**Table 1.**

**35**

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

Atlantic herring or their by-products [13].

*3.1.1 Wet reduction process*

main stages:

Marine oils are produced from whole fish mainly small pelagic species, but also from by-products generated by the transformation industry. In 2018, it was estimated that between 25 and 35% of the total volume of fishmeal and fish oil produced came from by-products [2]. In production of marine oils, different techniques can be

employed such as wet reduction process [52, 91, 92], solvent extraction [37, 87, 89, 106], supercritical fluid extraction [50, 90, 97, 98, 107, 108], urea complexation [108], cold

The traditional process for the production of marine oils is coupled with the production process of fishmeal. It is based on a heat treatment of the raw material which allows breaking the cell membrane to liberate the oil, pressing and separation [15, 106]. The principle of this process is based on the separation of the lipid phase from various fish compounds. The oil is separated by a decanter that separates insoluble compounds from the liquid phase, and a separator, separating oil and water. Another possibility is to use a tricanter, which separates solid, water phase and oil in one operation [8]. This process is mainly used in the treatment of fatty species containing high levels of fats such as anchovies, menhaden, sardines,

On an industrial scale, the wet extraction technique is the most used method to obtain the oil and a substrate rich in proteins [106]. This process consists of 3

Cooking, where the biomass is heated in a continuous screw cooker. Oil and water are separated from the solid protein. In order to recover an oil of high quality, the temperature and pressure must be adapted to the type and size of used biomass. Cooking can be done by direct injection of steam or by indirect steam heating in

Pressing, a screw press squeezes the oil and water from the cooked biomass to separate the liquid phase containing the oil and the solid material. Press juice contains a considerable amount of suspended solids in the form of coagulated proteins, scales, edge fragments which have escaped the pressing. These particles are added to

Centrifugation, which is now preferable compared to decantation, separates the oil from the aqueous phase. Draining and press water can be treated with live steam, allowing better separation of the oily phase on a horizontal centrifugal decanter [116]. In this step, water can be added also to wash the oil from any remaining impurities. At the end of this operation, the oil and stick water are obtained. The stickwater is evaporated to a concentrate before to add it to the pressing cake [36, 47, 117]. Quality and stability of the oil produced depend on process conditions. On one hand, the quality of oil depends on temperature, pumping speed and centrifugation. The marine oil is more stable if high pumping speed is used [118]. On the other hand, Chantachum and co-authors studied the influence of precooking on the separation and quality of tuna heads oil [92]. The results obtained have showed that

press allows better release of the oily fractions which would be slowed down when cooked at a higher temperature due to the coagulation of proteins. The process conditions applied normally depend also on the type and quality of raw material used [117]. Comparison of quality of herring oils produced from three different types of by-products: heads, mixed by-products (heads, frames, skin, viscera, etc.), and headless byproducts showed that heads by-products and its oil presented the highest oxidation levels and the lowest R-tocopherol content. Heads contained the lowest PUFAs level and the highest amount of saturated fatty acids (SFAs) [55].

using a hydraulic

order to denature cell proteins and facilitate oil extraction [113, 114].

the fishmeal while the liquid phase undergoes centrifugation [115].

cooking at 85°C for 30 min, followed by pressing at 140 tons/m2

pressing [99] and enzymatic treatment [9, 93–96, 101, 104, 109–112].

*EPA and DHA content (% of total fatty acid) in oil produced from marine by-products by different methods of extraction and purification.*

#### *Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

Marine oils are produced from whole fish mainly small pelagic species, but also from by-products generated by the transformation industry. In 2018, it was estimated that between 25 and 35% of the total volume of fishmeal and fish oil produced came from by-products [2]. In production of marine oils, different techniques can be employed such as wet reduction process [52, 91, 92], solvent extraction [37, 87, 89, 106], supercritical fluid extraction [50, 90, 97, 98, 107, 108], urea complexation [108], cold pressing [99] and enzymatic treatment [9, 93–96, 101, 104, 109–112].

#### *3.1.1 Wet reduction process*

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**34**

**Species** Salmon Salmon (*Salmo salar*)

Rainbow trout (*Oncorhynchus mykiss*)

Rainbow Trout (Oncorhynchus mykiss)

Nile perch (*Lates niloticus*)

Nile perch (Lates niloticus)

Salmon (Salmo salar)

Sardine

**Table 1.**

Bones with leftover fish meat,

skin, scales, fins

Viscera

Heads Heads Heads *EPA and DHA content (% of total fatty acid) in oil produced from marine by-products by different methods of extraction and purification.*

3.0 3.4 6.1 10.95

9.0 7.7 8.4

13.01

CO2 supercritical extraction

[105]

Enzymatic hydrolysis

Enzymatic hydrolysis

[103]

[104]

Roe

**By-products** head, skin, viscera, backbone,

frames, cuts off

Heads

7.7 8.4 11.3 11.5 6.49–6.89

11.9 12.1 19.0 24.0 14.76–5.72

Enzymatic hydrolysis

Solvent extraction

Enzymatic hydrolysis

Solvent extraction [88]

Isoelectric solubilization/precipitation

[102]

[101]

[100]

**EPA** 2.46

**DHA**

2.97

**Process** Cold pressing

**Ref**

[99]

The traditional process for the production of marine oils is coupled with the production process of fishmeal. It is based on a heat treatment of the raw material which allows breaking the cell membrane to liberate the oil, pressing and separation [15, 106]. The principle of this process is based on the separation of the lipid phase from various fish compounds. The oil is separated by a decanter that separates insoluble compounds from the liquid phase, and a separator, separating oil and water. Another possibility is to use a tricanter, which separates solid, water phase and oil in one operation [8]. This process is mainly used in the treatment of fatty species containing high levels of fats such as anchovies, menhaden, sardines, Atlantic herring or their by-products [13].

On an industrial scale, the wet extraction technique is the most used method to obtain the oil and a substrate rich in proteins [106]. This process consists of 3 main stages:

Cooking, where the biomass is heated in a continuous screw cooker. Oil and water are separated from the solid protein. In order to recover an oil of high quality, the temperature and pressure must be adapted to the type and size of used biomass. Cooking can be done by direct injection of steam or by indirect steam heating in order to denature cell proteins and facilitate oil extraction [113, 114].

