Food Bioprocess Technol (2009) 2:127 DOI 10.

1007/s11947-008-0088-4

REVIEW PAPER

The Acid and Alkaline Solubilization Process for the Isolation of Muscle Proteins: State of the Art
Helgi Nolse & Ingrid Undeland

Received: 2 July 2007 / Accepted: 17 April 2008 / Published online: 19 June 2008 # Springer Science + Business Media, LLC 2008

Abstract The acid and alkaline solubilization processes for isolating muscle protein from ground fish raw materials are investigated by scrutinizing the literature. Following an introduction to the processes, with some underlying chemistry, patents related to the acid and alkaline solubilization process are described together with previously patented methods for processing of protein isolates. Focus is then placed on comparing a range of factors important in fish muscle protein isolation between the acid and alkaline solubilization processes, and classic washing-based surimi technology. The factors addressed were: protein yield, gel quality, color, lipid reduction, lipid oxidation, microbial stability, and frozen storage stability. A long series of studies made with different fish/shellfish species have been used for this purpose. Certain results are summarized in table form (protein yields, gel strength, and whiteness), others only in text form. From this part of the review, it is obvious that the acid process often has certain advantages (e.g., protein yield) and the alkaline process other ones (gel strength, whiteness, lipid removal, lipid oxidation, and total microbial count). Thus, the choice of method depends on the application. It is clear that most species respond differently to acid and alkaline solubilization, which is why the two methods are initially compared. In a section about new processing attempts, the use of the acid and alkaline methodology for isolating proteins from whole
H. Nolse (*) Faroe Fisheries Laboratory, Natn 1, 110 Trshavn, Faroe Islands e-mail: helgino@frs.fo I. Undeland Department of Chemical and Biological EngineeringFood Science, Chalmers University of Technology, 412 96 Gteborg, Sweden

fish/shellfish and fish by-products is reviewed together with attempts to recover waste water proteins and attempts to modify the process. Tentative uses of the new protein isolates, e.g., as coatings, protein brines, and emulsifiers are finally described together with some conclusions and future opportunities for the acid and alkaline processes. Keywords Protein . Isolate . Acid . Alkaline . Solubilization . Fish . Processing

Introduction Along with an increasing awareness that our marine resources are not endless, numerous efforts are currently ongoing to better utilize fish by-products and small underutilized fish species. A common feature of these raw materials is that they have a complex bone structure rendering physical separation of the muscle challenging. The complexity also applies to the composition, since there is often an abundance of blood, fat, and pigments which makes stability of the separated muscle a challenge. Among previous efforts to improve the utilization of these kinds of raw materials are inventions involving different types of deboning equipment. Several suitable mechanical methods of separation are known. Most well known are the deboning machines from Baader and Bibun in which the flesh is pressed through perforations in a rotating drum, by means of a powerful rubber belt, leaving bone, etc. behind. In fish processing, the deboners are mainly used in production of mince from beheaded and eviscerated fish, from whole fillets or from off-cuts from the trimming of fillets. However, it can also be used for separating the meat left on the backbones, collarbones and heads, etc. To obtain a good yield from such low cost raw

