Research Article Received: 7 April 2014
Revised: 5 June 2014
Accepted article published: 13 June 2014
Published online in Wiley Online Library: 21 July 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6783
Application of response surface methodology to optimise microbial inactivation of shrimp and conch by supercritical carbon dioxide Manhua Chen,a Xiao Sui,b Xixiu Ma,a Xiaomei Fenga and Yuqian Hana* Abstract BACKGROUND: Supercritical carbon dioxide (SC-CO2 ) has been shown to have a good pasteurising effect on food. However, very few research papers have investigated the possibility to exploit this treatment for solid foods, particularly for seafood. Considering the microbial safety of raw seafood consumption, the study aimed to explore the feasibility of microbial inactivation of shrimp (Metapenaeus ensis) and conch (Rapana venosa) by SC-CO2 treatment. RESULTS: Response surface methodology (RSM) models were established to predict and analyse the SC-CO2 process. A 3.69-log reduction in the total aerobic plate count (TPC) of shrimp was observed by SC-CO2 treatment at 53∘ C, 15 MPa for 40 min, and the logarithmic reduction in TPC of conch was 3.31 at 55∘ C, 14 MPa for 42 min. Sensory scores of the products achieved approximately 8 (desirable). The optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55∘ C, 15 MPa and 40 min. CONCLUSION: SC-CO2 exerted a strong bactericidal effect on the TPC of shrimp and conch, and the products maintained good organoleptic properties. This study verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment. © 2014 Society of Chemical Industry Keywords: supercritical carbon dioxide; microbial inactivation; response surface methodology; optimisation; Metapenaeus ensis; Rapana venosa
INTRODUCTION
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Supercritical carbon dioxide (SC-CO2 ) pasteurisation has shown advantages compared with other innovative pasteurisation methods and has gained great interest in the field of food science.1,2 Since the 1980s, this method has been shown to have a good pasteurising effect on food, while simultaneously maintaining the nutritional and organoleptic properties of the food. Moreover, CO2 is relatively inert, inexpensive, non-toxic, non-flammable, recyclable and readily available in high purity, leaving no residue when removed after the pasteurisation process.3 This process employs milder conditions for achieving satisfactory microbial inactivation, with much lower pressures (generally below 20 MPa) compared to the pressures applied in high hydrostatic pressure treatment (nearly 300–600 MPa). Therefore, in addition to the relative ease of the control and management of pressure in SC-CO2 pasteurisation, this method allows considerable reductions in capital expenditure.4 SC-CO2 pasteurisation is included in both dense-phase carbon dioxide treatment and high-pressure carbon dioxide treatment, which requires the operating temperature and pressure to be above the critical point values (T c = 31∘ C, Pc = 7.4 MPa). SC-CO2 showed enhanced microbial lethality compared to either liquid or gaseous carbon dioxide, which may be attributed to its unique physico-chemical properties (liquid-like high solvating and extraction power and gas-like high diffusion rate).1,5 Although the exact mechanism of microbial inactivation by SC-CO2 remains to be unraveled, the models under discussion can be summarised as J Sci Food Agric 2015; 95: 1016–1023
follows: (1) solubilisation of pressurised CO2 in the external liquid phase, (2) cell membrane modification, (3) decreased intracellular pH, (4) inactivation of key enzymes and inhibition of cellular metabolism due to lowered intracellular pH, (5) direct (inhibitory) effects of molecular CO2 and HCO3 − on metabolism, (6) disordering of the intracellular electrolyte balance and (7) removal of vital components from cells and cell membranes.1,4,6 – 8 It is noted that most of these steps occur simultaneously in a complex and interrelated manner.1 The antimicrobial effect of SC-CO2 has been demonstrated mainly for liquid foodstuffs (e.g. physiological saline, sterile water, milk, fruit juice), but very few research papers have investigated the possibility of exploiting this treatment for solid foods, particularly for seafood.1,2,6 To date, there has been no report on microbial inactivation of conch with SC-CO2 . Shrimp and conch are common seafood in China, and they are often consumed raw with wine, vinegar and minced garlic in the
∗
Correspondence to: Yuqian Han, College of Food Science and Engineering, Ocean University of China, P.O. Box 266003, Qingdao, China. E-mail: [email protected]
a College of Food Science and Engineering, Ocean University of China, P.O. Box 266003, Qingdao, China b Biology Department of Medical College, Qingdao University, P.O. Box 266071, Qingdao, China
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Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide coastal areas of southern China. Consumption of raw seafood is considered to be extraordinarily nutritious and delicious, and it has become a fashion as a superior cuisine, with increasing popularity among the Chinese. Nevertheless, a worrisome increase in the number of outbreaks of food-borne illnesses and infections associated with raw seafood consumption has recently been observed.9 – 11 Microbial contamination is one of the main concerns associated with raw seafood consumption in China.9,11 Other risks include pathogenic microorganisms, viruses, parasites, toxic metals, biotoxins and chemical environmental contaminants.9 To that end, the aim of our work was to explore the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment. Response surface methodology (RSM) is a useful statistical tool used in analytical optimisation, and it consists of a group of mathematical and statistical techniques that are based on the fit of empirical models to experimental data obtained in relation to the experimental design.12 Toward this objective, linear or square polynomial functions are employed to describe the system studied and, consequently, to explore and optimise the experimental conditions.12 In this study, RSM was used to optimise process parameters (temperature, pressure and exposure time) of microorganism inactivation of shrimp and conch by SC-CO2 . The total aerobic plate counts (TPCs) indicate the level of microbial contamination in food, which is an important critical control point for microbial safety attributes of seafood products. International organisations such as the National Advisory Committee on Microbiological Criteria for Foods13 and the International Commission on Microbiological Specification for Foods14 have recommended TPC as a means of assessing the effectiveness of the HACCP programme. It has been reported that, in most types of seafood, the mean TPC of proximately 60% of the raw seafood in China was greater than 105 CFU g−1 , which was not in accordance with the current food safety standards and generates concern for the microbial safety of seafood that is eaten raw.11 Therefore, TPC
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was used in this study as an index to optimise the parameters of the SC-CO2 pasteurisation process. This study explored the feasibility of microbial inactivation of shrimp and conch by SC-CO2 and innovatively applied RSM in modeling this process to optimise process parameters (temperature, pressure and exposure time).