Pressing, a screw press squeezes the oil and water from the cooked biomass to separate the liquid phase containing the oil and the solid material. Press juice contains a considerable amount of suspended solids in the form of coagulated proteins, scales, edge fragments which have escaped the pressing. These particles are added to the fishmeal while the liquid phase undergoes centrifugation [115].

Centrifugation, which is now preferable compared to decantation, separates the oil from the aqueous phase. Draining and press water can be treated with live steam, allowing better separation of the oily phase on a horizontal centrifugal decanter [116]. In this step, water can be added also to wash the oil from any remaining impurities. At the end of this operation, the oil and stick water are obtained. The stickwater is evaporated to a concentrate before to add it to the pressing cake [36, 47, 117].

Quality and stability of the oil produced depend on process conditions. On one hand, the quality of oil depends on temperature, pumping speed and centrifugation. The marine oil is more stable if high pumping speed is used [118]. On the other hand, Chantachum and co-authors studied the influence of precooking on the separation and quality of tuna heads oil [92]. The results obtained have showed that cooking at 85°C for 30 min, followed by pressing at 140 tons/m2 using a hydraulic press allows better release of the oily fractions which would be slowed down when cooked at a higher temperature due to the coagulation of proteins. The process conditions applied normally depend also on the type and quality of raw material used [117]. Comparison of quality of herring oils produced from three different types of by-products: heads, mixed by-products (heads, frames, skin, viscera, etc.), and headless byproducts showed that heads by-products and its oil presented the highest oxidation levels and the lowest R-tocopherol content. Heads contained the lowest PUFAs level and the highest amount of saturated fatty acids (SFAs) [55].

Oil recovery yield varies during the year depending on fat content of by-products. Extraction of oil from cooked by-products of *Sardina pilchardus* (skin, meat, bones and cooking condensate), using wet hydraulic pressing at 85°C for 30 min and centrifugation, gave an important oil yield varying between 6.0% and 32.9% depending on the fishing period [52]. Another study reported that the percentage of oil extracted from cooked by-products of *Sardinella gibbosa* was 8.96% [119].

Wet reduction process is more suitable to extract oil from fatty fish producing oil with improved quality and high level of n-3 PUFAs [50]. However, the main disadvantage of this technique is the use of high temperature during cooking that degrades oxidative quality of the oil and causes a loss of EPA and DHA contents due to hydrolysis and oxidation reactions [12, 111, 120].

In addition to the conventional extraction process called also wet reduction or hydraulic pressing, the extraction of marine oils could be obtained with several methods, such as enzymatic hydrolysis, physical fractionation, low-temperature solvent fractionation, supercritical fluid extraction and pH adjustement method to the isoelectric point (**Table 1**).

#### *3.1.2 Enzymatic hydrolysis*

The principle of enzymatic hydrolysis is based on the action of specific proteases at low temperature on protein tissue without use of solvents and high temperature [13, 50]. Firstly, the by-products are hydrolyzed by the use of commercial proteolytic enzymes and endogenous enzymes. These enzymes destroy the structure of cells walls. They broke down protein molecules to small peptides and amino acids which allows releasing the oil contained. After inactivation of the enzymes, the oil, hydrolysate and the insoluble residue are separated. The released oil can be centrifuged as previously described in the conventional process. For example, Batista and co-authors extracted oil from black scabbardfish (*Aphanopus carbo*) by-products using enzymatic hydrolysis with 1% Protamex [95]. The percentage of free oil released from the by-products has reached 36% of the total amount.

The enzymatic extraction is influenced by several operating factors, namely: nature of enzymes, temperature, pH, concentration of enzymes during hydrolysis, method and quality of grinding, and water content of the raw material [93, 110, 111, 120]. Optimal conditions of hydrolysis of sardine heads by Protamex were studied (temperature, hydrolysis time and enzyme-substrate ratio) [93]. Results have showed that optimum conditions were found to be similar to recover lipids and phospholipids (29 min, 31°C with 2.6 g.kg-1 enzyme). The same study has showed that hydrolysis could increase the extraction of lipids and phospholipids by 27% and 50%, respectively compared to classical extraction.

Šližyte and co-authors studied the enzymatic hydrolysis of mixtures of cod (*Gadus morhua*) by-products using Flavoenzyme and Neutrase [110]. The results demonstrated that the most important factor influencing the extraction yield is the added water regardless the type of enzyme. On the other hand, using an enzymatic process based on a proteolytic extraction of oil from crude tuna heads followed by a urea complexation (−5°C, 20 h) has allowed to obtain a mixture of DHA and EPA with a purity of 85.02% and a liquid recovery yield of 25.10% [121].

The enzymatic hydrolysis of salmon frames with Protamex, was able to separate the salmon frames into an aqueous fraction rich in soluble nitrogen (fish protein hydrolysate), an insoluble nitrogen fraction, an emulsion fraction, salmon oil and a bone fraction rich in protein and minerals. This process allows to separate oil from protein fraction that can be valorized to recover peptides, essential amino acids or other molecules [94].

**37**

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

yields compared with the traditional process [8].

*3.1.3 Supercritical carbon dioxide*

becomes a gas and evaporates [120].

some arsenic products [50].

researchers [100, 101].

Batista and co-authors studied the use of 0.5% of three commercial enzymes (Alcalase, Neutrase, and Protamex TM) and a water/fish ratio of 1:1 for production of protein hydrolysates and oil from raw and cooked sardine by-products from the canning industry [96]. Results have showed that the highest nitrogen solubilization and degree of hydrolysis were obtained with Alcalase and Protamex. The raw byproducts were more easily hydrolyzed by these enzymes than the cooked sardine. The highest percentage of oil released was obtained from raw sardine, and Alcalase and Protamex were the most efficient. In another study, enzymatic extraction of oil from ground salmon heads at 55°C using different commercial enzymes (Alcalase, Neutrase, and Flavourzyme) showed that the highest oil recovery (17.4%) was obtained after 2 h by using Alcalase [111]. Same results were obtained later by other

