, the TA varied from 1.29% to 2.48% in the Suan yu during the long fermentation process, whereas the pH and TA values were in the ranges of 6.58e6.72 and 0.27e0.32 respectively in the raw material. The highly significant negative correlation (r ¼ 0.882 to 0.976, P < 0.01) between pH and TA values shows that the increase in TA is generally accompanied by a decrease in pH. Generally, pH values should be set below 4.6 to inhibit pathogenic and spoilage bacteria. This value is considered safe for consumption. However, the pH of brand D with high-salt content did not decrease to lower than 4.6, indicating the delayed LAB growth. The content of organic acids is also an important factor ensuring product safety. The levels of lactic acid and acetic acid were under detection limits in the raw material. Nevertheless, lactic acid was a dominant organic acid in Suan yu, and acetic acid was also found in all Suan yu samples (data not shown). The levels of lactic acid ranged from 1.89% to 2.99% in brands A, B, C, E, and F. Moreover, 1.00% lactic acid was observed in brand D because of the high salt content, which inhibited lactic acid growth. Butyric, Table 4 TCA-soluble peptides and TBARS in Suan yu. Sample
TCA-soluble peptides (mg tyrosine/kg)
Common Carp Grass Carp A B C D E F
67.2 63.4 792 884 926 658 895 973
2.13a 3.17a 29.3c 31.6d 43.4de 26.4b 41.7de 48.2e
TBARS (mg malonaldehyde/kg) 0.16 0.17 2.49 3.04 2.79 3.63 2.71 8.35
0.01a 0.01a 0.12b 0.15d 0.11c 0.14e 0.08c 0.21f
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test.
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Fig. 1. SDS-PAGE pattern of muscle proteins in Suan yu: S, molecular weight standard; MC, Common Carp; MG, Grass Carp; MHC, myosin heavy chain; AeF brands of Suan yu.
succinic, or other acids were under the detection limit in the samples. The same is reported by Riebroy et al. (2004), indicating that lactic acid contents are in the range of 1.21e2.17% in Som-fug. In previous studies, 2.2e2.5% lactic acid in fermented meat produced the highest sensory score (Ostergaard & Ben, 1998). Among all tested brands, the Suan yu samples containing high concentrations of lactic acid and acetic acids generally showed higher sensory acceptance for consumers. Furthermore, the samples contained 0.88e0.92% of Aw, showing that microbial growth was inhibited and product safety was improved because of lower water activity.
among samples were associated with the differences in initial raw mixed ingredients and proteolysis induced by acidic condition (Visessanguan et al., 2004). The initial hydrolysis of muscle proteins is attributed mainly to endogenous cathepsin and followed by the action of microbial peptidases, which further degrades protein fragments into small peptides and free amino acids (Molly et al., 1997). Greater degradation of muscle proteins contributes to the development of the flavor and aroma of the fermented products. This result is similar to that of Valyasevi and Rolle (2002), who reported that strong protease activity, produced by staphylococci and bacilli, was responsible for the breakdown of fish proteins into peptides and free amino acids, which contribute to the unique flavor of the products. The electrophoretic patterns of muscle proteins in the six brands and raw material are shown in Fig. 1. As presented in Fig. 1, the amounts of protein hydrolysis in Suan yu and the raw material are compared, the muscle proteins (200, 116, 97.2, 66.4, and 44.3 kDa) markedly disappear or decrease. The bands at 29.2 and 20.1 kDa were more intense after a lengthy fermentation. The low intensity of myosin heavy chain and actin chain bands observed in all the samples indicates that the degradation of proteins took place as ripening progressed. The bands 29.2 and 20.1 kDa were more intense because of the possible co-migration of degradation products from larger proteins. Similar findings have been reported by García de Fernando and Fox (1991) and Verplaetse, Demeyer, Gerard, and Buys (1992). These phenomena of proteolysis in Suan yu was probably influenced by factors, such as product formulation, indigenous microorganism, and processing condition because of the action of proteinases from staphylococcus or LAB as well as the hydrolysis of muscle proteins caused by acids (Astiasarán, Villanueva, & Bello, 1990).
3.3. Lipid oxidation 3.5. Biogenic amines of Suan yu TBARS has been widely used to indicate lipid oxidation, which results in rancid smell and taste as well as alterations in texture, color and nutrition in meat and meat products (Jo & Ahn, 2000). The TBARS values of Suan yu ranged from 2.49 to 8.36 mg/kg, indicating the different extents of lipid oxidation (Table 4). The results are consistent with those of Riebroy et al. (2004) concerning the lipid oxidation in Som-fug. It has been previously reported that LAB exhibited antioxidant effects on unsaturated fatty acids (Wang, 2000). The generated LAB lowered the TBARS values in the samples. 3.4. Protein degradation of Suan yu The levels of TCA-soluble peptide ranged from 63.4 to 67.2 mg tyrosine/kg in the raw material. However, the TCA-soluble peptide content varied from 658 to 973 mg tyrosine/kg in Suan yu (Table 4), which indicates muscle proteins were severely degraded during fermentation. The differences in TCA-soluble peptides observed
The contents of BA in Suan yu and the raw material are shown in Table 5. Different concentrations of BA were detected in all samples. Among all samples, the levels of tyramine, histamine, cadaverin, and putrescine were higher in the six brands of Suan yu compared to the raw material. To the best of our knowledge, BA (approximately at 1000 ppm in food) is supposed to induce toxicity in humans (Nout, 1994). The allowable levels of tyramine and histamine are 100e800 mg/kg and 50e100 mg/kg respectively in foods (Nout, 1994; Shalaby, 1996). In this study, the BA contents of the samples are significantly lower than the recommended hazardous levels, suggesting the accumulation of BA was inhibited in Suan yu. Furthermore, the levels of BA in Suan yu were lower compared to those of other naturally fermented meat products, such as tyramine and histamine (up to 1100 and 350 mg/kg, respectively) found in some Turkish sausage Sucuks (S¸enöz, Is¸ikli, & Çoksöyler, 2000). The total concentration of BA reached 1633.02 mg/kg (DW) in Egyptian
Table 5 Biogenic amines in Suan yu. Sample
Biogenic amine (mg/kg) Phenylehtylamine
Tyramine
Common Carp Grass Carp A B C D E F
ND ND ND ND ND ND ND ND
15.6 17.4 91.9 23.1 38.4 6.98 80.4 30.7
0.92b 0.86c 2.92h 1.25d 1.43f 0.13a 1.21g 1.47e
Histamine
Cadaverin
Putrescine
NDa NDa 4.66 7.51 7.91 0.18 5.48 11.1
NDa NDa 36.9 1.05g 5.37 0.22b 7.09 0.33d 9.7 0.33e 19.7 1.56f 6.42 0.32c
0.46 0.37 8.26 0.88 0.88 0.88 18.3 113
0.12c 0.33e 0.43e 0.01b 0.12d 0.41f
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test. ND: under the detection limit.