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materials, it is necessary to use a high belt pressure which implies drawbacks like decolorization by blood and other pigments as well as other unwanted substances like skin, small bones, cartilage, etc. To remove unwanted compounds abundant in minces, e.g., from dark muscle fish, efforts have been carried out to produce surimi. The traditional surimi process involves three washes with three volumes of water or a slightly alkaline solution. In the washes, water-soluble compounds are diluted and some of the neutral fat is removed, and cryprotectants are added before freezing in blocks. Unfortunately, storage stabiliy can remain a problem also after washing most likely because of severe dilution of the natural antioxidants in the fish raw material (Undeland et al. 1998). Also, the loss of sarcoplasmic proteins and some of the myofibrillar proteins into the wash water reduces the total protein yield of the surimi process. Some efforts have however been made to avoid this. Niki et al. (1985) for example adjusted the pH of the first wash water effluent to 10. Insoluble black proteins were then removed from the effluent by centrifugation. The pH of the effluent was then adjusted to pH 5 whereafter the effluent was heated at 80 C. This allowed the coagulated protein to be separated from the effluent. The yield of the recovered proteins was about 20% of the surimi products. Further, Huang et al. (1997) made a study where ohmic heat was used for coagulation of fish proteins from frozen wash water used to wash Pacific whiting mince. They were able to recover 33% of the proteins in the wash water. In 1999, there was a major technology breakthrough regarding isolation of muscle proteins from low value raw materials. Hultin and Kelleher patented the acid solubilization process as a way of improving yield and stability of muscle protein isolates. A few years later, a similar process, but based on alkaline solubilization was patented. Three main advantages with the acid and alkaline technologies need to be highlighted. The first one is that the muscle must not to be mechanically removed from bones/skin prior to processing. Crushed or minced raw materials can be directly subjected to acid or alkaline protein solubilization since all contaminating materials with a density different from the proteins can be removed by gravity, e.g., through centrifugation. The other advantage is that also sarcoplasmic proteins are recovered, raising the protein yield even further. Thirdly, both neutral lipids and membrane lipids can under favorable circumstances be efficiently removed in the process, something which minimizes the risk for lipid oxidation during subsequent storage. Since the whole process is carried out under cold conditions, the proteins retain the capacity to form a gel. The protein isolates can therefore be converted into a surmi and used in the same way, e.g., in shellfish analogues and kamaboko production. An additional advantage worthwile mentioning is the

finding that the waste water from acid and alkaline processing contained lower solids, N content, and chemical oxygen demand compared to the waste water originating from conventional surimi production (Park et al. 2003a). In the 8 years that have passed since the acid solubilization technique was first presented, a long series of attempts have been carried out to apply both this process and the alkaline process on various kinds of raw materials, both whole fish, fish fillets, and fish by-products. Some trials have also been made with shellfish, oysters, and blue mussels. Based on parameters like total protein yield, gelation capacity, color, and stability towards lipid oxidation, the acid and alkaline processes have been compared with each other, and also with traditional washing-based surimi processing. Utilization of the proteins has also been largely broadened beyond just surimi production. Attempts have for example been made to dry the isolates into protein powders, to resolubilize and inject them into fish fillets for better water holding capacity, and to use them as a batter to create low-fat fried seafood products. The process can certainly also be applied into other meat processing areas. For example in the poultry industry, utilization of by-products is currently of great importance. In this connection, separation of skin, fat, and meat is necessary, utilizing the meat fraction of the by-products for healthy low-fat products. This can partly be done by mechanical separation, but in cases with difficulties separating the meat from the fat fractions, it is a good opportunity to utilize the acid and alkaline processing methods for protein isolates. In this paper, it has been the aim to summarize the main outcomes from published studies using the acid and alkaline processing methods for protein isolation. After describing the principle of the process with some underlying chemistry, the patents forming the basis for this technology are presented along with some older patents utilizing similar principles. Thereafter, the outcome on key parameters that are most commonly used as success criteria when applying the acid and alkaline processes are reviewed, and comparisons are in some cases made with the outcome from traditional surimi processing. The key parameters include protein yield, gel quality, color, lipid reduction, lipid oxidation, microbial stability, and frozen storage stability. It should be stressed that although the processes are particularly suitable for complex raw materials, the majority of the papers published so far have dealt with fairly clean raw materials like fish fillets or even separated fish light muscle. In the later years, more challenging raw materials have however been addressed, which are summarized in a section focusing on new processing attempts. This section also highlights new additions/changes made to the process, attempts for upscaling and uses of the protein isolates.