The enzymatic process can be chosen to extract the oil for many reasons. First, it is conducted under mild conditions protecting PUFAs from oxidation. Second, this technique does not use chemical solvents in addition to short time of hydrolysis [120, 122]. However, this process focuses more on the yield and production of protein hydrolysates than on the production of high quality oil. This disadvantage is due to the presence of lipase in the by-products which still active during the hydrolysis therefore it affects the oil quality [96]. Furthermore, this effect is more important if the hydrolysis is done in the presence of oxygen which increases significantly the free fatty acids in the oil, compared with the conventional method [8]. The main product from this process is the protein hydrolysates that are produced with higher

Extraction with supercritical carbon dioxide (SC-CO2) is a promising technique, as CO2 is nontoxic gas, nonflammable and clean solvent [123]. This technique can be carried out under mild operating conditions in an oxygen-free environment and at a moderate temperature, preventing degradation of PUFAs [12, 50, 90, 97, 98, 105]. CO2 used is a green solvent. At or above critical temperature and pressure (31.1°C, 7.39 MPa), CO2 is in a liquid state while at ambient temperature and pressure, CO2

This technique allows the extraction of lipids of low polarity, avoids the extraction of impurities and reduces the heavy metal content [12, 50, 90]. Rubio-Rodriguez and co-authors proposed to couple this technique with extractionfractionation process to remove free fatty acids and improve fish oil quality, alternatively to physical and chemical refining [50]. Same authors compared different oil extraction methods from fish by-products, cold extraction or centrifuging, wet reduction, enzymatic extraction and supercritical fluid extraction. This study has showed that SC-CO2 is an interesting method, operating conditions are suitable to prevent lipid oxidation and to reduce the amount of certain pollutants such as

Another research has also compared 3 different oil extraction methods (supercritical carbon dioxide, n-hexane and traditional pressing) from Atlantic salmon by-products (belly part, trimmed muscle, frame bone and skin). The maximum oil yield was obtained by n-hexane extraction (total oil), followed by supercritical CO2 extraction (highly selective technique extracting non-polar compounds) and the traditional pressing that has showed the lowest yield. Likewise, differences were noted in the oil quality parameters between the 3 studied techniques, the longer oxidative stability was obtained in the oil extracted by supercritical fluid CO2 [97]. This technique was also compared to soxhlet extraction using hexane to produce oil from skins, bones and scales of bigeye tuna (*Thunnus obesus*). This study

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

oil extracted from cooked by-products of *Sardinella gibbosa* was 8.96% [119].

to hydrolysis and oxidation reactions [12, 111, 120].

and 50%, respectively compared to classical extraction.

with a purity of 85.02% and a liquid recovery yield of 25.10% [121].

the isoelectric point (**Table 1**).

*3.1.2 Enzymatic hydrolysis*

amount.

Oil recovery yield varies during the year depending on fat content of by-products. Extraction of oil from cooked by-products of *Sardina pilchardus* (skin, meat, bones and cooking condensate), using wet hydraulic pressing at 85°C for 30 min and centrifugation, gave an important oil yield varying between 6.0% and 32.9% depending on the fishing period [52]. Another study reported that the percentage of

Wet reduction process is more suitable to extract oil from fatty fish producing oil with improved quality and high level of n-3 PUFAs [50]. However, the main disadvantage of this technique is the use of high temperature during cooking that degrades oxidative quality of the oil and causes a loss of EPA and DHA contents due

In addition to the conventional extraction process called also wet reduction or hydraulic pressing, the extraction of marine oils could be obtained with several methods, such as enzymatic hydrolysis, physical fractionation, low-temperature solvent fractionation, supercritical fluid extraction and pH adjustement method to

The principle of enzymatic hydrolysis is based on the action of specific proteases at low temperature on protein tissue without use of solvents and high temperature [13, 50]. Firstly, the by-products are hydrolyzed by the use of commercial proteolytic enzymes and endogenous enzymes. These enzymes destroy the structure of cells walls. They broke down protein molecules to small peptides and amino acids which allows releasing the oil contained. After inactivation of the enzymes, the oil, hydrolysate and the insoluble residue are separated. The released oil can be centrifuged as previously described in the conventional process. For example, Batista and co-authors extracted oil from black scabbardfish (*Aphanopus carbo*) by-products using enzymatic hydrolysis with 1% Protamex [95]. The percentage of free oil released from the by-products has reached 36% of the total

The enzymatic extraction is influenced by several operating factors, namely: nature of enzymes, temperature, pH, concentration of enzymes during hydrolysis, method and quality of grinding, and water content of the raw material [93, 110, 111, 120]. Optimal conditions of hydrolysis of sardine heads by Protamex were studied (temperature, hydrolysis time and enzyme-substrate ratio) [93]. Results have showed that optimum conditions were found to be similar to recover lipids and phospholipids (29 min, 31°C with 2.6 g.kg-1 enzyme). The same study has showed that hydrolysis could increase the extraction of lipids and phospholipids by 27%

Šližyte and co-authors studied the enzymatic hydrolysis of mixtures of cod (*Gadus morhua*) by-products using Flavoenzyme and Neutrase [110]. The results demonstrated that the most important factor influencing the extraction yield is the added water regardless the type of enzyme. On the other hand, using an enzymatic process based on a proteolytic extraction of oil from crude tuna heads followed by a urea complexation (−5°C, 20 h) has allowed to obtain a mixture of DHA and EPA

The enzymatic hydrolysis of salmon frames with Protamex, was able to separate the salmon frames into an aqueous fraction rich in soluble nitrogen (fish protein hydrolysate), an insoluble nitrogen fraction, an emulsion fraction, salmon oil and a bone fraction rich in protein and minerals. This process allows to separate oil from protein fraction that can be valorized to recover peptides, essential amino acids or

**36**

other molecules [94].

Batista and co-authors studied the use of 0.5% of three commercial enzymes (Alcalase, Neutrase, and Protamex TM) and a water/fish ratio of 1:1 for production of protein hydrolysates and oil from raw and cooked sardine by-products from the canning industry [96]. Results have showed that the highest nitrogen solubilization and degree of hydrolysis were obtained with Alcalase and Protamex. The raw byproducts were more easily hydrolyzed by these enzymes than the cooked sardine. The highest percentage of oil released was obtained from raw sardine, and Alcalase and Protamex were the most efficient. In another study, enzymatic extraction of oil from ground salmon heads at 55°C using different commercial enzymes (Alcalase, Neutrase, and Flavourzyme) showed that the highest oil recovery (17.4%) was obtained after 2 h by using Alcalase [111]. Same results were obtained later by other researchers [100, 101].