0.02b 0.02a 0.33d 0.05c 0.04c 0.04c 1.48e 2.82f
X. Zeng et al. / Food Control 30 (2013) 590e595
salt-fermented fish (Feseekh) (Rabie, Simon-Sarkadi, Siliha, El-seedy, & El Badawy, 2009). Previous studies have shown that BA formation in foods is a result of microbial action, especially Enterobacteriaceae and Pseudomonas during fermentation and storage. They are generally considered as microorganisms conducting high decarboxylase activity, which is related to the production of BA. In this work, Enterobacteriaceae and Pseudomonas were under detection limit in samples, indicating a crucial factor and leading to low BA production. 4. Conclusion In summary, this paper is the first report on the chemical and microbial properties of Suan yu, a Chinese traditional low-salt fermented fish product. LAB, staphylococcus, and yeast were the dominant microorganisms in the six different Suan yu brands studied. Enterobacteria and Pseudomonads were under the detection limit in the samples. This result is in contrast with those on many spontaneously fermented fish products reported previously. The chemical compositions were different among all samples. Low moisture content and high lactic acid and protein contents were observed, and greater muscle protein degradation was detected during the ripening fermentation, all of which contribute to the nutrition and unique flavor of Suan yu. Furthermore, low BA concentrations and TBARS values were found among all samples compared with other fermented meat products, indicating that Suan yu is a high-quality product of improved safety and reduced hygienic risks. Further studies are necessary to determine the specific microbial, flavor succession, and other biochemical and physical reactions, which occur during ripening and fermentation. Acknowledgements This research was financially supported by the earmarked fund for China Agriculture Research System, PCSIRT0627, 111 ProjectB07029, NSFC, 31171709, and CXZZ11_0495. References Achinewhu, S., & Oboh, C. (2002). Chemical, microbiological, and sensory properties of fermented fish products from Sardinellasp. in Nigeria. Journal of Aquatic Food Product Technology, 11, 53e59. Anihouvi, V., Sakyi-Dawson, E., Ayernor, G., & Hounhouigan, J. (2007). Microbiological changes in naturally fermented cassava fish (Pseudotolithus sp.) for lanhouin production. International Journal of Food Microbiology, 116, 287e291. AOAC. (1997). Official methods of analysis of AOAC international (16th). Arlington, VA, USA: Association of Official Analytical Chemists. Asiedu, M., & Sanni, A. I. (2002). Chemical composition and microbiological changes during spontaneous and starter culture fermentation of Enam Ne-Setaakye, a West African fermented fish-carbohydrate product. European Food Research and Technology, 215, 8e12. Astiasarán, I., Villanueva, R., & Bello, J. (1990). Analysis of proteolysis and protein insolubility during the manufacture of some varieties of dry sausage. Meat Science, 28, 111e117. Ben-Gigirey, B., de Sousa, J. V. B. M., Villa, T. G., & Barros-Velazquez, J. (1999). Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga). Journal of Food Protection, 62, 933e939.