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The Acid and Alkaline Solubilization Process The acidic/alkaline solubilization process was developed at the University of Massachusetts Marine Station, Gloucester, MA, USA. The process utilizes the principle that the solubility of a comminute protein-containing material homogenized in water is affected by the pH of the mixture. At extreme acid or alkaline conditions, strong positive and negative changes, respectively, on the myofibrillar and cytoskeletal proteins drive them apart by repulsion whereby, interactions with water can take place, and thereby solubilization. The diagram in Fig. 1 illustrates the acid/alkaline solubilization process as it is usually performed in the laboratory (Hultin and Kelleher 2000a). In step 1, ground fish raw material, which can be, e.g., minced fillets, whole fish, or backbones (unwashed or pre-washed), is mixed with 69 parts of water and homogenized. In step 2, myofibrillar and cytoskeletal proteins are dissolved by adjusting the pH of the mixture to either pH 2.53.5 or 10.811.5. The sarcoplasmic proteins remain dissolved under these conditions. The only proteins not dissolved are some of the connective tissue proteins, membrane proteins, and possibly some severely denatured myofibrillar/cytoskeletal proteins (Hultin and Kelleher 2000a). With
1. Homogenized mince/water mixture

2. Solubilize with an acid (pH 2.5 - 3.5) or base (pH 10.8 - 11.5) 3a. Bottom layer: skin, bones, impurities and sometimes a gel fraction consisting of almost solubilized proteins

3. Separate undissolved from dissolved matter

4. Adjust pH to isoelectric point

fresh fish, the solubility of the muscle proteins can be greater than 95% of the total proteins. It needs to be stressed that the protein solubility can be high at less extreme pHs than those indicated above. However, just when the protein solubilization starts around pH 34 and 1011, the viscosity of the fish homogenate can in certain cases be very high, which has two major disadvantages. The first is that removal of impurities through centrifugation becomes more difficult (step 3), and secondly, large extra sediments are formed during the centrifugation which entraps a lot of the proteins and thus reduces the protein yields. The high viscosity is thought to arise from expansion and partial solvation of protein aggregates. Below pH 3 and above pH 10.511, the aggregates however usually dissociate into smaller units, and the viscosity declines dramatically (Undeland et al. 2003). In step 3 and step 5, there are processes separating undissolved from dissolved material. Usually these separations are performed by centrifugation (Kristinsson and Demir 2003; Hultin and Kelleher 2000a). In step 3/3a, which in laboratory scale processing is performed in a batch centrifuge, a bottom layer which can be composed of skin, bones cartilage, and impurities is formed. In addition, a soft gel fraction can also be formed on top of the regular sediment. Some of this gel fraction can also be floating. In both cases, the sediments entrap a lot of the solubilized proteins, and are thus unwanted. In the case of fatty fish, a top layer of more or less emulsified oil also forms and has to be removed in step 3a. In step 4, the pH of the separated fluid is adjusted to the isoelectric point of the proteins (pH 5.16) in order to induce precipitation of both myofibrillar and sarcoplasmic proteins. In step 5, the precipitated proteins are recovered by centrifugation and decanting. In the laboratory, also this step is done using a batch centrifuge. Finally, the protein-containing sediment is isolated. The moisture of the protein isolates which at this point often is quite high (often over 90%) can be reduced by squeezing the isolate in a filter cloth or by a second centrifugation. The pH of the sample can then be adjusted to the desired value before, during, or after the addition of potential cryoprotectants. It should be stressed that the acidand alkali-produced protein isolates have a generally regarded as safe (GRAS) status in the US (FDA 2004).

5. Recover precipitated proteins by centrifugation and decanting

Protein Conformational Changes Taking Place During the Acid and Alkaline Process An important feature with the acid and alkaline processes is that when muscle proteins are subjected to the extreme pH values, the proteins are partly unfolding. This partial unfolding leads to substantial changes in the conformation and structure of the proteins which in turn leads to different

Fig. 1 The acid and alkaline solubilization process for protein isolates as performed in the laboratory. In step 1, minced muscle and water are homogenized. In step 2, the homogenate is solubilized by adding a base or an acid. In steps 3 and 3a, the homogenate is centrifuged and undissolved material is separated from dissolved material. In step 4, the pH of the dissolved material is adjusted to the isoelectric point. In step 5, the precipitated proteins are recovered