MATERIALS AND METHODS Materials Metapenaeus ensis sized at 50–60 shrimps kg−1 and Rapana venosa sized at 10–20 conch kg−1 were purchased from a wholesale seafood market in Qingdao, China. The shrimp were kept in cold sea water, peeled and decapitated to obtain shrimp meat. Conch were knocked out, and the contents of the shell, including meat and internal organs, were obtained. Materials were then quickly transported on ice to the laboratory, where they were washed in cold water and stored at −18∘ C until use. The recipe for plate count agar was as follows (w/v): 0.1% glucose (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), 0.25% yeast extracts (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China), 0.5% peptone (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China), 1.5% agar (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China) and to adjust pH to 7.0–7.2 by adding NaOH (aq). All of the chemical reagents used were of analytical grade. The carbon dioxide was 99.9% pure, purchased from Huanyu Gas Company in Qingdao, China. The SC-CO2 treatment system The apparatus was assembled and installed in our laboratory. It consists of a 50-mL and a 800-mL stainless steel pressure vessels, temperature controllers, pressure gauges and two plunger-type pumps (a schematic diagram of the apparatus is shown in Fig. 1).
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Figure 1. Schematic diagram of the SC-CO2 pasteurisation apparatus. (1) CO2 tank. (2) Condenser. (3) Pump. (4) Vessel. (5) Pasteurisation unit. (6) Heating system. A, a valve; P, a piezometer; and T, a thermometer.
www.soci.org The system pressure was controlled by a back-pressure regulator and indicated by pointer manometers. An electrical heating jacket was placed around the vessel. Another thermocouple, connected to a temperature controller, controls and maintains a constant temperature. The pressure and temperature were controlled to an accuracy of ±0.4 MPa and ±0.5∘ C, respectively. SC-CO2 pasteurisation Before pasteurisation of each sample, the supercritical carbon dioxide apparatus was washed using 75% ethanol, heated to 55∘ C and sanitised for 20 min; it was then rinsed three times with sterile water. Shrimp and conch were defrosted until they reached room temperature (20∘ C). Then shrimp or conch were weighed and enclosed in the vessel. The liquid carbon dioxide source was opened filling the vessel and venting for 5 s to purge the vessel of any air. The samples were subjected to different pressure levels (8 MPa, 14 MPa, 20 MPa) and combined with different temperatures (35∘ C, 45∘ C, 55∘ C) for various time lengths (5 min, 35 min, 65 min). The average pressurisation rate was 1 MPa min−1 . The exposure time of SC-CO2 was started to measure when temperature and pressure both reached required condition. The sample was held at constant pressure and temperature during the treatment. At the end of the treatment, the vessel was quickly depressurised within 1 min. Samples were removed aseptically and immediately cooled in a 4∘ C water bath. The experiments were performed in triplicate. Microbial analysis A 5.0 g sample of shrimp or conch meat was weighed aseptically and homogenised in a sterile homogeniser. Sterile saline solution (45 mL, 0.85% NaCl, w/v) was added, and the mixture was homogenised. Ten-fold serial dilutions of the homogenate were performed (up to 10−7 ), and 1 mL from each dilution was spread in duplicate plates, using plate count agar (PCA) to determine the total aerobic plate counts (TPC). Inoculated plates were incubated at 30∘ C ± 1∘ C for two days, according to the national food security standard for food microbiological examination GB47892.2010. The plates of different dilutions yielding 30–300 colony forming units (CFU) per plate were counted, and CFU g−1 was calculated. The initial TPC of untreated shrimp and conch were 3.51 ± 0.4 × 105 CFU g−1 and 3.61 ± 0.3 × 105 CFU g−1 respectively before they were stored at −18∘ C. Inactivation effect was expressed as −log(N/N0 ),where N and N0 represent the TPC of treated and untreated samples, respectively. The initial TPC of untreated shrimp and conch were measured for each run of the experiments.
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Sensory assessment The sensory analysis of the shrimp and conch treated by supercritical carbon dioxide was performed by a panel of eight people. These panellists (untrained), were presented with standards of fresh and spoiled shrimp and conch (left at 20∘ C for 3 days) assisting them in the grading process. The features evaluated included physical appearance, smell and texture of samples, and the samples were not tasted. They evaluated the organoleptic properties of the treated and untreated samples on a scale of 0–10 (from the most undesirable to extremely desirable). Samples were labelled using unrelated numbers to blind the panel to the treatments. Shrimp and conch must have obtained a score ≥ 5.0 to be judged as acceptable ones.
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Table 1. Levels of independent factors
Factor
Ranges and levels of independent factors
Symbol
Temperature (∘ C) Pressure (MPa) Exposure time (min)
x1 x2 x3
35 8 5
45 14 35
55 20 65
Experimental design and statistical analysis The parameters for microbial inactivation by SC-CO2 were optimised by RSM. The process parameters investigated in this study were temperature, pressure and exposure time, all of which have major effects in SC-CO2 treatment. Box–Behnken experimental design with three variables (temperature, pressure and exposure time) in three levels and three replications at the centre point was applied. Temperature, pressure and exposure time were coded as x 1 , x 2 and x 3 , respectively. The levels of independent factors are presented in Table 1. The second-order polynomial equation was used as a mathematical model for the microbial inactivation process by SC-CO2 [Eqn (1)]: Yi = 𝛽0 + 𝛽1 x1 + 𝛽2 x2 + 𝛽3 x3 + 𝛽11 x12 + 𝛽22 x22 + 𝛽33 x32 + 𝛽12 x1 x2 + 𝛽13 x1 x3 + 𝛽23 x2 x3
(1)
In this equation, Y i , the dependent variable, is the logarithmic reduction of microorganisms. 𝛽 0 is a constant, 𝛽 1 , 𝛽 2 and 𝛽 3 are the linear coefficients, 𝛽 11 , 𝛽 22 and 𝛽 33 are the quadratic coefficients; 𝛽 12 , 𝛽 13 and 𝛽 23 are the interaction coefficients; and x 1 , x 2 and x 3 are the coded values of independent variables. All of the experiments were performed in triplicate, and the data are expressed as the mean ± SD. Design-expert software (Version 8.0.6, Stat-ease Inc., Minneapolis, MN, USA) was used for data analysis and optimation.