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Ben-Giglrey, B., Vieites Baptista de Sousa, J. M., & Villa, T. G. (1998). Changes in biogenic amines and microbiological analysis in albacore (Thunnus alalunga) muscle during frozen storage. Journal of Food Protection, 61, 608e615. Buckenhüskes, H. J. (1993). Selection criteria for lactic acid bacteria to be used as starter cultures for various food commodities. FEMS Microbiology Reviews, 12, 253e271. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302e310. Casiraghi, E., Pompei, C., Dellaglio, S., Parolari, G., & Virgili, R. (1996). Quality attributes of Milano salami, an Italian dry-cured sausage. Journal of Agricultural and Food Chemistry, 44, 1248e1252. Comi, G., Urso, R., Iacumin, L., Rantsiou, K., Cattaneo, P., Cantoni, C., et al. (2005). Characterisation of naturally fermented sausages produced in the North East of Italy. Meat Science, 69, 381e392. García de Fernando, G. D., & Fox, P. F. (1991). Study of proteolysis during the processing of a dry fermented pork sausage. Meat Science, 30, 367e383. Gilbert, R., De Louvois, J., Donovan, T., Little, C., Nye, K., Ribeiro, C., et al. (2000). Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. PHLS Advisory Committee for Food and Dairy Products. Communicable Disease and Public Health/PHLS, 3, 163. Jo, C., & Ahn, D. (2000). Volatiles and oxidative changes in irradiated pork sausage with different fatty acid composition and tocopherol content. Journal of Food Science-Chicago, 65, 270e275. Kose, S., & Hall, G. M. (2010). Sustainability of fermented fish products. Fish Processing, 138e166. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680e685. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biologilal Chemistry, 193, 265e275. Lücke, F. K. (2000). Utilization of microbes to process and preserve meat. Meat Science, 56, 105e115. Molly, K., Demeyer, D., Johansson, G., Raemaekers, M., Ghistelinck, M., & Geenen, I. (1997). The importance of meat enzymes in ripening and flavour generation in dry fermented sausages. First results of a European project. Food Chemistry, 59, 539e545. Nout, M. (1994). Fermented foods and food safety. Food Research International, 27, 291e298. Ostergaard, A., & Ben, E. (1998). Fermentation and spoilage of som fak, a Thai lowsalt fish product. Tropical Science, 38, 105e112. Phithakpol, B. (1995). The traditional fermented foods of Thailand: Institute of food research and product development. Kasetsart University. Rabie, M., Simon-Sarkadi, L., Siliha, H., El-seedy, S., & El Badawy, A. A. (2009). Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish (Feseekh) during ripening and storage. Food Chemistry, 115, 635e638. Rawles, D. D., Flick, G. J., & Martin, R. E. (1996). Biogenic amines in fish and shellfish. Advances in Food and Nutrition Research, 39, 329e365. Riebroy, S., Benjakul, S., Visessanguan, W., Kijrongrojana, K., & Tanaka, M. (2004). Some characteristics of commercial Som-fug produced in Thailand. Food Chemistry, 88, 527e535. Roseiro, L., Santos, C., Sol, M., Borges, M., Anjos, M., Gon alves, H., et al. (2008). Proteolysis in Painho de Portalegre dry fermented sausage in relation to ripening time and salt content. Meat Science, 79, 784e794. S¸enöz, B., Is¸ikli, N. N., & Çoksöyler, N. (2000). Biogenic amines inTurkish sausages (Sucucks). Journal of Food Science, 65, 764e767. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29, 675e690. Valyasevi, R., & Rolle, R. S. (2002). An overview of small-scale food fermentation technologies in developing countries with special reference to Thailand: scope for their improvement. International Journal of Food Microbiology, 75, 231e239. Verplaetse, A., Demeyer, D., Gerard, S., & Buys, E. (1992). Endogenous and bacterial proteolysis in dry sausage fermentation. Proceedings of 38th International Congress of Meat Science and Technology, 38, 851e854. Visessanguan, W., Benjakul, S., Riebroy, S., & Thepkasikul, P. (2004). Changes in composition and functional properties of proteins and their contributions to Nham characteristics. Meat Science, 66, 579e588. Wang, F. S. (2000). Effects of three preservative agents on the shelf life of vacuum packaged Chinese-style sausage stored at 20 C. Meat Science, 56, 67e71.
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Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu Xuefeng Zeng, Wenshui Xia*, Qixing Jiang, Fang Yang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2012 Received in revised form 16 July 2012 Accepted 24 July 2012
This paper reports the first study on the chemical and microbial properties of Suan yu, a sanitary Chinese traditional low-salt fermented whole fish product of high nutrition, unique flavor, and long-storage capability. Six brands of Suan yu from different locations in China were studied. Significant differences were observed in some characteristics of this traditional fermented fish, raw material and other spontaneously fermented fish. The values of Enterobacteria and Pseudomonads were under the detection limits in six different brands. In addition, the products contained higher protein and lower moisture ranging from 17.2% to 22.9% and 52.9%e58.1%, respectively. The pH values ranged between 4.27 and 5.18, and lactic acid was the major organic acid. The proteolysis of the myosin heavy chain and actin chain were observed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Moreover, low concentrations of biogenic amine (BA) and thiobarturic acid (TBARS) values were found in all samples. The characteristics of the six Suan yu brands were all influenced by raw materials, ingredients, and fermentation processes and conditions. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Suan yu Fermented fish Composition Protein Biogenic amines
1. Introduction Low-salt fermented whole fish or fish pieces products, such as Plaa-som (Riebroy, Benjakul, Visessanguan, Kijrongrojana, & Tanaka, 2004) and Enam NeeSetaakye (Asiedu & Sanni, 2002), are mainly prepared and consumed in Southeast Asia and subSaharan Africa. These products are highly nutritious and of relatively firm and springy texture, providing an ideal and cheap protein source. However, the relatively short shelf life (four to five days) under ambient conditions and the strong fishy odor of these traditional fermented fish pose major limitations with respect to marketing (Valyasevi & Rolle, 2002). The high amounts of biogenic amine (BA) (mainly histamine) and pathogenic bacteria in these foods also increase concern on food safety. Suan yu is also a Chinese low-salt fermented whole fish snack with a characteristic flavor. It is stable during storage and free of fishy odor and taste and retains all the nutritional advantages of fish compared with other traditional fermented fish products. It is manufactured using traditional technologies without the addition of starter cultures in small-scale processing units. Fish used are mainly freshwater fish mixed with cooked carbohydrate, salt, and