The enzymatic process can be chosen to extract the oil for many reasons. First, it is conducted under mild conditions protecting PUFAs from oxidation. Second, this technique does not use chemical solvents in addition to short time of hydrolysis [120, 122]. However, this process focuses more on the yield and production of protein hydrolysates than on the production of high quality oil. This disadvantage is due to the presence of lipase in the by-products which still active during the hydrolysis therefore it affects the oil quality [96]. Furthermore, this effect is more important if the hydrolysis is done in the presence of oxygen which increases significantly the free fatty acids in the oil, compared with the conventional method [8]. The main product from this process is the protein hydrolysates that are produced with higher yields compared with the traditional process [8].

#### *3.1.3 Supercritical carbon dioxide*

Extraction with supercritical carbon dioxide (SC-CO2) is a promising technique, as CO2 is nontoxic gas, nonflammable and clean solvent [123]. This technique can be carried out under mild operating conditions in an oxygen-free environment and at a moderate temperature, preventing degradation of PUFAs [12, 50, 90, 97, 98, 105]. CO2 used is a green solvent. At or above critical temperature and pressure (31.1°C, 7.39 MPa), CO2 is in a liquid state while at ambient temperature and pressure, CO2 becomes a gas and evaporates [120].

This technique allows the extraction of lipids of low polarity, avoids the extraction of impurities and reduces the heavy metal content [12, 50, 90]. Rubio-Rodriguez and co-authors proposed to couple this technique with extractionfractionation process to remove free fatty acids and improve fish oil quality, alternatively to physical and chemical refining [50]. Same authors compared different oil extraction methods from fish by-products, cold extraction or centrifuging, wet reduction, enzymatic extraction and supercritical fluid extraction. This study has showed that SC-CO2 is an interesting method, operating conditions are suitable to prevent lipid oxidation and to reduce the amount of certain pollutants such as some arsenic products [50].

Another research has also compared 3 different oil extraction methods (supercritical carbon dioxide, n-hexane and traditional pressing) from Atlantic salmon by-products (belly part, trimmed muscle, frame bone and skin). The maximum oil yield was obtained by n-hexane extraction (total oil), followed by supercritical CO2 extraction (highly selective technique extracting non-polar compounds) and the traditional pressing that has showed the lowest yield. Likewise, differences were noted in the oil quality parameters between the 3 studied techniques, the longer oxidative stability was obtained in the oil extracted by supercritical fluid CO2 [97].

This technique was also compared to soxhlet extraction using hexane to produce oil from skins, bones and scales of bigeye tuna (*Thunnus obesus*). This study has confirmed improved quality parameters of oil obtained by supercritical CO2 technique extraction (low heavy metal content in the oil) [90].

Using the ground skin of Indian mackerel (*Rastrelliger kanagurta*), various techniques of supercritical CO2 were studied by varying pressure (20-35 MPa) and temperature (45–75°C). This study has showed that oil yield increased with pressure and temperature and the highest yields were 24.7, 53.2, 52.8, and 52.3/100 g sample (dry basis) for the continuous, cosolvent, soaking, and pressure swing techniques, respectively, at 35 MPa and 75°C [98].

Supercritical fluid technology coupled with membrane, enzymatic or adsorption process have been shown to produce high-quality oil with best reduction of levels of contaminants compared to traditional refining of oils [124, 125].

Faced with all these advantages, the main drawback of this method is the high cost of the application on an industrial scale [12, 50, 124]. In this contest, the effect of supercritical CO2 techniques on CO2 consumption was studied. The results have showed that the total amount of CO2 consumption decreases significantly with temperature and increases with pressure in all extraction modes using supercritical CO2 method. A higher amount of CO2 was needed for the continuous technique, compared to the techniques of cosolvent, soaking and pressure swing regardless of levels of pressure and temperature. Consequently, the best extraction technique of the oil with least amount of CO2 consumption was achieved with pressure swing mode at 35 MPa and 75 C [98].

#### *3.1.4 Solvent extraction*

Solvent extraction methods are numerous. They have been studied by several researchers applied on marine by-products [37, 87, 89, 106]. Unfortunately, these techniques have many disadvantages. Large amount of hazardous solvent and important energy are required. Besides, marine oils are oxidized exhibiting a strong red-brown or brown color when extraction of oil is done at high temperature and longtime [90]. Generally, extraction by solvent is only carried out on a laboratory scale for analytical purposes [124]. Among the widely used techniques, Bligh and Dyer method is the most recommended for the total extraction of lipids from biological tissues [126]. Most of the published data on lipid content are related to this method [105, 127–129]. The effectiveness of the Bligh and Dyer method was evaluated compared to Soxhlet method. The results have showed that the Bligh and Dyer extraction method is more effective in extracting polar and non-polar lipids from fish compared to the Soxhlet technique [130].

#### *3.1.5 Cold pressing extraction*

Other innovative processes are coming to the market such as cold pressing extraction, a patented process originating from the olive oil production industry [13]. This technique allows protection of PUFAs content, producing high quality marine oil from different types of by-products [50, 99]. This process is well known to produce a lower-yield, but higher quality of oil [131].

Due to its high degree in PUFAs, marine oils are very sensitive to oxidation. The degree of oxidation increases in the presence of air (oxygen), light and heat during extraction and storage [8, 47]. This phenomenon mainly reduces the shelf life of marine oils [53]. All techniques using high-temperature or toxic solvents can induce degradation and loss of nutritional qualities of marine oil.

For this, looking for gentle extraction of marine lipids or using non-heat processes might generate more stable lipid fractions [8]. It is also necessary to protect the oil by stopping or slowing down the oxidation process during the production

**39**

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

with supplements n-3 fatty acids ready to consume.