RESULTS AND DISCUSSION Establishment of the RSM model for microbial inactivation of shrimp and conch by SC-CO2 The Box–Behnken experimental design and results of microbial inactivation of shrimp and conch by SC-CO2 are shown in Table 2. The process parameters investigated in this study were temperature (x 1 ), pressure (x 2 ) and exposure time (x 3 ), which all have major effects in SC-CO2 treatment. Logarithmic reduction in TPC of shrimp (Y 1 ) and conch (Y 2 ) were measured. Second-order polynomial regression equations were established by RSM to evaluate the relationships between logarithmic reduction in TPC and the process parameters. They are shown below as Eqn (2) and Eqn (3): Y1 = −12.71 + 0.37x1 + 0.35x2 + 0.095x3 − 0.002x12 − 0.006x22 − 0.0004x32 − 0.003x1 x2 − 0.001x1 x3 + 0.001x2 x3 (2)
Y2 = −9.65 + 0.28x1 + 0.27x2 + 0.075x3 − 0.001x12 − 0.005x22 − 0.0003x32 − 0.003x1 x2 − 0.001x1 x3 + 0.0008x2 x3 (3) The plots of actual experimental results versus predicted results for −log(N/N0 ) of shrimp and conch are shown in Fig. 2a and b, respectively. The effectiveness of the model and its adaptability to
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide
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Table 2. Box–Behnken experimental design and results
Temperature, Run x 1 (∘ C) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
45 35 35 35 45 45 45 55 45 45 55 55 45 55 35
Pressure, x 2 (MPa)
Exposure time, x 3 (min)
14 20 8 14 8 14 8 14 20 20 20 8 14 14 14
35 35 35 5 65 35 5 65 5 65 35 35 35 5 65
−log (N/N0 ), Y 1 a (shrimp) Y 2 a (conch) 2.98 ± 0.09 2.05 ± 0.14 0.95 ± 0.03 0.22 ± 0.15 2.57 ± 0.12 2.89 ± 0.07 1.67 ± 0.05 3.77 ± 0.10 1.83 ± 0.02 3.44 ± 0.06 3.68 ± 0.11 3.41 ± 0.07 3.10 ± 0.15 3.61 ± 0.11 2.12 ± 0.04
2.61 ± 0.02 1.87 ± 0.11 0.99 ± 0.14 0.40 ± 0.09 2.28 ± 0.15 2.54 ± 0.06 1.56 ± 0.08 3.24 ± 0.16 1.69 ± 0.03 2.98 ± 0.13 3.17 ± 0.18 2.95 ± 0.07 2.71 ± 0.06 3.11 ± 0.10 1.92 ± 0.06
a All the experiments were performed in triplicate, and the data are expressed as the mean ± SD.
the experimental data was verified by the coefficient of determination (R2 ).14 The value of R2 was found to be 0.9898 and 0.9900 for shrimp and conch, respectively, which demonstrates that the models are effective and proficient in the range of the experimental conditions. Adequate precision requires measurement of the signal-to-noise ratio.12 The signal-to-noise ratios for shrimp and conch were 23.935 and 24.156, respectively, indicating an adequate signal. The results of analysis of variance (ANOVA) showed the models F-values of 54.14 and 54.74 and P-value of 0.0002 (75∘ C) deteriorate the organoleptic properties of foods.2 When the pressure was increased to 14 MPa and the temperature was raised to 45∘ C (with an exposure time of 35 min), a 3.1-log reduction in TPC of shrimp was observed, while the logarithmic reduction in TPC of conch was 2.71. As shown in Fig. 5, the logarithmic reduction in TPC of shrimp and conch sharply increased by increases in exposure time and temperature (with a pressure of 14 MPa). Moreover, the interaction between exposure time and temperature was also obvious. Exposure time is considered to be the most significant factor of microbial inactivation by SC-CO2 , based on the ANOVA of the models. A contradictory conclusion was reported by Ferrentino et al., who found that treatment time of SC-CO2 treatment did not significantly influence microbial inactivation.20 Nevertheless, that conclusion was limited by the range of time explored, which was
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide
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Table 3. Results of validation experiment and sensory assessment −log (N/N0 ) Material Shrimp Untreated Conch Untreated
Temperature (∘ C)
Pressure (MPa)
Time (min)
53 35 – 55 40 –
15 20 – 14 20 –
40 60 – 42 60 –
from 5 to 15 min. The results of the present study were also limited by the experimental conditions, including ranges of independent variables, target microorganisms and substrates. Meurehg reported that only 0.83-log reduction of TPC was observed in beef trimmings after SC-CO2 treatment for 15 min at 10.3 MPa and 36∘ C.21 Total inactivation in TPC was observed in cocoa powder treated with SC-CO2 at 30 MPa and 65∘ C for 40 min.22 Notably, temperature and exposure time play quite important roles in microbial inactivation of solid foods by SC-CO2 . Due to the complexity of the matrix of solid food, it is more difficult for SC-CO2 to dissolve, diffuse and penetrate in the medium and hence act as a bactericide.2 Therefore, the combination of moderate temperatures (45–55∘ C) and proper exposure time (30–50 min) is reasonable and effective to increase the microbial inactivation rate in a solid matrix. Ji et al. thought that SC-CO2 processing of seafood had a more promising outlook than that for other meat products, because seafood includes more water and less fat than terrestrial livestock.17 The first attempt to apply SC-CO2 for microbial inactivation of seafood was conducted by Wei et al. In that study, a 99% reduction of Listeria in shrimp was obtained after SC-CO2 treatment at 13.7 MPa and 35∘ C for 2 h.23 Recently, Ji et al. reported that a 3-log reduction in TPC of shrimp (Litopenaeus vannamei) was observed after SC-CO2 treatment at 15 MPa and 45∘ C for 35 min.17 Meujo et al. reported that the level of total bacterial inactivation (TPC) of oysters achieved a 2–3-log reduction with SC-CO2 treatment (10 MPa, 37∘ C for 30 min or 17.2 MPa, 60∘ C for 60 min).18 The results of the present study are consistent with the previous similar studies.