* Corresponding author. Tel./fax: þ86 510 85919121. E-mail address: [email protected] (W. Xia). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.07.037
spice. Long-term spontaneous fermentation is conducted to develop flavor in anaerobic conditions. Studies have shown that the spontaneous fermentation of meat is characterized by the participation of lactic acid bacteria (LAB), Gram-positive and catalase-positive cocci, yeasts, and molds (Buckenhüskes, 1993). Among them, LAB is mainly responsible for acidification, ensuring the safety of products by reducing the pH level through sugar fermentation (Lücke, 2000). Coagulasenegative staphylococci and yeast participate in the development of aroma, flavor, and color of fermented products, such as cassava fish (Anihouvi, Sakyi-Dawson, Ayernor, & Hounhouigan, 2007) and Plaa-som (Riebroy et al., 2004). Proteolysis is an important biochemical change during the ripening of fermented meats. This process influences both texture and flavor development through the formation of several low-molecular weight compounds, mainly peptides, amino acids, aldehydes, organic acids, and amines, which are known as important flavor compounds or precursors of flavor compounds (Roseiro et al., 2008). Besides, the occurrence of high amounts of BA in these products is of great concern in food safety issues. When high amounts of BA are consumed or normal pathways of amine catabolism are inhibited, various physiological effects, namely, hypotension (in the case of histamine, putrescine, cadaverine) or hypertension (in the case of tyramine), nausea, headache, rash, dizziness, cardiac palpitation, and even intracerebral hemorrhage and death, may occur in very severe cases (Rawles,
X. Zeng et al. / Food Control 30 (2013) 590e595
Flick, & Martin, 1996). Thus, these complex chemical and biochemical reactions probably contributed to the unique flavor and safety properties of Suan yu. However, the characteristics of this product have not been widely studied. Therefore, the aim of the present study was to investigate the chemical and microbial properties of Suan yu. 2. Materials and methods 2.1. Sampling Six types of high-quality commercial Suan yu produced through traditional fermentation technology were purchased from handicraft shops in four Chinese counties, namely, Fenghuang, Jishou, Huayuan, and Baojin. It is traditionally prepared from freshwater fish, thawed in running tap water and then eviscerated, scale-removed and gills-removed. Fish, salt and various spices were added according to the consumer preferences. After being mixed thoroughly, the mixtures were cured at ambient temperature for 12 h. Next, they were partially dried in the sun or oasthouse. Then they were manually mixed with ground roasted carbohydrates, such as cornmeal or glutinous rice flour (COFCO, Shanghai, China), and some of the roasted carbohydrates were stuffed into the fish bellies. The mixtures were placed in smallsized Pickle Pot with lids and then sealed tightly by surrounded water or wet mud. The products were fermented at different seasons until the desirable taste and aroma were produced. The special manufacturing conditions of the six different Suan yu are illustrated in Table 1. Prior to analyses, the bone and cornmeal or glutinous rice flour in Suan yu were removed using a sterilized knife. Samples were cut and ground in a meat grinder (MX-T2G National, Tokyo, Japan) for 3 min and kept at 4 C for further analysis.
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Manitol Salt Agar (MSA) plates incubated at 37 C for 48 h. The Enterobacteriaceae count was determined using violet red bile glucose agar (VBGA) was incubated at 37 C for 24 h. The Pseudomonas count was determined using a pseudomonas-aeromonasselective (GSP) agar base incubated at 26 C for 72 h. Yeast and mold were grown on PDA (nature pH) containing 0.01% (w/v) chloramphenicol (selective supplement, Oxoid) at 25 C for 3e4 days, and single colonies and ivory strains were enumerated and identified as yeast. The results are reported as colony-forming units per gram (cfu/g). 2.3. Proximate chemical analyses Protein content was analyzed using the Kjeldahl Method AOAC (1997). A factor 6.25 was used for conversion content of nitrogen to crude protein. Crude fat content was measured through an extraction technique using petroleum ether AOAC (1997). Ash content was determined from the weight of the sample burned at 550 C for 4 h. Moisture content was calculated from the weight loss of the sample (5 g) after being oven-dried at 105 C for 2 h until constant weight. 2.4. Determination of salt content Salt content was determined according to the AOAC (1997) procedure. Accurately weighed 1 g of the sample was treated with 10 ml of 0.1 M AgNO3 and 10 ml of HNO3. The mixture was heated gently on an induction-cooker for 10 min. Then the mixture was cooled with running water, to which were added distilled water (50 ml) and ferric alum indicator (5 ml). The mixture was titrated with standard 0.1 M KSCN until the solution became permanent brownish-red. The results were calculated and expressed as % NaCl.
2.2. Microbiological analysis
2.5. Determination of pH, total acidity, and Aw
From each Suan yu, samples (25 g) were taken aseptically, transferred to sterile plastic pouches, and homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) for 120 s with 225 ml of 0.1% peptone water. Appropriate decimal dilutions of the samples were made using the same diluents, and 0.1 ml of each dilution was inoculated in duplicate in different growth media to estimate microbial counts. LAB was grown anaerobically on DeMan Rogosa Sharpe (MRS) agar at 30 C for two to three days. The Micrococcaceae count was determined using
The pH levels of the samples were determined according to the procedure proposed by Wang (2000). Ten-gram samples were homogenized with 90 ml of deionized water at 12,000 rpm for 1 min. The pH levels were then measured using a digital pH meter (Mettler Toledo 320-s, Shanghai, China). Titrable acidity (TA) was determined via AOAC (1997), and the results were expressed as lactic acid percentage. Water activity (Aw) was measured using a digital water activity meter (Rotronic Hygroskop DT, Zurich, Switzerland) after equilibrium at 25 C.
