Food Science and Technology Research
Online ISSN : 1881-3984
Print ISSN : 1344-6606
ISSN-L : 1344-6606
Review
Advantages and challenges of sous vide cooking
ZhenKun CuiHan YanTatiana ManoliHaiZhen MoJiCai BiHao Zhang
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2021 年 27 巻 1 号 p. 25-34

詳細
Abstract

Abstract: In recent years, sous vide (SV), or vacuum cooking, has been used to prepare foods and beverages by both food industries and restaurants worldwide. SV is considered a transformation of traditional cooking into a more nutritionally, healthier cooking. SV has the advantages in precise control of heating temperature and time, to improve quality, color, flavor, and nutritional value of food. In this review, we first describe the research progress in SV technology, concerning food quality (e.g., texture, color, flavor), nutrients (e.g., lipids, proteins, vitamins) food safety, and feasibility. Meanwhile, we also address the challenges and perspectives of this new food processing technique.

Introduction

In the 1970s, a Hungarian physicist, Professor Nicholas Kurti at the University of Oxford, United Kingdom, and a French physical chemist, Herve, organized a team of scientists and professional chefs, including Pierre Gilles de Gennes, a Nobel Prize winner in physics, to study cooking (Brenner and Sorensen, 2015) which led to the proposal of the concept of molecular gastronomy in 1988. As a result, molecular gastronomy research leading to the discovery of sous vide (SV) was initially applied to dinners served at the world's top restaurants. Later, it widely uses in catering service providers (food factories, restaurants), fast food industries, and supermarkets. SV, due to its particular form of thermal processing, can still maintain food quality by reheating after cold storage or cryopreservation. In the past 20 years, studies on SV cover various research interests, such as food safety (Karyotis et al., 2017), storage time (Hernandez et al., 2017, Kato et al., 2017), quality improvement (Cadun, et al., 2016, Kosewski et al., 2018), effects on nutrients (Rondanelli et al., 2017), nutritional bioavailability (da Silva et al., 2017), and various other technical approaches (Renna et al., 2017). The research has also conducted in different types of meat and plant products (Table 1).

Table 1. Synopsis of studies investigating sous vide in different types of meat and plant products.
Reference Cooking conditions Ingredients Highlights
Temperature (°C) Holding Time
Meat products
Roldan et al., 2015a 60 °C and 80 °C 3–24 h lamb loin It seems that sous vide cooking of lamb meat at either 60°C or 80°C, for either 6 h or 24 h, leads to different meat features, enough to ensure a shelf-life of at least one month of refrigerated storage.
Increasing cooking temperatures led to higher weight losses and lower moisture content. The sous vide cooking of lamb loins dramatically reduced the microbial population even with the less intense heat treatment studied (60°C, 6 h).
Regarding functionality and palatability, sous vide is more ideal to promote the use of venison as a functional food.
Lemon juice can reduce protein degradation, fat oxidation, prolong shelf life.
Citric acid can prevent the redness of meat products in the vacuum process. At the concentration of 2%–5% citric acid, the quality and physicochemical properties of chicken breast meat are greatly affected.
The treatment at 95°C for 15 min was the most effective treatment for shrimp to extend the shelf life.

Sous vide processing, which extends shelf life, however, storage temperature has to be strictly controlled.