**3.2 Production of n-3 fatty acids concentrates**

enrichment in EPA and DHA.

and during storage. Adding antioxidants to the oil is one of the most used methods [132]. Using a modified atmosphere packaging [133], or encapsulation, which keeps marine oil away from oxygen and light [133, 134] can be also used. In addition to protecting the oil, the use of microencapsulation technology provides consumers

Another valorization of marine oils (produced from whole fish or from fish by-products) is their use in production of concentrated n-3 fatty acids in the form of free fatty acids, methyl and ethyl esters or acylglycerols [135]. Several processes can be used, the most important are urea complexation [108, 121, 136], molecular distillation [137], supercritical fluid extraction [98, 108], winterization [138, 139], fractionation by chromatography [120] and by enzymatic processes [111, 112, 140]. These techniques have been reviewed by many authors [13, 124, 135] and recently by [12]. The main challenge in the choice of concentration technique at industrial level is to reach higher yield and purity at lower cost [13, 124, 138]. **Table 2** outlines some methods to produce n-3 fatty acids concentrates with levels achieved of

Concentration by winterization allows elimination of SFAs present in the oil, which crystallize at low temperatures [139]. Winterization is primarily designed for oils with a high content of SFAs. It allows elimination of stearic phase, by cooling the oil to 0–4°C. The degree of concentration of PUFAs by this process is evaluated as low as these interesting fatty acids could be lost in the stearic fraction [13]. However, this method produces n-3 PUFAs concentrate in natural form [138].

An alternative solvent winterization and enzymatic interesterification was studied to concentrate n-3 fatty acids in cod liver oil [138]. The optimization parameters considered were separation method, solvent, oil concentration, time and temperature of winterization. Likewise, enzymes used were examined for interesterification efficiency under different system air condition, time and temperature. Authors proposed the optimal conditions of the technique via winterization (0.1 g/mL oil/acetone, 24 h, −80°C, precooled Büchner filtration) and interesterification (Lipozyme TL IM, N2 flow, 2.5 h, 40°C) improving n-3 fatty acid content to 43.20 mol%.

In another study, winterization was carried out on a bleached oil by a progressive cooling (30–5°C) in three phases. The effect of solvent type, solvent proportion, and agitation in the second cooling stage was studied. The results have demonstrated that using hexane has improved content of PUFA of 64.3% with 13% as a decrease percentage in level of SFAs compared to the fatty profile of bleached oil [139].

In addition to what is explained previously for CO2 supercritical fluid extraction, this technique could be used to concentrate fatty acids. It's based on the use of CO2 which, in the supercritical state, behaves like an extraction fluid and entrains fatty acids. In several passages, their concentrations therefore increase [13]. This method

Supercritical carbon dioxide was used for simultaneous extraction and fractionation of fish oil from Tuna by-products [143]. The obtained oil was divided into six fractions based on molecular weight and the chain length of triglycerides in terms of fatty acid constituents. The results showed that the three first separated fractions were rich in SFAs followed by monounsaturated fatty acids (MUFAs), then PUFAs.

Melgosa and co-authors studied the use of supercritical CO2 as solvent in the lipase-catalyzed ethanolysis of fish oil. The effect of initial substrate ethanol/oil molar ratio (2–38), pressure (7.5–30 MPa), and temperature (323.15–353.15 K) on equilibrium conversion, reaction rate and oxidative status of the products were

While the three last fractions contained high levels of MUFAs and PUFAs.

can achieve high concentration levels of n-3 PUFAs.

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

technique extraction (low heavy metal content in the oil) [90].

contaminants compared to traditional refining of oils [124, 125].

respectively, at 35 MPa and 75°C [98].

mode at 35 MPa and 75 C [98].

*3.1.5 Cold pressing extraction*

from fish compared to the Soxhlet technique [130].

to produce a lower-yield, but higher quality of oil [131].

degradation and loss of nutritional qualities of marine oil.

*3.1.4 Solvent extraction*

has confirmed improved quality parameters of oil obtained by supercritical CO2

Using the ground skin of Indian mackerel (*Rastrelliger kanagurta*), various techniques of supercritical CO2 were studied by varying pressure (20-35 MPa) and temperature (45–75°C). This study has showed that oil yield increased with pressure and temperature and the highest yields were 24.7, 53.2, 52.8, and 52.3/100 g sample (dry basis) for the continuous, cosolvent, soaking, and pressure swing techniques,

Supercritical fluid technology coupled with membrane, enzymatic or adsorption process have been shown to produce high-quality oil with best reduction of levels of

Faced with all these advantages, the main drawback of this method is the high cost of the application on an industrial scale [12, 50, 124]. In this contest, the effect of supercritical CO2 techniques on CO2 consumption was studied. The results have showed that the total amount of CO2 consumption decreases significantly with temperature and increases with pressure in all extraction modes using supercritical CO2 method. A higher amount of CO2 was needed for the continuous technique, compared to the techniques of cosolvent, soaking and pressure swing regardless of levels of pressure and temperature. Consequently, the best extraction technique of the oil with least amount of CO2 consumption was achieved with pressure swing

Solvent extraction methods are numerous. They have been studied by several researchers applied on marine by-products [37, 87, 89, 106]. Unfortunately, these techniques have many disadvantages. Large amount of hazardous solvent and important energy are required. Besides, marine oils are oxidized exhibiting a strong red-brown or brown color when extraction of oil is done at high temperature and longtime [90]. Generally, extraction by solvent is only carried out on a laboratory scale for analytical purposes [124]. Among the widely used techniques, Bligh and Dyer method is the most recommended for the total extraction of lipids from biological tissues [126]. Most of the published data on lipid content are related to this method [105, 127–129]. The effectiveness of the Bligh and Dyer method was evaluated compared to Soxhlet method. The results have showed that the Bligh and Dyer extraction method is more effective in extracting polar and non-polar lipids

Other innovative processes are coming to the market such as cold pressing extraction, a patented process originating from the olive oil production industry [13]. This technique allows protection of PUFAs content, producing high quality marine oil from different types of by-products [50, 99]. This process is well known

Due to its high degree in PUFAs, marine oils are very sensitive to oxidation. The degree of oxidation increases in the presence of air (oxygen), light and heat during extraction and storage [8, 47]. This phenomenon mainly reduces the shelf life of marine oils [53]. All techniques using high-temperature or toxic solvents can induce

For this, looking for gentle extraction of marine lipids or using non-heat processes might generate more stable lipid fractions [8]. It is also necessary to protect the oil by stopping or slowing down the oxidation process during the production

**38**

and during storage. Adding antioxidants to the oil is one of the most used methods [132]. Using a modified atmosphere packaging [133], or encapsulation, which keeps marine oil away from oxygen and light [133, 134] can be also used. In addition to protecting the oil, the use of microencapsulation technology provides consumers with supplements n-3 fatty acids ready to consume.