Table 1 Product manufacture conditions of the six different types of Suan yu. Manufacture condition
Sample A
B
C
D
E
F
Origin of product Ingredientsa Fish Roasted cornmeal Glutinous rice flour NaCl Sucrose Glucose Spiceb Fermentation container Processing time Ripening temperature Ripening time (days)
Fenghuang
Fenghuang
Jishou
Baojin
Jishou
Huayuan
70% Common Carp 22% 0 3% 2% 0 3% Spices A Ceramic pots Summer 30 C 55
75% Grass Carp 18% 0 3% 2% 0 2% Spices A Glass pot Autumn 20 C 46
72% Common Carp 22% 0 3% 0 1% 2% Spices A Ceramic pots Autumn 20 C 54
75% Grass Carp 15% 0 6% 2% 0 2% Spices A Glass pot Autumn 20 C 62
70% Common Carp 23% 0 3% 2% 0 2% Spices B Ceramic pots Autumn 20 C 44
75%Common Carp 0 18% 3% 2% 0 2% Spices A Ceramic pots Winter 10 C 394
a
The weight percentages of ingredients for fermented fish was expressed as w % w (wet weight). 2% or 3% Spices A: 1e2% chili powder, 0.1e0.3% cumin, 0.1e0.3% cinnamon, 0.1e0.3% pepper; 2% Spices B: 1%e2% chili powder, 0.1e0.3% cumin, 0.1e0.3% cinnamon, 0.1e0.3% pepper, 0.1e0.3% bay leaves. The content of species A or B for fermented fish was controlled by the local acceptance, without any strict regulation. b
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2.6. Determination of organic acids
Shanghai, China). The results were expressed as mg malonaldehyde/kg sample.
Organic acids were extracted using the method proposed by Riebroy et al. (2004). The samples (15 g) were homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) with 35 ml of distilled water at 8000 rpm for 1 min and centrifuged (Sigma Laborzentrifugen, Model 4K15, Osterode, Germany) at 4500 g for 15 min. The supernatant (0.9 ml) was mixed with 0.1 ml of 5% (w/v) butyric acid as an internal standard. The mixture was added with 0.5 M of perchloric acid and allowed to stand for 5 min. Then, it was centrifuged at 12,000 g for 30 min at room temperature to remove precipitated proteins. The supernatant was filtered through a 0.45 mm membrane (Minisart RC-15, Satorious, Goettingen, Germany) prior to high-performance liquid chromatography (HPLC) analysis (Agilent1100, water, USA) with an Ecomsil C18 (4.6 250 mm). A gradient elution program was employed, and a mixture of methanol:water:phosphoric acid (5:95:0.05 v/v) was used as the mobile phase. Column temperature and flow rate were set at 30 C and 0.8 ml/min, respectively. Twenty microliters of the mobile phase was injected into the HPLC, and a photo diode array (Model Waters 996) set at 210 nm wavelength was used as the detector. The data were processed and analyzed using Agilent Chemstations. The different organic acids were identified by comparing their retention times with those of standard solutions (Sigma). Organic acid concentration was expressed as the percentage of each organic acid in the samples (w/w). 2.7. Determination of TCA-soluble peptides TCA-soluble peptides were determined according to the methods described by Visessanguan, Benjakul, Riebroy, and Thepkasikul (2004). Samples (3 g) were homogenized with 27 ml of 5% TCA (w/v) and kept at 4 C for 1 h and then centrifuged at 12,000 g for 5 min at 4 C (Sigma Laborzentrifugen, Model 4K15, Osterode, Germany). TCA-soluble peptides in the supernatant were determined via the method proposed by Lowry, Rosebrough, Farr, and Randall (1951). Bovine serum albumin was used as standard. The TCA-soluble peptides in the samples were expressed as mg tyrosine/kg sample. 2.8. Determination of thiobarbituric acid reactive substances Thiobarbituric acid (TBARS) was determined according to the method described by Buege and Aust (1978). Samples (5 g) were homogenized in 25 ml of TBARS solution (0.375% TBA, 15% TCA, and 0.25 M HCl), and the mixture was heated for 10 min in boiling water (95e100 C) to obtain a pink color. After being cooled with running water, the mixture was centrifuged at 5500 g for 25 min. Finally, the absorbance of the supernatant was determined at 532 nm using a spectrophotometer (Model 135 WFZ-UV-2100, UNICOTM,
2.9. SDS-PAGE Samples (3 g) were homogenized (Ultra Turrax homogenizer, IKA Labortechnik, Selangor, Malaysia) with 27 ml of solubilizing agent (TriseHCl 8.0 buffer, 2% SDS, 8 M urea, and 2% b-mercaptoethanol). The homogenate was heated at 85 C for 60 min and centrifuged at 10,000 g for 15 min at room temperature. Protein concentration was determined using the method proposed by Lowry et al. (1951). The supernatant was mixed (1:1, v/v) with the SDS-PAGE sample buffer (0.125 M TriseHCl, pH 6.8, 4% SDS, 10% glycerol, and 0.005% bromophenol blue). According to the method of Laemmli (1970), SDS-PAGE was performed in a vertical gel electrophoresis unit (Mini-Protean-3 Cell, Bio-Rad, Richmond, CA, USA). A 10% separating gel with 4% stacking gel was used, and aliquots of 10 ml were injected in each well. Electrophoresis was performed at 100e120 V. After electrophoresis, the gels were stained with Comassie Brilliant Blue R-250 (0.125%) in 25% methanol and 10% acetic acid. The gels were destained using 40% methanol and 10% acetic acid. 2.10. Determination of BA BA was extracted according to a slightly modified version of the procedure proposed by Ben-Gigirey, de Sousa, Villa, and BarrosVelazquez (1999). Two-gram samples with 10 ml of 0.4 M perchloric acid were homogenized at 3000 rpm using an Ultra Turrax homogenizer (IKA Labortechnik, Selangor, Malaysia) for 10 min. The homogenate was centrifuged at 12,000 g for 10 min at 4 C using a Sorvall Model RC-5B Plus centrifuge (Newtown, CT, USA). Extraction was performed twice. The supernatants were combined with 0.4 M perchloric acid to reach 25 ml. BA was derived according to the method proposed by Ben-Giglrey, Vieites Baptista de Sousa, and Villa (1998). A 1-ml extract or a standard solution was mixed with 200 ml of 2 M sodium hydroxide and 300 ml of saturated sodium bicarbonate. Two milliliters of dansyl chloride (10 mg/ml) was mixed with each sample. The mixture was incubated for 45 min at 40 C. Residual dansyl chloride was removed by adding 100 ml of 25% ammonium hydroxide. After 30 min at room temperature, the mixture was complemented to 5 ml with acetonitrile. Finally, the supernatant was filtered (0.45 mm Millipore membrane), and 5 ml of filtrate was injected into the HPLC (Agilentll00, USA). Liquid chromatographic separations were performed using Zorbax XDB C18 (4.6 250 mm). A gradient-elution program was used with the mobile phase containing 50% acetonitrile as solvent A and 90% acetonitrile as solvent B at a flow rate of 1.0 ml/min and column temperature of 30 C. Fluorescence was detected at an emission wavelength of 515 nm using an excitation wavelength of 340 nm.