In order to ensure its shelf life and quality, the fat content and fatty acid composition of fish should be considered.
Sous vide has a significant effect on the retention rate of tuna umami, and can preserve the taste and juiciness.
Sous vide cooking temperature of 60°C was found to be suitable to obtain the optimum texture and good sensory quality of seabream.
Roldan et al., 2013 60 °C, 70 °C, and 80 °C 6 h, 12 h, and 24 h lamb loin
Yoshimura et al., 2014 80 °C and 100 °C 30 min Venison
Hong et al., 2016 60 °C 35 min chicken breast
Kim et al., 2015 61 °C 30 min chicken breast
Cadun et al., 2016 75 °C or 95 °C 10 min or 15 min pink shrimp (Parapenaeus longirostris)
Mohan et al., 2017 70 °C–80 °C 5 min Indian white shrimp (Fenneropenaeus Indicus)
Garcialinares et al., 2004 90 °C 10 min salmon and trout
Llave et al., 2018 50 °C or 59 °C, 30 min or 39 tuna
Espinosa et al., 2016 50 °C, 60 °C, and 70 °C 2 h seabream (Sparus aurata)
Plant products
Iborra-Bernad et al., 2013 92 °C 20 min–1 h green bean pod The best cooking times were 28 and 14 min for 1 and 7 days of storage by sous vide treatment (92°C) of green bean pods, respectively. Sous vide treatment is better preferred than traditional cooking.
Sous vide treated carrots were harder than cooked ones, and the red color of carrots increased during storage in any way. Steamed Brussels sprouts were softer and greener than sous vide processed ones, but softening and loss of green of the latter increased under vacuum storage.
The characteristic color of the raw vegetable and exhibits lower degradation of chlorophylls (total, a, and b) compared to the boiled product. After sous vide treatment, lettuce stems and chlorophyll were well preserved.
After a long period of heating, the hardness of carrots decreased significantly.
Rinaldi et al., 2013 100 °C 5 min carrots and brussels sprouts
Alcuson, et al., 2017 90 °C 949 s borage (Borago officinalis L.) stems
Hong et al., 2014 70 °C, 80 °C, 90 °C 10 min, 20 min carrot

SV refers to a cooking method to place food in a vacuum bag and cooked under strictly controlled temperature and time conditions (Schellekens, 1996). SV differs from traditional cooking in various aspects. First, the ingredients are sealed in a vacuum bag and cooked at low temperatures. Such pasteurization conditions can avoid the risk of bacterial contamination while inhibits the growth of anaerobic bacteria in food during storage (Botinestean et al., 2016). Thus, the cooked food can be stored for a more extended period and can rapidly cool down after cooking. Second, the heating temperature and time can be precisely controlled. Vacuum-sealed packaging can effectively transfer heat while preventing oxidation and loss of volatile substances and moisture (Church and Parsons, 2000), resulting in a betterflavor of foods (Diaz et al., 2008).

The precisely controlled temperature and time not only reduce the negative effect of cooking on nutrients (e.g., proteins, lipids, vitamins) but also increase total phenolic content and antioxidant activity (Alcuson et al., 2017) and improve the overall texture and color of foods (Creed, 1998, Garcialinares et al., 2004, Ghazala et al., 1996, Lassen et al., 2002, Schellekens, 1996).

Here, we systematically review the effects of SV, a new cooking method, on food eating quality, nutritional value, safety of foods, and also discuss the possible mechanisms. Provide a reference for the development of food industrialization.

Improve the food eating quality

Texture    Cooking temperature and mode of heat transfer can affect the physicochemical properties of food, such as hardness, elasticity, and chewiness. Heat can denature pectin in fruits and vegetables, thus change their hardness (Sila et al., 2009). The cooking temperature can improve the texture of food: prolonging cooking time and increasing cooking temperature can promote the dissolution of pectin, thus reduce the hardness of fruits and vegetables (Iborra-Bernad et al., 2015, Van Buggenhout et al., 2009); a cooking medium, water, can also increase the dissolution rate of pectin (Greve et al., 1994).

The heating temperature has significant effects on shear strength, springiness, cohesiveness, and chewiness of foods of animal origin. Bramblett (1964) addressed the core temperature of the roasts maintained at 65 °C for better eating quality (tenderness and overall appearance). Cooking at a temperature close to 60 °C for a long time not only avoids the increase in meat toughness observed at higher temperatures but also improves the tenderness of the meat after being kept for 4 hours (Laakkonen et al., 1970). Shrinkage of collagen fiber occurs at 57 °C (Bruggemann et al., 2010). Heating to 60–70 °C exacerbates shrinkage and then denature, dissolve, and gel (Palka, 2003, Tornberg, 2005). It should emphasize that cooking time in SV of foods of animal origin should be sufficient for the complete dissolution of collagen in food, in turn, reduces the hardness of the finished product. These observations have been further confirmed (Becker et al., 2015, Christensen et al., 2013, Roldan et al., 2013). However, the effects of heating time are not significant statistically (Garciasegovia et al., 2007, Roldan et al., 2013). Besides, SV technology can replace the pretreatment step of tenderized meat products. A study has shown that beef treated at 56 °C for 24 h has a tenderness that is equivalent to the tenderness of beef treated with bromelain while it has a more evenly distributed tenderization effect (Suriaatmaja and Lanier, 2014).