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3.69 ± 0.10 3.58 ± 0.07 – 3.31 ± 0.11 2.85 ± 0.14 –
Predicted 3.81 3.65 – 3.25 3.00 –
Sensory score 8.1 ± 0.7 7.1 ± 0.3 7.2 ± 0.5 7.7 ± 0.6 8.0 ± 0.9 8.2 ± 1.1
These results are shown in Table 3. There was no significant difference (P > 0.05) between the experimental values and the predicted values of −log(N/N0 ), reflecting the capability of the RSM models to predict and analyse the SC-CO2 process of microbial inactivation of shrimp and conch. When the temperature was as high as 53 or 55∘ C, lower pressures (such as 15 or 14 MPa) and shorter exposure times (such as 40 or 42 min) were required to achieve satisfactory logarithmic reduction in TPC of shrimp and conch. The physical appearance, texture and smell of the shrimp treated with higher temperature (53∘ C) were more desirable (sensory score was 8.1). These shrimp had reddish colour, a slightly cooked smell and elastic texture (see Fig. 6b and d). The phenomenon was also observed by Ji et al. in shrimp (Litopenaeus vannamei) treated with SC-CO2 at 55∘ C, 15 MPa for 26 min. Shrimp colour is dependent largely upon the amount of astaxanthin, which present as carotene–proteins, particularly in the exoskeleton. The colour change of shrimp indicated that astaxanthin was dissociated from the protein by SC-CO2 treatment. Though shrimp presented a cooked colour, the meat of shrimp remained fresh, transparent and elastic (see Fig. 6a and b). Shrimp treated with lower temperature (35∘ C) were less pleasing in the sensory assessment than the high-temperature group. It was thought the reddish colour of shrimp appeared to be more acceptable for Chinese dietary customs.17 Despite the colour change, it was thought that there would be less adverse effects of thermal destruction when shrimp was treated with SC-CO2 . Conch treated with SC-CO2 at 40∘ C, 20 MPa for 60 min did not show a notable colour change, and their smell and texture were fresh and were well accepted by most of the panellists (see Fig. 7c and d). Conch exposed to higher temperature (55∘ C) showed slight cooked smell and texture (see Fig. 7a and b). Although they were less desirable than the lower-temperature group, but were still acceptable (sensory score was 7.7 > 5.0). The effect of SC-CO2 on nutritional and organoleptic properties of shrimp and conch requires future research. It can be observed from Fig. 4 that an increase of pressure from 15 to 20 MPa did not appreciably influence the bactericidal effect of SC-CO2 , though it increased the operating and investment costs. Additionally, moderate temperature (45–55∘ C) combined with proper time (30–50 min) could accelerate the inactivation rate without deteriorating the food quality. Overall, the optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55∘ C, 15 MPa and 40 min. The results of optimisation in the present study are close to the optimal parameters Ji et al.17 concluded (55∘ C, 15 MPa, 26 min) on microbial inactivation of shrimp (Litopenaeus vannamei) using neural network model, which verified the feasibility and applicability of the processing parameters in different types of seafood.
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Optimisation of SC-CO2 process parameters on microbial inactivation of shrimp and conch by RSM models Aimed at maximising −log(N/N0 ) of shrimp and conch in the experimental ranges, RSM analysis software provided 43 combinations of different levels of temperature, pressure and exposure time (not shown) respectively for shrimp and conch. The optimised log reductions were 3.8–3.9-log for shrimp and 3.2–3.3-log for conch in TPC. Specifically, the residual bacterial counts were less than 300 CFU g−1 , thereby achieving the level of microbial inactivation required by law.17 Among the 86 combinations of optimised parameters, temperature was 53–55∘ C, pressure was 11–20 MPa, exposure time was 35–65 min. The combination of 53∘ C, 15 MPa and 40 min was selected for shrimp, 55∘ C, 14 MPa and 42 min was selected for conch in terms of maximising −log(N/N0 ). In order to investigate the possibility of a lower temperature compared with 55∘ C, the combination of 35∘ C, 20 MPa and 60 min was selected for shrimp, 40∘ C, 20 MPa and 60 min was selected for conch. The four selected combinations for shrimp and conch were for experiments to verify the RSM models and assess sensory analysis.
Experimental
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M Chen et al.
Figure 6. Appearance of untreated shrimp and shrimp treated with SC-CO2 .
Figure 7. Appearance of untreated conch and conch treated with SC-CO2 .
CONCLUSION
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SC-CO2 exerted a strong bactericidal effect on TPC of shrimp and conch, and microorganisms in shrimp were more sensitive to this inactivation than those in conch. RSM analysis showed that exposure time and temperature within the experimental ranges exerted highly significant effects on microbial inactivation and, these two parameters interacted with each other. The pressure was also significant for the inactivation process. An increase of pressure from 15 to 20 MPa did not appreciably influence the bactericidal
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effect of SC-CO2 . Moderate temperature (45–55∘ C) combined with proper time (30–50 min) could accelerate the inactivation rate without deteriorating the food quality. It might be appropriate to employ a moderate temperature (55∘ C), a relatively low pressure (15 MPa) and a proper exposure time (40 min) for SC-CO2 treatment of shrimp and conch. This study focused on microbial contamination of seafood and verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 , but future studies are necessary for applying
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J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide SC-CO2 as an innovative pasteurising agent to raw-eaten seafood and for possible future industrial applications. Pathogens are a serious concern of raw-eaten seafood consumption, especially for shellfish, therefore, the efficacy of SC-CO2 treatment must also be tested on common pathogenic microorganisms in seafood (e.g. V. parahaemolyticus, E. coli, Salmonella, Shigella spp., Hepatitis viruses, Clostridium perfringens, C. botulinum, Yersinia enterocolitica, Listeria, etc.). Nutritional analysis and quality evaluation of the treated seafood are required. Moreover, to improve the SC-CO2 effectiveness of microbial inactivation, combinations of techniques and pretreatments (pulsed electric field, ultrasonic treatment, etc.) should also be investigated.
ACKNOWLEDGEMENT The authors are grateful for the financial support provided by the National Natural Science Funds (Project Number 31071541) and program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT1188).