Table 2 Microbiological properties in Suan yu. Sample
Lactic acid bacteria (log cfu/g)
Common Carp Grass Carp A B C D E F
3.51 3.73 7.36 7.85 7.62 5.53 7.81 7.13
0.16a 0.18a 0.26cd 0.38d 0.42cd 0.27b 0.38d 0.22c
Micrococcaceae (log cfu/g) 4.34 4.23 4.13 4.53 4.17 5.25 4.11 4.57
0.22a 0.19a 0.11a 0.23a 0.21a 0.53b 0.24a 0.32a
Yeast (log cfu/g) 2.47 2.68 6.36 6.72 6.47 5.13 6.14 6.48
0.11a 0.12a 0.31c 0.43c 0.47c 0.36b 0.35c 0.52c
Enterobacteria (log cfu/g)
Pseudomonads (log cfu/g)
3.37 0.15b 3.29 0.16b <1a <1a <1a <1a <1a <1a
3.62 0.18b 3.51 0.21b <1a <1a <1a <1a <1a <1a
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test. <1: Under the detection limit.
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Table 3 Chemical composition (%) of Suan yu. Sample
Parameter Protein (%)
Common Carp Grass Carp A B C D E F
17.3 16.3 22.8 20.5 22.9 20.6 20.3 17.2
0.43b 0.47a 0.58d 0.40c 0.62d 0.67c 0.45c 0.41b
Lipid (%) 1.58 4.46 1.13 0.97 1.08 1.48 0.91 0.88
0.07d 0.21e 0.03c 0.01ab 0.03bc 0.04d 0.02a 0.03a
Moisture (%) 79.4 78.6 52.9 56.8 55.8 53.9 58.1 57.4
2.04d 1.79d 0.82a 0.76bc 0.85b 0.75a 0.92c 0.81bc
Ash (%) 3.07 1.06 4.71 6.83 5.22 6.78 4.18 8.18
0.11b 0.04a 0.22d 0.33f 0.20e 0.33f 0.22c 0.3d
Salt (%) 0.14 0.13 3.03 2.85 3.17 6.86 3.24 2.94
pH
0.01a 0.01a 0.13bc 0.15b 0.16c 0.31d 0.18c 0.12bc
6.58 6.79 4.51 4.41 4.28 5.18 4.27 4.40
TA (%)
0.27c 0.31c 0.17a 0.12a 0.13a 0.29b 0.13a 0.22a
0.41 0.38 2.24 1.4 2.48 1.29 2.48 2.40
0.02a 0.02a 0.13c 0.19b 0.16c 0.11b 0.22c 0.14c
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test.
2.11. Statistical analysis The data obtained were subjected to the analysis of variance (ANOVA) and mean differences were evaluated by the least square difference (LSD) test (P < 0.05). Statistical analysis was performed using the SPSS statistic program (Version 13.0) for Window (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Microbiological analysis LAB, CNS, and yeast are actively involved in the development of safety, texture, color, and flavor (Ostergaard & Ben, 1998). Microbial properties of the raw materials (Common Carp and Grass Carp) and Suan yu are presented in Table 2. A large number of LAB, staphylococcus, and yeast were found in the spontaneously fermented fish products compared to the raw material (P < 0.05). Besides, the LAB cell numbers in brand D were lower (P < 0.05) than the other fermented samples, which contained similar counts. According to Achinewhu and Oboh (2002), salt concentration is likely to have a pronounced influence on the microbial growth and rate of fermentation. Therefore, in the present work, the growth of LAB was inhibited by the high salt concentrations in brand D. The result of this study is similar to those reported on other fermented fish products by Anihouvi et al. (2007) and Ostergaard and Ben (1998). Enterobacteria and Pseudomonads were under the detection limit in the fermented samples. This test met the guidelines for the microbiological quality of fermented meats published by Gilbert et al. (2000), indicating that these products are of good hygienic quality. The growth inhibition effects of Enterobacteriaceae and Pseudomonas on Suan yu might be attributed to the actions of bacteriocins and adequate acidification. 3.2. Chemical composition of Suan yu The chemical composition of the Suan yu and the raw material is shown in Table 3. Significant differences were found between Suan yu and raw material (P < 0.05). In addition, samples have typically higher protein concentrations than Thai Som-fug (11.4e16.2%) and Ghanaian Enam NeeSetaakye (15.6e15.8%), indicating that Suan yu is nutrient-rich because of the high protein and low fat content. The high protein content in the samples was most likely ascribed to the exclusion of carbohydrate and bones during Suan yu preparation. Furthermore, higher protein content probably affected by the different species, sizes, originations, and removal extent of water prior to processing (Casiraghi, Pompei, Dellaglio, Parolari, & Virgili, 1996; Comi et al., 2005). Moisture content is one of the main preservation factors in fermented fish products. High moisture content can greatly reduce shelf life. In this study, lower moisture