#### **3.2 Production of n-3 fatty acids concentrates**

Another valorization of marine oils (produced from whole fish or from fish by-products) is their use in production of concentrated n-3 fatty acids in the form of free fatty acids, methyl and ethyl esters or acylglycerols [135]. Several processes can be used, the most important are urea complexation [108, 121, 136], molecular distillation [137], supercritical fluid extraction [98, 108], winterization [138, 139], fractionation by chromatography [120] and by enzymatic processes [111, 112, 140]. These techniques have been reviewed by many authors [13, 124, 135] and recently by [12]. The main challenge in the choice of concentration technique at industrial level is to reach higher yield and purity at lower cost [13, 124, 138]. **Table 2** outlines some methods to produce n-3 fatty acids concentrates with levels achieved of enrichment in EPA and DHA.

Concentration by winterization allows elimination of SFAs present in the oil, which crystallize at low temperatures [139]. Winterization is primarily designed for oils with a high content of SFAs. It allows elimination of stearic phase, by cooling the oil to 0–4°C. The degree of concentration of PUFAs by this process is evaluated as low as these interesting fatty acids could be lost in the stearic fraction [13]. However, this method produces n-3 PUFAs concentrate in natural form [138].

An alternative solvent winterization and enzymatic interesterification was studied to concentrate n-3 fatty acids in cod liver oil [138]. The optimization parameters considered were separation method, solvent, oil concentration, time and temperature of winterization. Likewise, enzymes used were examined for interesterification efficiency under different system air condition, time and temperature. Authors proposed the optimal conditions of the technique via winterization (0.1 g/mL oil/acetone, 24 h, −80°C, precooled Büchner filtration) and interesterification (Lipozyme TL IM, N2 flow, 2.5 h, 40°C) improving n-3 fatty acid content to 43.20 mol%.

In another study, winterization was carried out on a bleached oil by a progressive cooling (30–5°C) in three phases. The effect of solvent type, solvent proportion, and agitation in the second cooling stage was studied. The results have demonstrated that using hexane has improved content of PUFA of 64.3% with 13% as a decrease percentage in level of SFAs compared to the fatty profile of bleached oil [139].

In addition to what is explained previously for CO2 supercritical fluid extraction, this technique could be used to concentrate fatty acids. It's based on the use of CO2 which, in the supercritical state, behaves like an extraction fluid and entrains fatty acids. In several passages, their concentrations therefore increase [13]. This method can achieve high concentration levels of n-3 PUFAs.

Supercritical carbon dioxide was used for simultaneous extraction and fractionation of fish oil from Tuna by-products [143]. The obtained oil was divided into six fractions based on molecular weight and the chain length of triglycerides in terms of fatty acid constituents. The results showed that the three first separated fractions were rich in SFAs followed by monounsaturated fatty acids (MUFAs), then PUFAs. While the three last fractions contained high levels of MUFAs and PUFAs.

Melgosa and co-authors studied the use of supercritical CO2 as solvent in the lipase-catalyzed ethanolysis of fish oil. The effect of initial substrate ethanol/oil molar ratio (2–38), pressure (7.5–30 MPa), and temperature (323.15–353.15 K) on equilibrium conversion, reaction rate and oxidative status of the products were


*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**Table 2.** *Some methods of production of concentrated n-3 fatty acids in oil.*

**41**

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

EPA and DHA esters in fish oil [147, 148].

ester concentrate [141].

tion of triglycerides [124].

DHA from oxidation [140, 153].

PUFAs oxidation due to displacement of oxygen [144].

tested. The results have revealed the importance role of employing CO2 in improving reaction kinetics by reduction of mass transfer limitations and prevention of n-3

The results of supercritical fluid extraction applied on Rainbow sardine oil have showed that the highest decrease in SFAs and MUFAs were obtained at 50–60°C and 350 bars [108]. This technique was evaluated selective even in the fractionation of fish oil with lower content of EPA (4.6%) and DHA (6.7%) under conditions of lower pressure (100 and 200 bar) [145]. In another study, conditions (pressure, temperature and supercritical CO2 flow rate) influencing concentration of fatty acids in fish oil by supercritical carbon dioxide were studied [146]. The results have demonstrated that fractionation by a supercritical fluid under optimal conditions: a pressure increase (at 5 kg/h flow rate) and flow rate increase (at 150 bar pressure), both determined a higher EPA + DHA concentration and decreased the EPA/DHA ratio. The same authors proposed to carry out a urea adduction during preparation to decrease the amount of SFAs in the starting oil before supercritical fractionation. With regard to optimization conditions, numerous researches have worked on modeling, simulation, and optimization of the CO2 supercritical fractionation of

Optimal conditions of application by using adjuvant material and modifying CO2 volumetric density and temperature were also investigated in other studies to get the highest fractionation yield of EPA and DHA [149]. Likewise, Antunes-Corrêa and co-authors and Davarnejad and co-authors studied the optimal operating conditions (pressure and temperature) to fractionate fish oil. In the first study [150], the best results based on oil solubility were obtained using 7.8 MPa and 301.15 K. While in the second research [151], the maximal solubility of the fish oil (0.921 g of oil/100 g of CO2) was obtained at optimum conditions of 40°C and 27.2 MPa. In both studies,

The experience of CO2 supercritical fluid chromatography was transferred to be used in laboratory in a pilot plant to produce EPA enriched mixtures. Fractionation was done on a silica adsorption column using CO2 as supercritical solvent [152]. This allowed to achieve best purity of 93% in EPA ethyl ester fraction with a 24.6% yield. The study of the technical and economic feasibility to produce n-3 PUFAs ethyl ester concentrates from trans esterified fish oil using CO2 supercritical fluid extraction has revealed that process cost is around 550 U.S \$/kg DHA and EPA ethyl

Other investigations have studied the use of enzymatic hydrolysis in production of n-3 PUFAs concentrates. This technique involves the use of specific enzymes (lipases), able to catalyze reactions such hydrolysis, ethanolysis or transesterifica-

Concentration of Pacific sardines (*Sardinops sagax*) oil was carried out using lipase-catalyzed hydrolysis [140]. The results of this study have showed that hydrolysis with 250 U from *Candida rugosa* lipase has increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62% to 29.94% with 500 U after 9 h. This technique uses mild conditions (neutral pH and low temperatures), very important to preserve EPA and

Salmon oil produced from by-products of this species by controlled enzymatic procedure with Neutrase has followed a selective enzymatic hydrolysis under mild conditions, using Novozyme SP398 to enrich the n-3 PUFAs. The process used consist of a lipolysis, filtration in flat membrane device and enzymatic re-esterification with glycerol and Immobilized 1,3-specific lipase IM60 (Lipozym IM). This method induced a significant increase in the amount of PUFAs from 39.20 mol% of total fatty acids in the crude oil to 43.29 mol% in the re-esterified permeate [112].