Color    Color is a direct and one of the most important indicators in sensory evaluation of food. SV uses vacuum packaging that can effectively prevent direct contact between foods of plant origin and oxygen, thus reduce oxidation of pigments, such as chlorophyll and carotenoids, leading to better food color. The vacuum packaging can also retain organic acids in fruits and vegetables, resulting in lower pH, while delaying the degradation of chlorophyll (Gan and Wang, 2002, Koca et al., 2007) and preventing hydrophilic pigments from dissolution in water. Borage cooked using SV (90 °C, 949 s) had 82.4% green retention and 62.0% retention of total chlorophyll content compared to traditional cooking (Alcuson, et al., 2017). The color of SV purple potato (80 °C, 25 min) assembled that of raw potato, while the color of traditionally cooked boiled purple potato was largely different from that of raw potato due to the dissolution of hydrophilic anthocyanins in water (Iborra-Bernad et al., 2015).

In the heating process of foods of animal origin, the change of meat color is due to the oxidation of heme. During the SV process, myoglobin in muscle is in the form of deoxymyoglobin, which is relatively resistant to heat-induced denaturation (Naveena et al., 2017). Therefore, SV can significantly improve food color, which is superior to the color of traditionally cooked food. The degree of redness (a* value) of SV meat products is inversely proportional to the degree of denaturation of myoglobin. Additionally, with increasing cooking temperature, the degree of denaturation of myoglobin increases while the degree of redness (a* value) decreases. The degree of redness (a* value) of chicken sausage cooked using SV was significantly higher than that of chicken sausage cooked using the traditional cooking method (Naveena et al., 2017). The degree of redness (a* value) of SV lamb meat decreased with increasing cooking temperature (60, 70, and 80 °C). The degree of redness (a* value) found to be highest at a temperature of 60 °C (Roldan et al., 2013). Coincidentally, Sanchez del Pulgar (2012) and his team found that the degree of redness (a* value) of SV pork cooked at temperatures between 60 and 80 °C was higher than that of traditionally cooked pork. The degree of redness (a* value) of beef cooked by SV at 70 °C for 15–60 min is higher than that cooked under normal pressure at 70 °C for 15–60 min (Garciasegovia et al., 2007).

Flavor    The heat treatment of foods of animal origin produces more than 1,000 volatile compounds. These volatile compounds can come from two main reactions, thermal degradation of lipids and degradation of amino acid components (Elmore and Mottram, 2006), including the Maillard reaction. Cooking conditions are critical to the formation of flavor volatiles in cooked meat products. Roldán (2015b) identified a total of 57 volatile compounds in SV mutton soup; only 11 of these compounds found in raw mutton samples. While certain main volatile compounds, which are formed by amino acid degradation, can be produced at low temperatures, oxidation of lipids can take place at high temperatures with long cooking time. Naveena (2017) found that prolonged cooking time led to the release of metal ions that, in turn, enhanced the oxidation of metallic odors. Long cooking time at a low temperature of SV is more conducive to hydrolysis, which produces the abundance of umami taste-active compounds. With increasing temperature and time, the hydrolysis of proteins and peptides, producing water-soluble free amino acids that can then be released (from animal raw materials) into a gravy, resulted in increased concentrations of amino acids and adenosine monophosphate in the broth.

In contrast, the concentration of inosinic acid was unaffected (Rotola - Pukkila et al., 2015). However, heating under vacuum conditions inhibits the oxidation of fats while reducing the production of flavor substances. Also, it generally accepted that it is challenging to produce aromatic compounds at lower temperatures (Calkins and Hodgen, 2007). Therefore, in order to make up for the lack of flavor of SV, it may be considered to add a flavor precursors before cooking or to add a step of cooking after SV cooking.

Reduce nutrient loss

Although cooking can improve the maturation of food, kill microorganisms, and facilitate digestion and absorption, it can also destruct nutrients in food. Such destruction can occur through the degradation of heat-labile vitamins (e.g., vitamin C and B) and the destruction of some essential amino acids, minerals, and other water-soluble vitamins, which can mostly lead to the decrease in the nutritional value of foods (Garciasegovia et al., 2007, Martínez-Hernández et al., 2013). SV, which has the characteristics of low heating temperature and heat insulation, can provide different degrees of protection to nutrients in food.