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J Sci Food Agric 2015; 95: 1016–1023
© 2014 Society of Chemical Industry
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Revised: 5 June 2014
Accepted article published: 13 June 2014
Published online in Wiley Online Library: 21 July 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6783
Application of response surface methodology to optimise microbial inactivation of shrimp and conch by supercritical carbon dioxide Manhua Chen,a Xiao Sui,b Xixiu Ma,a Xiaomei Fenga and Yuqian Hana* Abstract BACKGROUND: Supercritical carbon dioxide (SC-CO2 ) has been shown to have a good pasteurising effect on food. However, very few research papers have investigated the possibility to exploit this treatment for solid foods, particularly for seafood. Considering the microbial safety of raw seafood consumption, the study aimed to explore the feasibility of microbial inactivation of shrimp (Metapenaeus ensis) and conch (Rapana venosa) by SC-CO2 treatment. RESULTS: Response surface methodology (RSM) models were established to predict and analyse the SC-CO2 process. A 3.69-log reduction in the total aerobic plate count (TPC) of shrimp was observed by SC-CO2 treatment at 53∘ C, 15 MPa for 40 min, and the logarithmic reduction in TPC of conch was 3.31 at 55∘ C, 14 MPa for 42 min. Sensory scores of the products achieved approximately 8 (desirable). The optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55∘ C, 15 MPa and 40 min. CONCLUSION: SC-CO2 exerted a strong bactericidal effect on the TPC of shrimp and conch, and the products maintained good organoleptic properties. This study verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment. © 2014 Society of Chemical Industry Keywords: supercritical carbon dioxide; microbial inactivation; response surface methodology; optimisation; Metapenaeus ensis; Rapana venosa
INTRODUCTION
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Supercritical carbon dioxide (SC-CO2 ) pasteurisation has shown advantages compared with other innovative pasteurisation methods and has gained great interest in the field of food science.1,2 Since the 1980s, this method has been shown to have a good pasteurising effect on food, while simultaneously maintaining the nutritional and organoleptic properties of the food. Moreover, CO2 is relatively inert, inexpensive, non-toxic, non-flammable, recyclable and readily available in high purity, leaving no residue when removed after the pasteurisation process.3 This process employs milder conditions for achieving satisfactory microbial inactivation, with much lower pressures (generally below 20 MPa) compared to the pressures applied in high hydrostatic pressure treatment (nearly 300–600 MPa). Therefore, in addition to the relative ease of the control and management of pressure in SC-CO2 pasteurisation, this method allows considerable reductions in capital expenditure.4 SC-CO2 pasteurisation is included in both dense-phase carbon dioxide treatment and high-pressure carbon dioxide treatment, which requires the operating temperature and pressure to be above the critical point values (T c = 31∘ C, Pc = 7.4 MPa). SC-CO2 showed enhanced microbial lethality compared to either liquid or gaseous carbon dioxide, which may be attributed to its unique physico-chemical properties (liquid-like high solvating and extraction power and gas-like high diffusion rate).1,5 Although the exact mechanism of microbial inactivation by SC-CO2 remains to be unraveled, the models under discussion can be summarised as J Sci Food Agric 2015; 95: 1016–1023
follows: (1) solubilisation of pressurised CO2 in the external liquid phase, (2) cell membrane modification, (3) decreased intracellular pH, (4) inactivation of key enzymes and inhibition of cellular metabolism due to lowered intracellular pH, (5) direct (inhibitory) effects of molecular CO2 and HCO3 − on metabolism, (6) disordering of the intracellular electrolyte balance and (7) removal of vital components from cells and cell membranes.1,4,6 – 8 It is noted that most of these steps occur simultaneously in a complex and interrelated manner.1 The antimicrobial effect of SC-CO2 has been demonstrated mainly for liquid foodstuffs (e.g. physiological saline, sterile water, milk, fruit juice), but very few research papers have investigated the possibility of exploiting this treatment for solid foods, particularly for seafood.1,2,6 To date, there has been no report on microbial inactivation of conch with SC-CO2 . Shrimp and conch are common seafood in China, and they are often consumed raw with wine, vinegar and minced garlic in the
∗
Correspondence to: Yuqian Han, College of Food Science and Engineering, Ocean University of China, P.O. Box 266003, Qingdao, China. E-mail: [email protected]
a College of Food Science and Engineering, Ocean University of China, P.O. Box 266003, Qingdao, China b Biology Department of Medical College, Qingdao University, P.O. Box 266071, Qingdao, China
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Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide coastal areas of southern China. Consumption of raw seafood is considered to be extraordinarily nutritious and delicious, and it has become a fashion as a superior cuisine, with increasing popularity among the Chinese. Nevertheless, a worrisome increase in the number of outbreaks of food-borne illnesses and infections associated with raw seafood consumption has recently been observed.9 – 11 Microbial contamination is one of the main concerns associated with raw seafood consumption in China.9,11 Other risks include pathogenic microorganisms, viruses, parasites, toxic metals, biotoxins and chemical environmental contaminants.9 To that end, the aim of our work was to explore the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment. Response surface methodology (RSM) is a useful statistical tool used in analytical optimisation, and it consists of a group of mathematical and statistical techniques that are based on the fit of empirical models to experimental data obtained in relation to the experimental design.12 Toward this objective, linear or square polynomial functions are employed to describe the system studied and, consequently, to explore and optimise the experimental conditions.12 In this study, RSM was used to optimise process parameters (temperature, pressure and exposure time) of microorganism inactivation of shrimp and conch by SC-CO2 . The total aerobic plate counts (TPCs) indicate the level of microbial contamination in food, which is an important critical control point for microbial safety attributes of seafood products. International organisations such as the National Advisory Committee on Microbiological Criteria for Foods13 and the International Commission on Microbiological Specification for Foods14 have recommended TPC as a means of assessing the effectiveness of the HACCP programme. It has been reported that, in most types of seafood, the mean TPC of proximately 60% of the raw seafood in China was greater than 105 CFU g−1 , which was not in accordance with the current food safety standards and generates concern for the microbial safety of seafood that is eaten raw.11 Therefore, TPC
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was used in this study as an index to optimise the parameters of the SC-CO2 pasteurisation process. This study explored the feasibility of microbial inactivation of shrimp and conch by SC-CO2 and innovatively applied RSM in modeling this process to optimise process parameters (temperature, pressure and exposure time).