content was detected in Suan yu compared with raw material, Thai Som-fug and Ghanaian Enam NeeSetaakye, which contained 79.4e78.6%, 69.66e77.08% and 72.2e77.8% moisture, respectively. The low levels of the moisture in the samples may have resulted from the drying and curing processes prior to fermentation. The salt content of Suan yu is similar to Plaa-som, a traditional fermented fish product with 2.5e5.5% NaCl (Phithakpol, 1995). The different salt concentrations in different fermented fish have a pronounced influence on the microbial growth and the rate of fermentation and, thereby, on the sensory quality and safety of the product (Kose & Hall, 2010). Low levels of ash content was found in the raw material, which indicated that the variation in ash content possibly resulted from the different fermentation times of the samples and the salt and other additives Visessanguan et al. (2004). pH value,TA, Aw, and organic acid. Significantly different pH and TA values were observed between Suan yu and the raw material (P < 0.05). The pH values were between 4.27 and 5.18, the TA varied from 1.29% to 2.48% in the Suan yu during the long fermentation process, whereas the pH and TA values were in the ranges of 6.58e6.72 and 0.27e0.32 respectively in the raw material. The highly significant negative correlation (r ¼ 0.882 to 0.976, P < 0.01) between pH and TA values shows that the increase in TA is generally accompanied by a decrease in pH. Generally, pH values should be set below 4.6 to inhibit pathogenic and spoilage bacteria. This value is considered safe for consumption. However, the pH of brand D with high-salt content did not decrease to lower than 4.6, indicating the delayed LAB growth. The content of organic acids is also an important factor ensuring product safety. The levels of lactic acid and acetic acid were under detection limits in the raw material. Nevertheless, lactic acid was a dominant organic acid in Suan yu, and acetic acid was also found in all Suan yu samples (data not shown). The levels of lactic acid ranged from 1.89% to 2.99% in brands A, B, C, E, and F. Moreover, 1.00% lactic acid was observed in brand D because of the high salt content, which inhibited lactic acid growth. Butyric, Table 4 TCA-soluble peptides and TBARS in Suan yu. Sample
TCA-soluble peptides (mg tyrosine/kg)
Common Carp Grass Carp A B C D E F
67.2 63.4 792 884 926 658 895 973
2.13a 3.17a 29.3c 31.6d 43.4de 26.4b 41.7de 48.2e
TBARS (mg malonaldehyde/kg) 0.16 0.17 2.49 3.04 2.79 3.63 2.71 8.35
0.01a 0.01a 0.12b 0.15d 0.11c 0.14e 0.08c 0.21f
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test.
594
X. Zeng et al. / Food Control 30 (2013) 590e595
Fig. 1. SDS-PAGE pattern of muscle proteins in Suan yu: S, molecular weight standard; MC, Common Carp; MG, Grass Carp; MHC, myosin heavy chain; AeF brands of Suan yu.
succinic, or other acids were under the detection limit in the samples. The same is reported by Riebroy et al. (2004), indicating that lactic acid contents are in the range of 1.21e2.17% in Som-fug. In previous studies, 2.2e2.5% lactic acid in fermented meat produced the highest sensory score (Ostergaard & Ben, 1998). Among all tested brands, the Suan yu samples containing high concentrations of lactic acid and acetic acids generally showed higher sensory acceptance for consumers. Furthermore, the samples contained 0.88e0.92% of Aw, showing that microbial growth was inhibited and product safety was improved because of lower water activity.
among samples were associated with the differences in initial raw mixed ingredients and proteolysis induced by acidic condition (Visessanguan et al., 2004). The initial hydrolysis of muscle proteins is attributed mainly to endogenous cathepsin and followed by the action of microbial peptidases, which further degrades protein fragments into small peptides and free amino acids (Molly et al., 1997). Greater degradation of muscle proteins contributes to the development of the flavor and aroma of the fermented products. This result is similar to that of Valyasevi and Rolle (2002), who reported that strong protease activity, produced by staphylococci and bacilli, was responsible for the breakdown of fish proteins into peptides and free amino acids, which contribute to the unique flavor of the products. The electrophoretic patterns of muscle proteins in the six brands and raw material are shown in Fig. 1. As presented in Fig. 1, the amounts of protein hydrolysis in Suan yu and the raw material are compared, the muscle proteins (200, 116, 97.2, 66.4, and 44.3 kDa) markedly disappear or decrease. The bands at 29.2 and 20.1 kDa were more intense after a lengthy fermentation. The low intensity of myosin heavy chain and actin chain bands observed in all the samples indicates that the degradation of proteins took place as ripening progressed. The bands 29.2 and 20.1 kDa were more intense because of the possible co-migration of degradation products from larger proteins. Similar findings have been reported by García de Fernando and Fox (1991) and Verplaetse, Demeyer, Gerard, and Buys (1992). These phenomena of proteolysis in Suan yu was probably influenced by factors, such as product formulation, indigenous microorganism, and processing condition because of the action of proteinases from staphylococcus or LAB as well as the hydrolysis of muscle proteins caused by acids (Astiasarán, Villanueva, & Bello, 1990).