Proteins and fats    Cooking temperature and time can affect free radicals in animal-derived foods, and the oxidation of lipids and proteins can lead to changes in nutritional values (Estévez, et al., 2011). The oxidation of fat in animal-derived foods can be significantly affected by cooking temperature and time: higher cooking temperature and longer cooking time can lead to a higher degree of oxidation of lipid (Roldan et al., 2014, Sanchez Del Pulgar et al., 2012). When the meat heat at high temperature for an extended time, lipid peroxide can react more easily with other compounds (such as proteins, phospholipids, or amino acids) that present in the meat. As a result, the oxidation of secondary lipid, and the thiobarbituric acid reactive substance value (TBARS) decrease, but the formation of Strecker aldehydes increase. Roldan (2014) discovered that the TBARS value in SV lamb meat was significantly affected by cooking temperature and time. Sanchez (2012) reported that higher cooking temperatures and times led to a higher degree of lipid oxidation. However, Nieva-Echevarria (2017) found that SV (core temperature 83 ± 2 °C, did not provoke noticeable changes in sea bass lipids, including cholesterol, phospholipids.

Proteins denature at a low temperature of 35 to 40 °C, and as the temperature increases, the structure will shrink (Warner, et al., 2017). Compared with traditional cooking, SV produces less longitudinal shrinkage of protein, which may be the cause of less cooking loss (Dominguez-Hernandez et al., 2018). With low-temperature heating for a long time, proteolytic enzymes with higher thermal stability undergo protein hydrolysis, thereby affecting cooking losses (Christensen et al., 2011). With prolonged heating time, total protein, and carbonyl compounds in animal-derived foods were increased (Roldan et al., 2014). Additionally, the denaturation of myofibrillar protein is more prominent, and temperature-time-dependent denaturation becomes more sensitive, causing the disappearance of low- and high-molecular-weight protein bands, as well as low- and high-molecular-weight protein pairs (Murphy and Marks, 2000).

Vitamins    Appropriate cooking methods can have protective effects on nutrients. SV technology has demonstrated to be superior to traditional cooking, by which vitamin C, vitamin B6, and folic acid in broccoli, and anthocyanin in purple potato are protected (Petersen, 1993), and vitamin B12 (Rinaldi et al., 2014), in addition to the protection of vitamin C and other nutrients in green soybean meal. Nonetheless, traditionally boiled carrots found to contain higher beta-carotene content compared with SV carrots (Iborra-Bernad et al., 2015), which suggests that SV may not have protective effects on all types of vitamins.

Others    Traditional boiling methods can result in a 20 to 40% loss of mineral content in foods (Engler-Stringer, 2010, Kimura et al., 1990, Meiners et al., 1976, Severi et al., 1998). Because SV prevents direct contact between food and water (the cooking medium), it can effectively prevent mineral loss in SV beef liver - the losses of minerals, including Ca, Fe, K, Mg, Zn, were less than 14%, which is below that of Cu (27.7%) (Rondanelli et al., 2017). Mineral contents in SV legumes (red lentils, peas, and borlotti beans) and cereals (pearl barley and cereal soup), except the content of K content in cereal soup, Fe in borlotti beans, and Mg in the pearl barley, were found higher than those cooked using the traditional boiling method (Rondanelli et al., 2017). Because high temperatures can cause oxidative cleavage of the porphyrin ring, leading to the release of heme-iron complex (Schricker and Miller, 1983), the traditionally boiled beef liver had the Fe loss of > 20%, while the SV beef liver (cooked at 65 °C for 2 h) had the Fe loss of only 2%. Considering the mineral bioavailability, the bioavailabilities of Cu and Fe, which were 8.78% and 8.80%, respectively, in the raw bovine liver, were increased to 26.9% and 36.5%, respectively in SV bovine liver compared with 14.9% and 11.2%, respectively in the traditionally boiled liver (Rondanelli et al., 2017). It indicates that the mineral bioavailability increases with prolonged low-temperature cooking time.