MATERIALS AND METHODS Materials Metapenaeus ensis sized at 50–60 shrimps kg−1 and Rapana venosa sized at 10–20 conch kg−1 were purchased from a wholesale seafood market in Qingdao, China. The shrimp were kept in cold sea water, peeled and decapitated to obtain shrimp meat. Conch were knocked out, and the contents of the shell, including meat and internal organs, were obtained. Materials were then quickly transported on ice to the laboratory, where they were washed in cold water and stored at −18∘ C until use. The recipe for plate count agar was as follows (w/v): 0.1% glucose (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), 0.25% yeast extracts (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China), 0.5% peptone (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China), 1.5% agar (Shuangxuan Microbial Culture Medium Products Factory, Beijing, China) and to adjust pH to 7.0–7.2 by adding NaOH (aq). All of the chemical reagents used were of analytical grade. The carbon dioxide was 99.9% pure, purchased from Huanyu Gas Company in Qingdao, China. The SC-CO2 treatment system The apparatus was assembled and installed in our laboratory. It consists of a 50-mL and a 800-mL stainless steel pressure vessels, temperature controllers, pressure gauges and two plunger-type pumps (a schematic diagram of the apparatus is shown in Fig. 1).
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Figure 1. Schematic diagram of the SC-CO2 pasteurisation apparatus. (1) CO2 tank. (2) Condenser. (3) Pump. (4) Vessel. (5) Pasteurisation unit. (6) Heating system. A, a valve; P, a piezometer; and T, a thermometer.
www.soci.org The system pressure was controlled by a back-pressure regulator and indicated by pointer manometers. An electrical heating jacket was placed around the vessel. Another thermocouple, connected to a temperature controller, controls and maintains a constant temperature. The pressure and temperature were controlled to an accuracy of ±0.4 MPa and ±0.5∘ C, respectively. SC-CO2 pasteurisation Before pasteurisation of each sample, the supercritical carbon dioxide apparatus was washed using 75% ethanol, heated to 55∘ C and sanitised for 20 min; it was then rinsed three times with sterile water. Shrimp and conch were defrosted until they reached room temperature (20∘ C). Then shrimp or conch were weighed and enclosed in the vessel. The liquid carbon dioxide source was opened filling the vessel and venting for 5 s to purge the vessel of any air. The samples were subjected to different pressure levels (8 MPa, 14 MPa, 20 MPa) and combined with different temperatures (35∘ C, 45∘ C, 55∘ C) for various time lengths (5 min, 35 min, 65 min). The average pressurisation rate was 1 MPa min−1 . The exposure time of SC-CO2 was started to measure when temperature and pressure both reached required condition. The sample was held at constant pressure and temperature during the treatment. At the end of the treatment, the vessel was quickly depressurised within 1 min. Samples were removed aseptically and immediately cooled in a 4∘ C water bath. The experiments were performed in triplicate. Microbial analysis A 5.0 g sample of shrimp or conch meat was weighed aseptically and homogenised in a sterile homogeniser. Sterile saline solution (45 mL, 0.85% NaCl, w/v) was added, and the mixture was homogenised. Ten-fold serial dilutions of the homogenate were performed (up to 10−7 ), and 1 mL from each dilution was spread in duplicate plates, using plate count agar (PCA) to determine the total aerobic plate counts (TPC). Inoculated plates were incubated at 30∘ C ± 1∘ C for two days, according to the national food security standard for food microbiological examination GB47892.2010. The plates of different dilutions yielding 30–300 colony forming units (CFU) per plate were counted, and CFU g−1 was calculated. The initial TPC of untreated shrimp and conch were 3.51 ± 0.4 × 105 CFU g−1 and 3.61 ± 0.3 × 105 CFU g−1 respectively before they were stored at −18∘ C. Inactivation effect was expressed as −log(N/N0 ),where N and N0 represent the TPC of treated and untreated samples, respectively. The initial TPC of untreated shrimp and conch were measured for each run of the experiments.
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Sensory assessment The sensory analysis of the shrimp and conch treated by supercritical carbon dioxide was performed by a panel of eight people. These panellists (untrained), were presented with standards of fresh and spoiled shrimp and conch (left at 20∘ C for 3 days) assisting them in the grading process. The features evaluated included physical appearance, smell and texture of samples, and the samples were not tasted. They evaluated the organoleptic properties of the treated and untreated samples on a scale of 0–10 (from the most undesirable to extremely desirable). Samples were labelled using unrelated numbers to blind the panel to the treatments. Shrimp and conch must have obtained a score ≥ 5.0 to be judged as acceptable ones.
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Table 1. Levels of independent factors
Factor
Ranges and levels of independent factors
Symbol
Temperature (∘ C) Pressure (MPa) Exposure time (min)
x1 x2 x3
35 8 5
45 14 35
55 20 65
Experimental design and statistical analysis The parameters for microbial inactivation by SC-CO2 were optimised by RSM. The process parameters investigated in this study were temperature, pressure and exposure time, all of which have major effects in SC-CO2 treatment. Box–Behnken experimental design with three variables (temperature, pressure and exposure time) in three levels and three replications at the centre point was applied. Temperature, pressure and exposure time were coded as x 1 , x 2 and x 3 , respectively. The levels of independent factors are presented in Table 1. The second-order polynomial equation was used as a mathematical model for the microbial inactivation process by SC-CO2 [Eqn (1)]: Yi = 𝛽0 + 𝛽1 x1 + 𝛽2 x2 + 𝛽3 x3 + 𝛽11 x12 + 𝛽22 x22 + 𝛽33 x32 + 𝛽12 x1 x2 + 𝛽13 x1 x3 + 𝛽23 x2 x3
(1)
In this equation, Y i , the dependent variable, is the logarithmic reduction of microorganisms. 𝛽 0 is a constant, 𝛽 1 , 𝛽 2 and 𝛽 3 are the linear coefficients, 𝛽 11 , 𝛽 22 and 𝛽 33 are the quadratic coefficients; 𝛽 12 , 𝛽 13 and 𝛽 23 are the interaction coefficients; and x 1 , x 2 and x 3 are the coded values of independent variables. All of the experiments were performed in triplicate, and the data are expressed as the mean ± SD. Design-expert software (Version 8.0.6, Stat-ease Inc., Minneapolis, MN, USA) was used for data analysis and optimation.