EPA fractionation was recorded not possible and low, respectively.

#### *Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**40**

**Species** Tuna (*Thunnus albacares*)

Rainbow sardine (*Dussumieria acuta*)

Pacific sardines (*Sardinops sagax*)

Atlantic salmon (*Salmo salar*)

Crude oil from Skin-on fillets)

Refined unhydrolyzed oil

Concentrate oil

Crude Salmon by-products oil

Salmon oil 40% hydrolysis

Permeate Esterified permeate

Oil from salmon heads

Permeate Permeate re-esterified

Arctic cod liver oil

Winterized oil

Tunafish oil Tunafish oil ethyl ester

Menhaden oil Menhaden oil after urea inclusion

Refined oil n-3 PUFAs concentrate

n-3 PUFAs concentrate

4.6 5.3 13.5 29.4 14.51 34.17 46.26

40.32

39.47

12.55

41.8

12.6

23.7

18.3

Salmon Arctic cod Tuna (*Thunnus thynnus*)

Menhaden Sardine (Sardinops sagax caeruleus)

**Table 2.**

*Some methods of production of concentrated n-3 fatty acids in oil.*

Oil from white muscle

Concentrate oil

15.39

—

19.47 17.74 28.2 26.86 33.74

3.71 4.72 5.16 5.64

3.6 5.6 5.06 10.53 21.08

17.83

7.63

11.90

11.6

9.9

10.30

Enzymatic re-esterification

Enzymatic hydrolysis

Lipolysis

Re-esterification

—

Alternate solvent winterization and enzymatic

interesterification

—

Supercritical fluid chromatography

—

Urea inclusion method

—

Chemical hydrolysis + urea complexation

Enzymatic hydrolysis + urea complexation

[142]

[136]

[141]

[138]

[111]

9.91

8.94

9.02

29.94

13.62

16.7

25.51

—

29.61

17.45

**Type of oil**

Tuna oil

**EPA**

**DHA**

EPA + DHA (85.02%)

recovery 25.10%

**Process** Concentration by urea complexation

Extraction [88]

CO2 Supercritical extraction

Urea complexation

Low-temperature crystallization with ethanol

—

Extraction and refining

Lipase-catalyzed hydrolysis

—

Lipolysis, filtration

[112]

[140]

[108]

**Ref**

[121]

tested. The results have revealed the importance role of employing CO2 in improving reaction kinetics by reduction of mass transfer limitations and prevention of n-3 PUFAs oxidation due to displacement of oxygen [144].

The results of supercritical fluid extraction applied on Rainbow sardine oil have showed that the highest decrease in SFAs and MUFAs were obtained at 50–60°C and 350 bars [108]. This technique was evaluated selective even in the fractionation of fish oil with lower content of EPA (4.6%) and DHA (6.7%) under conditions of lower pressure (100 and 200 bar) [145]. In another study, conditions (pressure, temperature and supercritical CO2 flow rate) influencing concentration of fatty acids in fish oil by supercritical carbon dioxide were studied [146]. The results have demonstrated that fractionation by a supercritical fluid under optimal conditions: a pressure increase (at 5 kg/h flow rate) and flow rate increase (at 150 bar pressure), both determined a higher EPA + DHA concentration and decreased the EPA/DHA ratio. The same authors proposed to carry out a urea adduction during preparation to decrease the amount of SFAs in the starting oil before supercritical fractionation.

With regard to optimization conditions, numerous researches have worked on modeling, simulation, and optimization of the CO2 supercritical fractionation of EPA and DHA esters in fish oil [147, 148].

Optimal conditions of application by using adjuvant material and modifying CO2 volumetric density and temperature were also investigated in other studies to get the highest fractionation yield of EPA and DHA [149]. Likewise, Antunes-Corrêa and co-authors and Davarnejad and co-authors studied the optimal operating conditions (pressure and temperature) to fractionate fish oil. In the first study [150], the best results based on oil solubility were obtained using 7.8 MPa and 301.15 K. While in the second research [151], the maximal solubility of the fish oil (0.921 g of oil/100 g of CO2) was obtained at optimum conditions of 40°C and 27.2 MPa. In both studies, EPA fractionation was recorded not possible and low, respectively.

The experience of CO2 supercritical fluid chromatography was transferred to be used in laboratory in a pilot plant to produce EPA enriched mixtures. Fractionation was done on a silica adsorption column using CO2 as supercritical solvent [152]. This allowed to achieve best purity of 93% in EPA ethyl ester fraction with a 24.6% yield. The study of the technical and economic feasibility to produce n-3 PUFAs ethyl ester concentrates from trans esterified fish oil using CO2 supercritical fluid extraction has revealed that process cost is around 550 U.S \$/kg DHA and EPA ethyl ester concentrate [141].

Other investigations have studied the use of enzymatic hydrolysis in production of n-3 PUFAs concentrates. This technique involves the use of specific enzymes (lipases), able to catalyze reactions such hydrolysis, ethanolysis or transesterification of triglycerides [124].

Concentration of Pacific sardines (*Sardinops sagax*) oil was carried out using lipase-catalyzed hydrolysis [140]. The results of this study have showed that hydrolysis with 250 U from *Candida rugosa* lipase has increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62% to 29.94% with 500 U after 9 h. This technique uses mild conditions (neutral pH and low temperatures), very important to preserve EPA and DHA from oxidation [140, 153].