Traditional cooking methods can also cause losses of antioxidants and phenolics in fruits and vegetables (Bushra et al., 2008, Sissi et al., 2008). While traditional cooking leads to the reduction in the antioxidant capacity of most vegetables, SV reduces dry matter, total phenolics, and antioxidant activity of green beans and Swedish cabbage (Baardseth et al., 2010). Compared with those of raw borage, the total phenolic content and antioxidant activity of the SV borage (90 °C, 949 s) were increased by 1.8 and 2.5 times, respectively, while the total phenolic content in traditional boiled borage was lost by 32% (Alcuson et al., 2017). Although SV vegetables found to have improved antioxidant capacity (Kosewski et al., 2018), a research conducted to determine the antioxidant capacity of fruits and vegetables that were cooked using other cooking methods suggested that further research, determining antioxidant capacity of which fruits and vegetables are protected by SV, should be carried out.

Any loss that occurs during cooking affects the cost of food production. SV can increase the product yield, and with the increased cooking time at a specific temperature range, it has no effects on cooking losses. Compared with traditional cooking methods, the production of SV chicken sausage (100 °C, 30 min) increased by 0.77%, whereas the cooking losses were decreased by 0.7%, and the ash content was increased by 0.27% (Naveena et al., 2017). The cooking loss in SV pork, which was cooked with increasing temperature from 53 °C to 58 °C, while the cooking time found not to affect cooking loss (Christensen et al., 2011).

Ensure food safety and stability

In contrast to the traditional cooking, SV, which can improve food quality, reduce the loss of nutrients, improve food color, is performed at a temperature of generally below 100 °C, its safety is, therefore, questioned (Hansen and Knøchel, 1996, Juneja and Marks, 2003). Consequently, many scholars have conducted research focusing on the safety of food prepared by sous vid. SV relies on low temperatures that are sufficient to cause the death of microorganisms (Rybkarodgers, 2001, Snyder et al., 1995) and vacuum packaging that can effectively inhibit microbial spoilage, prolonging storage time (to more extended than the storage time of conventional air-packed food). Mussels cooked by SV (85 °C, 10 min) can store in a freezer for 21 d, by contrast, traditionally cooked mussels (90 °C, 10 min) have a shelf life of only 14 d (Bongiorno et al., 2018). White shrimp cooked using SV has extended shelf life of up to 28 d, whereas the vacuum- and air-packaged samples have a shelf life of only 15 d and 8 d, respectively (Mohan et al., 2017). Numerous researched explicitly focusing on the studies of heat resistance of human pathogens in SV products (Vajda et al., 2016). These studies showed that Clostridium perfringens in SV samples the initial viable cell numbers were generally two orders of magnitude lower, which is partly the result of the death of cells sensitive to pressure changes. The samples SV at 55 °C for 20 min, at 60 °C for 4 min, and at 65 °C for 1.5 min to reduce the initial number of S. enteritidis cells. With detection limits of 10 CFU mL1 or less, SV found to exert a specific effect on some pathogenic microorganisms, and the SV samples found to have the antibacterial ability.

In addition to controlling the cooking temperature, numerous scholars have adopted other methods, including SV and non-heat-treated packages (Hernandez et al., 2017), irradiation (Dogruyol and Mol, 2017), ultra-high pressure processing (Sun et al., 2017), microwave (Renna et al., 2017), and the addition of essential oils (Gouveia et al., 2017) and tea powder (Juneja et al., 2009) to control pathogenic microorganisms. The number of mesophilic and psychrophilic bacteria and total volatile basic nitrogen values of irradiated SV fish were within the limit after eight weeks of refrigeration, whereas that of un-irradiated samples exceeded the limit after six weeks (Dogruyol and Mol, 2017). Treatment with ultra-high pressure (450 and 600 MPa) for 10 min was sufficient to kill E. coli in uncooked steak, and high pressure did not affect its quality (Sun et al., 2017). Escherichia coli and L. monocytogenes, which inoculated into fresh chicory, were completely inactivated when fresh chicory cooked in a vacuum microwave for 90 s (Renna et al., 2017). Spices contain antimicrobial substances that delay the growth of spoilage microorganisms, thus extending the shelf life of meat while improving its tenderness and palatability (Kargiotou et al., 2011, Nisiotou et al., 2013). The pickling juice provides an acidic environment that reduces the population density of the pathogens (Bremer and Osborne, 1995, Calicioglu et al., 2002). Pickling causes pathogens to become more sensitive to lethal thermal effects, thereby ensuring the vacuum processed products are free from microorganisms (Nisiotou et al., 2013, Pathania et al., 2010). Karyotis (2017) studied marinated chicken breast in Japanese-style teriyaki sauce. It showed that the D-values (the time required to reduce the microbial population by 90%) of L. monocytogenes and Salmonella in chicken breast meat after SV (55, 57.5 and 60 °C) were lower than those in control samples. SV-cooked fillet (65 °C, 12.5 min) marinated in the fish sauce has a shelf life of 49 d, whereas that of marinated in basil sauce has a shelf life of 42 d (Kato et al., 2017).