RESULTS AND DISCUSSION Establishment of the RSM model for microbial inactivation of shrimp and conch by SC-CO2 The Box–Behnken experimental design and results of microbial inactivation of shrimp and conch by SC-CO2 are shown in Table 2. The process parameters investigated in this study were temperature (x 1 ), pressure (x 2 ) and exposure time (x 3 ), which all have major effects in SC-CO2 treatment. Logarithmic reduction in TPC of shrimp (Y 1 ) and conch (Y 2 ) were measured. Second-order polynomial regression equations were established by RSM to evaluate the relationships between logarithmic reduction in TPC and the process parameters. They are shown below as Eqn (2) and Eqn (3): Y1 = −12.71 + 0.37x1 + 0.35x2 + 0.095x3 − 0.002x12 − 0.006x22 − 0.0004x32 − 0.003x1 x2 − 0.001x1 x3 + 0.001x2 x3 (2)
Y2 = −9.65 + 0.28x1 + 0.27x2 + 0.075x3 − 0.001x12 − 0.005x22 − 0.0003x32 − 0.003x1 x2 − 0.001x1 x3 + 0.0008x2 x3 (3) The plots of actual experimental results versus predicted results for −log(N/N0 ) of shrimp and conch are shown in Fig. 2a and b, respectively. The effectiveness of the model and its adaptability to
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J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide
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Table 2. Box–Behnken experimental design and results
Temperature, Run x 1 (∘ C) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
45 35 35 35 45 45 45 55 45 45 55 55 45 55 35
Pressure, x 2 (MPa)
Exposure time, x 3 (min)
14 20 8 14 8 14 8 14 20 20 20 8 14 14 14
35 35 35 5 65 35 5 65 5 65 35 35 35 5 65
−log (N/N0 ), Y 1 a (shrimp) Y 2 a (conch) 2.98 ± 0.09 2.05 ± 0.14 0.95 ± 0.03 0.22 ± 0.15 2.57 ± 0.12 2.89 ± 0.07 1.67 ± 0.05 3.77 ± 0.10 1.83 ± 0.02 3.44 ± 0.06 3.68 ± 0.11 3.41 ± 0.07 3.10 ± 0.15 3.61 ± 0.11 2.12 ± 0.04
2.61 ± 0.02 1.87 ± 0.11 0.99 ± 0.14 0.40 ± 0.09 2.28 ± 0.15 2.54 ± 0.06 1.56 ± 0.08 3.24 ± 0.16 1.69 ± 0.03 2.98 ± 0.13 3.17 ± 0.18 2.95 ± 0.07 2.71 ± 0.06 3.11 ± 0.10 1.92 ± 0.06
a All the experiments were performed in triplicate, and the data are expressed as the mean ± SD.
the experimental data was verified by the coefficient of determination (R2 ).14 The value of R2 was found to be 0.9898 and 0.9900 for shrimp and conch, respectively, which demonstrates that the models are effective and proficient in the range of the experimental conditions. Adequate precision requires measurement of the signal-to-noise ratio.12 The signal-to-noise ratios for shrimp and conch were 23.935 and 24.156, respectively, indicating an adequate signal. The results of analysis of variance (ANOVA) showed the models F-values of 54.14 and 54.74 and P-value of 0.0002 (75∘ C) deteriorate the organoleptic properties of foods.2 When the pressure was increased to 14 MPa and the temperature was raised to 45∘ C (with an exposure time of 35 min), a 3.1-log reduction in TPC of shrimp was observed, while the logarithmic reduction in TPC of conch was 2.71. As shown in Fig. 5, the logarithmic reduction in TPC of shrimp and conch sharply increased by increases in exposure time and temperature (with a pressure of 14 MPa). Moreover, the interaction between exposure time and temperature was also obvious. Exposure time is considered to be the most significant factor of microbial inactivation by SC-CO2 , based on the ANOVA of the models. A contradictory conclusion was reported by Ferrentino et al., who found that treatment time of SC-CO2 treatment did not significantly influence microbial inactivation.20 Nevertheless, that conclusion was limited by the range of time explored, which was
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J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide
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Table 3. Results of validation experiment and sensory assessment −log (N/N0 ) Material Shrimp Untreated Conch Untreated
Temperature (∘ C)
Pressure (MPa)
Time (min)
53 35 – 55 40 –
15 20 – 14 20 –
40 60 – 42 60 –
from 5 to 15 min. The results of the present study were also limited by the experimental conditions, including ranges of independent variables, target microorganisms and substrates. Meurehg reported that only 0.83-log reduction of TPC was observed in beef trimmings after SC-CO2 treatment for 15 min at 10.3 MPa and 36∘ C.21 Total inactivation in TPC was observed in cocoa powder treated with SC-CO2 at 30 MPa and 65∘ C for 40 min.22 Notably, temperature and exposure time play quite important roles in microbial inactivation of solid foods by SC-CO2 . Due to the complexity of the matrix of solid food, it is more difficult for SC-CO2 to dissolve, diffuse and penetrate in the medium and hence act as a bactericide.2 Therefore, the combination of moderate temperatures (45–55∘ C) and proper exposure time (30–50 min) is reasonable and effective to increase the microbial inactivation rate in a solid matrix. Ji et al. thought that SC-CO2 processing of seafood had a more promising outlook than that for other meat products, because seafood includes more water and less fat than terrestrial livestock.17 The first attempt to apply SC-CO2 for microbial inactivation of seafood was conducted by Wei et al. In that study, a 99% reduction of Listeria in shrimp was obtained after SC-CO2 treatment at 13.7 MPa and 35∘ C for 2 h.23 Recently, Ji et al. reported that a 3-log reduction in TPC of shrimp (Litopenaeus vannamei) was observed after SC-CO2 treatment at 15 MPa and 45∘ C for 35 min.17 Meujo et al. reported that the level of total bacterial inactivation (TPC) of oysters achieved a 2–3-log reduction with SC-CO2 treatment (10 MPa, 37∘ C for 30 min or 17.2 MPa, 60∘ C for 60 min).18 The results of the present study are consistent with the previous similar studies.