3.3. Lipid oxidation 3.5. Biogenic amines of Suan yu TBARS has been widely used to indicate lipid oxidation, which results in rancid smell and taste as well as alterations in texture, color and nutrition in meat and meat products (Jo & Ahn, 2000). The TBARS values of Suan yu ranged from 2.49 to 8.36 mg/kg, indicating the different extents of lipid oxidation (Table 4). The results are consistent with those of Riebroy et al. (2004) concerning the lipid oxidation in Som-fug. It has been previously reported that LAB exhibited antioxidant effects on unsaturated fatty acids (Wang, 2000). The generated LAB lowered the TBARS values in the samples. 3.4. Protein degradation of Suan yu The levels of TCA-soluble peptide ranged from 63.4 to 67.2 mg tyrosine/kg in the raw material. However, the TCA-soluble peptide content varied from 658 to 973 mg tyrosine/kg in Suan yu (Table 4), which indicates muscle proteins were severely degraded during fermentation. The differences in TCA-soluble peptides observed
The contents of BA in Suan yu and the raw material are shown in Table 5. Different concentrations of BA were detected in all samples. Among all samples, the levels of tyramine, histamine, cadaverin, and putrescine were higher in the six brands of Suan yu compared to the raw material. To the best of our knowledge, BA (approximately at 1000 ppm in food) is supposed to induce toxicity in humans (Nout, 1994). The allowable levels of tyramine and histamine are 100e800 mg/kg and 50e100 mg/kg respectively in foods (Nout, 1994; Shalaby, 1996). In this study, the BA contents of the samples are significantly lower than the recommended hazardous levels, suggesting the accumulation of BA was inhibited in Suan yu. Furthermore, the levels of BA in Suan yu were lower compared to those of other naturally fermented meat products, such as tyramine and histamine (up to 1100 and 350 mg/kg, respectively) found in some Turkish sausage Sucuks (S¸enöz, Is¸ikli, & Çoksöyler, 2000). The total concentration of BA reached 1633.02 mg/kg (DW) in Egyptian
Table 5 Biogenic amines in Suan yu. Sample
Biogenic amine (mg/kg) Phenylehtylamine
Tyramine
Common Carp Grass Carp A B C D E F
ND ND ND ND ND ND ND ND
15.6 17.4 91.9 23.1 38.4 6.98 80.4 30.7
0.92b 0.86c 2.92h 1.25d 1.43f 0.13a 1.21g 1.47e
Histamine
Cadaverin
Putrescine
NDa NDa 4.66 7.51 7.91 0.18 5.48 11.1
NDa NDa 36.9 1.05g 5.37 0.22b 7.09 0.33d 9.7 0.33e 19.7 1.56f 6.42 0.32c
0.46 0.37 8.26 0.88 0.88 0.88 18.3 113
0.12c 0.33e 0.43e 0.01b 0.12d 0.41f
Data are given as mean standard deviation. Values with different superscript letters in the same column indicate significant differences at p < 0.05 by LSD test. ND: under the detection limit.
0.02b 0.02a 0.33d 0.05c 0.04c 0.04c 1.48e 2.82f
X. Zeng et al. / Food Control 30 (2013) 590e595
salt-fermented fish (Feseekh) (Rabie, Simon-Sarkadi, Siliha, El-seedy, & El Badawy, 2009). Previous studies have shown that BA formation in foods is a result of microbial action, especially Enterobacteriaceae and Pseudomonas during fermentation and storage. They are generally considered as microorganisms conducting high decarboxylase activity, which is related to the production of BA. In this work, Enterobacteriaceae and Pseudomonas were under detection limit in samples, indicating a crucial factor and leading to low BA production. 4. Conclusion In summary, this paper is the first report on the chemical and microbial properties of Suan yu, a Chinese traditional low-salt fermented fish product. LAB, staphylococcus, and yeast were the dominant microorganisms in the six different Suan yu brands studied. Enterobacteria and Pseudomonads were under the detection limit in the samples. This result is in contrast with those on many spontaneously fermented fish products reported previously. The chemical compositions were different among all samples. Low moisture content and high lactic acid and protein contents were observed, and greater muscle protein degradation was detected during the ripening fermentation, all of which contribute to the nutrition and unique flavor of Suan yu. Furthermore, low BA concentrations and TBARS values were found among all samples compared with other fermented meat products, indicating that Suan yu is a high-quality product of improved safety and reduced hygienic risks. Further studies are necessary to determine the specific microbial, flavor succession, and other biochemical and physical reactions, which occur during ripening and fermentation. Acknowledgements This research was financially supported by the earmarked fund for China Agriculture Research System, PCSIRT0627, 111 ProjectB07029, NSFC, 31171709, and CXZZ11_0495. References Achinewhu, S., & Oboh, C. (2002). Chemical, microbiological, and sensory properties of fermented fish products from Sardinellasp. in Nigeria. Journal of Aquatic Food Product Technology, 11, 53e59. Anihouvi, V., Sakyi-Dawson, E., Ayernor, G., & Hounhouigan, J. (2007). Microbiological changes in naturally fermented cassava fish (Pseudotolithus sp.) for lanhouin production. International Journal of Food Microbiology, 116, 287e291. AOAC. (1997). Official methods of analysis of AOAC international (16th). Arlington, VA, USA: Association of Official Analytical Chemists. Asiedu, M., & Sanni, A. I. (2002). Chemical composition and microbiological changes during spontaneous and starter culture fermentation of Enam Ne-Setaakye, a West African fermented fish-carbohydrate product. European Food Research and Technology, 215, 8e12. Astiasarán, I., Villanueva, R., & Bello, J. (1990). Analysis of proteolysis and protein insolubility during the manufacture of some varieties of dry sausage. Meat Science, 28, 111e117. Ben-Gigirey, B., de Sousa, J. V. B. M., Villa, T. G., & Barros-Velazquez, J. (1999). Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga). Journal of Food Protection, 62, 933e939.
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