Salmon oil produced from by-products of this species by controlled enzymatic procedure with Neutrase has followed a selective enzymatic hydrolysis under mild conditions, using Novozyme SP398 to enrich the n-3 PUFAs. The process used consist of a lipolysis, filtration in flat membrane device and enzymatic re-esterification with glycerol and Immobilized 1,3-specific lipase IM60 (Lipozym IM). This method induced a significant increase in the amount of PUFAs from 39.20 mol% of total fatty acids in the crude oil to 43.29 mol% in the re-esterified permeate [112].

The proteolytic extraction of oil from salmon heads using three different types of enzymes (Alcalase, Neutrase and flavourzyme) and the lipolysis of this oil to concentrate PUFAs were carried out. Lipolysis was done with Novozym SP398 to obtain a mixture of free fatty acids and glycerol (24 hours 45% hydrolysis). The mixture was then filtered. This process has allowed an increase of the PUFAs content from 41.6% in the crude oil to 46.5% in the permeate. Likewise, DHA and EPA percentages have increased from 9.9% to 11.6%, and from 3.6 to 5.6%, respectively [111]. The same authors used a re-esterification in the permeate with Lipozyme IM which permitted obtention of 5.06% and 11.90% in EPA and DHA contents, accordingly [111]. Moreover, other authors proposed combination of enzymatic or chemical hydrolysis with urea complexation to produce high concentrates of n-3 PUFAs. The enzymatic hydrolysis followed with urea complexation of refined sardine oil has increased the level of EPA and DHA from 14.51% to 46.26%, and from 12.55% to 40.32%, respectively [142].

Another technique, short path distillation was tested to purify Alaskan Walleye Pollock (*Gadus chalcogrammus*) and New Zealand Hoki (*Macruronus novaezelandiae*) liver oils [154]. Certainly, this process has reduced free fatty acids and lipid oxidation parameters, which is appreciated to produce purified oils. Consequently, the conduct of this operation at high temperatures may cause degradation of PUFAs or development of new undesirable compounds. The short path distillation was coupled to a previous enzymatic glycerolysis of sardine oil with glycerol [155]. This work showed that short path distillation is able to concentrate n-3 PUFAs in monoacylglycerols at suitable evaporator temperature (125°C) Same technique aided by a working fluid was evaluated efficient in removal of persistent organic pollutants in marine oils (PCDD/PCDF, dl-PCB and ndl-PCB) [156].

When comparing the effect of using urea complexation on the concentration yield compared with dry fractionation and low temperature solvent crystallization, results revealed that n-3 fatty acids were enriched in liquid fractions of all methods except by dry fractionation. The highest enrichment was achieved with the urea complexation method (83.00%) [157]. In the same context of valorization of marine by-products, application of urea crystallization on tuna oil recovered from liquid waste by-product from a tuna canning process allowed an increase in the concentration of n-3 PUFAs [158]. In another study conducted on concentration of fatty acids in sardine oil, the highest PUFA concentrations in low-temperature crystallization with ethanol were attained at −5°C, with EPA and DHA purities equivalent to 17.74 and 25.51%, respectively [108].

These authors also compared three different concentration techniques, supercritical fluid extraction (T = 40, 50, 60°C and 150, 250, 350 bar), Urea complexation (T = 1, −5, −10°C) and low-temperature crystallization with ethanol solvent (T = 10, 0, −5°C). The optimal conditions for each technique were determined. Nevertheless, the highest reduction of SFA and MUFA, the best increase in PUFA and the highest n-3 yield (47.53%), were obtained at −10°C in urea complexation method [108].

There are still several techniques used for the concentration of n-3 PUFAs, among which there is the use of polymeric membrane separation [159]. Optimal conditions of this method were found to be at the temperature of 36.19°C, pressure of 4.82 bar and stirring rate of 43.01 rpm with a desirability value of 0.99. With these conditions, a concentration of n-3 PUFAs of 34.98% was achieved.

Synthesized poly-vinylidene fluoride (PVDF) asymmetric membranes are also tested in concentration of n-3 PUFAs [160]. Conditions of preparation of PVDF membranes influences significantly results. In this work, PVDF membrane prepared at a coagulation bath temperature of 0°C resulted in the best n-3 PUFAs enrichment (40.4%) at 5 bar and 30°C.

**43**

**Author details**

**Conflict of interest**

Amar Kaanane\* and Hind Mkadem

provided the original work is properly cited.

low unwanted environmental effects.

\*Address all correspondence to: a.kaanane@iav.ac.ma

The authors declare that there is no conflict of interest.

Institute of Agronomy and Veterinary Medicine Hassan II, Rabat, Morocco

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Valorization Technologies of Marine By-Products DOI: http://dx.doi.org/10.5772/intechopen.95031*

Marine by-products (viscera, heads, trimmings, bones, cartilage, tails, skin, scales, blood, shells, carcasses, damaged fish, eggs, milt or soft roe), generated by marine transformation industries, constitute a good opportunity of valorization into highly valuable products. Their characterization determines the choice of the most suitable and efficient valorization method among all possibilities available, production of marine proteins (fishmeal, silage and hydrolysates), oils rich in polyunsaturated fatty acids (PUFAs) and preparation of high value compounds such as vitamins, enzymes, minerals, gelatin, collagen, chitin and chitosan, taurine

In this context, several studies have been carried out to explore possible technologies that can be used in the valorization of the marine by-products into marine oils and concentrated fatty acids. In addition to the conventional extraction process called also wet reduction process or hydraulic pressing, solvent extraction, supercritical fluid extraction, urea complexation, cold pressing or enzymatic hydrolysis processes could be used to transform these by-products into marine oils highly rich in PUFAs very demanded by food, nutraceutical and pharmaceutical industries. For more advanced enhancement, the concentration of fatty acids in marine oils is also widely practiced. Several techniques can be used such as winterization, urea complexation, short path distillation, supercritical fluid extraction, low temperature solvent crystallization, fractionation by chromatography or by enzymatic processes. Combined methods were also tested like solvent winterization and enzymatic interesterification, urea adduction before a supercritical fractionation. Many studies have focused on comparison between these techniques to provide differences, advantages, disadvantages, or even optimal conditions of operating. The main challenge in the choice of extraction and concentration techniques at industrial level is to reach higher yield, purity, quality, stability at lower cost and

and creatine, hydroxyapatite, natural pigments, biodiesel and biogas.

**4. Conclusion**