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3.69 ± 0.10 3.58 ± 0.07 – 3.31 ± 0.11 2.85 ± 0.14 –
Predicted 3.81 3.65 – 3.25 3.00 –
Sensory score 8.1 ± 0.7 7.1 ± 0.3 7.2 ± 0.5 7.7 ± 0.6 8.0 ± 0.9 8.2 ± 1.1
These results are shown in Table 3. There was no significant difference (P > 0.05) between the experimental values and the predicted values of −log(N/N0 ), reflecting the capability of the RSM models to predict and analyse the SC-CO2 process of microbial inactivation of shrimp and conch. When the temperature was as high as 53 or 55∘ C, lower pressures (such as 15 or 14 MPa) and shorter exposure times (such as 40 or 42 min) were required to achieve satisfactory logarithmic reduction in TPC of shrimp and conch. The physical appearance, texture and smell of the shrimp treated with higher temperature (53∘ C) were more desirable (sensory score was 8.1). These shrimp had reddish colour, a slightly cooked smell and elastic texture (see Fig. 6b and d). The phenomenon was also observed by Ji et al. in shrimp (Litopenaeus vannamei) treated with SC-CO2 at 55∘ C, 15 MPa for 26 min. Shrimp colour is dependent largely upon the amount of astaxanthin, which present as carotene–proteins, particularly in the exoskeleton. The colour change of shrimp indicated that astaxanthin was dissociated from the protein by SC-CO2 treatment. Though shrimp presented a cooked colour, the meat of shrimp remained fresh, transparent and elastic (see Fig. 6a and b). Shrimp treated with lower temperature (35∘ C) were less pleasing in the sensory assessment than the high-temperature group. It was thought the reddish colour of shrimp appeared to be more acceptable for Chinese dietary customs.17 Despite the colour change, it was thought that there would be less adverse effects of thermal destruction when shrimp was treated with SC-CO2 . Conch treated with SC-CO2 at 40∘ C, 20 MPa for 60 min did not show a notable colour change, and their smell and texture were fresh and were well accepted by most of the panellists (see Fig. 7c and d). Conch exposed to higher temperature (55∘ C) showed slight cooked smell and texture (see Fig. 7a and b). Although they were less desirable than the lower-temperature group, but were still acceptable (sensory score was 7.7 > 5.0). The effect of SC-CO2 on nutritional and organoleptic properties of shrimp and conch requires future research. It can be observed from Fig. 4 that an increase of pressure from 15 to 20 MPa did not appreciably influence the bactericidal effect of SC-CO2 , though it increased the operating and investment costs. Additionally, moderate temperature (45–55∘ C) combined with proper time (30–50 min) could accelerate the inactivation rate without deteriorating the food quality. Overall, the optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55∘ C, 15 MPa and 40 min. The results of optimisation in the present study are close to the optimal parameters Ji et al.17 concluded (55∘ C, 15 MPa, 26 min) on microbial inactivation of shrimp (Litopenaeus vannamei) using neural network model, which verified the feasibility and applicability of the processing parameters in different types of seafood.
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Optimisation of SC-CO2 process parameters on microbial inactivation of shrimp and conch by RSM models Aimed at maximising −log(N/N0 ) of shrimp and conch in the experimental ranges, RSM analysis software provided 43 combinations of different levels of temperature, pressure and exposure time (not shown) respectively for shrimp and conch. The optimised log reductions were 3.8–3.9-log for shrimp and 3.2–3.3-log for conch in TPC. Specifically, the residual bacterial counts were less than 300 CFU g−1 , thereby achieving the level of microbial inactivation required by law.17 Among the 86 combinations of optimised parameters, temperature was 53–55∘ C, pressure was 11–20 MPa, exposure time was 35–65 min. The combination of 53∘ C, 15 MPa and 40 min was selected for shrimp, 55∘ C, 14 MPa and 42 min was selected for conch in terms of maximising −log(N/N0 ). In order to investigate the possibility of a lower temperature compared with 55∘ C, the combination of 35∘ C, 20 MPa and 60 min was selected for shrimp, 40∘ C, 20 MPa and 60 min was selected for conch. The four selected combinations for shrimp and conch were for experiments to verify the RSM models and assess sensory analysis.
Experimental
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Figure 6. Appearance of untreated shrimp and shrimp treated with SC-CO2 .
Figure 7. Appearance of untreated conch and conch treated with SC-CO2 .
CONCLUSION
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SC-CO2 exerted a strong bactericidal effect on TPC of shrimp and conch, and microorganisms in shrimp were more sensitive to this inactivation than those in conch. RSM analysis showed that exposure time and temperature within the experimental ranges exerted highly significant effects on microbial inactivation and, these two parameters interacted with each other. The pressure was also significant for the inactivation process. An increase of pressure from 15 to 20 MPa did not appreciably influence the bactericidal
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effect of SC-CO2 . Moderate temperature (45–55∘ C) combined with proper time (30–50 min) could accelerate the inactivation rate without deteriorating the food quality. It might be appropriate to employ a moderate temperature (55∘ C), a relatively low pressure (15 MPa) and a proper exposure time (40 min) for SC-CO2 treatment of shrimp and conch. This study focused on microbial contamination of seafood and verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 , but future studies are necessary for applying
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J Sci Food Agric 2015; 95: 1016–1023
Optimization on microbial inactivation of shrimp and conch by supercritical carbon dioxide SC-CO2 as an innovative pasteurising agent to raw-eaten seafood and for possible future industrial applications. Pathogens are a serious concern of raw-eaten seafood consumption, especially for shellfish, therefore, the efficacy of SC-CO2 treatment must also be tested on common pathogenic microorganisms in seafood (e.g. V. parahaemolyticus, E. coli, Salmonella, Shigella spp., Hepatitis viruses, Clostridium perfringens, C. botulinum, Yersinia enterocolitica, Listeria, etc.). Nutritional analysis and quality evaluation of the treated seafood are required. Moreover, to improve the SC-CO2 effectiveness of microbial inactivation, combinations of techniques and pretreatments (pulsed electric field, ultrasonic treatment, etc.) should also be investigated.
ACKNOWLEDGEMENT The authors are grateful for the financial support provided by the National Natural Science Funds (Project Number 31071541) and program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT1188).
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