Moreover, the addition of combined exogenous inhibitors can extend the shelf life of SV products, as has been demonstrated in the addition of rosemary diterpene mixtures, which have antimicrobial activity into SV chicken sausages (Klancnik et al., 2009). Such an approach was able to prolong the storage of chicken to 120 d at 4 ± C, and the chicken had relatively lower TBARs, bacterial plate count, and paedophilic plate count (Naveena et al., 2017). These observations show that the combination of various cooking methods can be a new approach leading to new food technology that does not change the quality of raw materials while controlling the number of microorganisms, ensuring food safety, and reducing production costs.

It is well known that when the food temperature exceeds 52.3 °C, all known food pathogens stop growing and begin to die. And we have already mentioned the application of some new technologies, and the addition of essential oils, salt, or spices can all decrease the number of active pathogens. However, many factors determine the rate of bacterial death, including meat species, muscle type, and fat content. Therefore, for food safety, sous vide cooking should usually be at 55 °C or higher—also, enough heating time to ensure the safety of food consumption.

Nevertheless, Nieva-Echevarria (2017) found that 2,4-dimethyl-1-heptene, detected in SV (85 °C) European sea bass, is a well-known by-product of polypropylene degradation (Suman, 2001). Other scholars have not reported similar food safety issues. Thus, it is also essential to avoid food safety hazards brought by other parties.

Effectiveness and convenience

As changing demographics have increased, growing dual-income families, singles and seniors, fast and convenient food has been paid more and more attention (Smith et al., 1990). Consumers demand foods that are convenient, efficient, easy to prepare, and high-quality food (Galimpin-Johan et al., 2007, Koo et al., 2008). SV cooking can provide both intermediate food (Kumari et al., 2016, Yang et al., 2020) in the cold chain and ready-to-eat food (Dominguez-Soberanes et al., 2017, Gonnella et al., 2018, Renna et al., 2017). Studies have shown that SV food is convenient and fast to make, low health risk, and has a long storage period (Nissen et al., 2002). In addition, SV cooking is suitable for a variety of raw materials, which can be served directly, or stored in refrigeration or frozen, and provide services after reheating (Figure 1). Therefore, SV cooking is suitable for many service scenarios, e.g., families, restaurants, hospitals, and old age welfare services (Botinestean et al., 2016, Briley, 1992).

Fig. 1.

Flow chart of sous vide cooking

Challenges and prospect

For decades, SV has proved to be safe by some scientists. The precisely controlled temperature and time of SV technology have demonstrated to improve flavor, texture, the color of food, in addition to preserving its nutritional values and prolonging its storage time, the quality that cannot obtain from the traditional cooking methods. However, vacuum and heating at a lower temperature reduce or hinder fat oxidation and Maillard reaction, thereby reducing the flavor of food. Besides, packaging also prevents the spillage of more inferior volatile components. The challenges for SV are to enrich the flavor of SV food and improve the appearance color of SV animal-derived food to replace traditional cooking methods. The future trend is from restaurants to food factories, adopting SV technology at all levels of catering services, and industrializing and standardizing it. Therefore, studies on SV in various aspects, such as structural changes of food and the mechanism of changes in food quality, to prolong food storage time while maintaining its quality can increasingly become new research interests.

Author contributions

Conceptualization, Hao Zhang and ZhenKun Cui; methodology, ZhenKun Cui and Han Yan; writing—original draft preparation, ZhenKun Cui. and Han Yan; writing—review and editing, HaiZhen Mo, JiCai Bi and Tatiana Manoli; funding acquisition, HaiZhen Mo and Hao Zhang; investigation, Hao Zhang. All authors have read and agree to the published version of the manuscript.

Acknowledgements    This research was supported by Henan Province Technology System for Conventional Freshwater Fish Industries (S2014-10-g02).

Conflicts of Interest    The authors declare that they do not have any conflict of interest.

References
 
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