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properties of the proteins after refolding (Kristinsson and Hultin 2003a, b). In the thesis of Kristinsson (2001), conformational, structural, and functional changes of two key muscle proteins, hemoglobin (Hb) and myosin, were studied during exposure to low and high pH, as well as after subsequent refolding at the isolelectric point. pHinduced changes taking place to Hb are crucial for lipid oxidation. Changes in the myosin molecule on the other hand largely influence the functionality of the protein isolates, e.g., water holding, gelation, and emulsification. The results obtained by Kristinsson (2001) indicated that Hb became fully dissociated at low pH (pH 1.53.5), and the heme group lost contact with the distal and proximate histidine although it was never detached. The globin became partially unfolded into the so-called molten globular stage. The more unfolded the Hb became, the more difficult it was to refold it propertly at pH 5.5. Misfolded Hb was more hydrophobic than properly refolded Hb. Interestingly enough, alkaline pH (pH 1012) had almost no influence on the conformation and assembly of Hb. Along with this, lipid oxidation studies using a washed cod model system showed that the acid-treated Hb was more prooxidative than the alkali-treated one, most likely due to the conformational changes recorded. The alkaline treatment in fact suppressed the prooxidative role of Hb, most likely due to strong coordination of the heme to distal histidine. These results support the use of the alkaline process with raw materials that are susceptible to lipid oxidation. Regarding myosin, it was partially or fully dissociated at pH 2.5, but not at pH 11. At both pHs, the tertiary structure was lost, suggesting the molten globular configuration of the head. From 60% to 80% of the light chains were lost at low and high pH. The refolding pattern of the myosin rod, head, and light chains made refolded myosin different from native myosin. There were also some refolding differences in the head group after acid vs. alkaline pH. The refolded species was more thermally unstable, which was ascribed the misfolded head. Myosin subjected to extreme pHs therefore gelled at lower temperatures. Also, the emulsification capacity of acid-/alkali-treated myosin was improved compared to native myosin. The solubility on 0600 mM KCl was however the same between pH-treated and native myosin. Results that were similar to those of Kristinsson (2001) were later also observed by Mohan et al. (2007). In their study, the head group of mullet (Mugil cephalus) myosin was more affected by acidification and alkalization than the rod part. Upon refolding, the tertiary structure of the acid-treated mysoin also remained partly disrupted, which was not seen after alkalization. When studying cod myofibrillar proteins that had been subjected to high and low pH, Kristinsson (2001) found that these had higher solubilities at 0600 mM KCl than

native myofibrillar proteins, possibly as some structural proteins normally preventing expansion of the myofibrillar structure had been solubilized. The emulsification capacity of myofibrillar proteins subjcted to extreme pHs was also improved, and gelation was observed at lower temperatures than for native myofibrillar proteins. Also the gelation mechanism differed. After cooling, gel strength was however similar whether or not the proteins had been subjected to the extreme pHs. A general observation was that the functionality of both myosin and myofibrillar proteins was slightly more improved after the alkaline than the acid process. Together with the results of alkaline treatment stabilization of Hb, these observations have caused a larger interest in the alkaline than acid process in later applications. In a study by Raghavan and Kristinsson (2007), changes in the acid-induced conformation of catfish (Ictalurus punctatus) myosin were found to be dependent on the type of anions and salt added. The relationship between conformation and storage modulus (G) of acid-treated myosin was studied. The three acids tested were HCl, H2SO4, and H3PO4, and refolding was carried out at pH 7.3. Results showed that salt (0.6 M NaCl) present during unfolding and refolding stabilized myosin and induced less denaturation than when salt was added after refolding. In the latter case, myosin exhibited a higher G. The lower the pH, the more the myosin was denatured. From testing different acids during acidification, it was found that the G of acid-treated myosin decreased in the order Cl > SO42 > PO43. Among the different pH treatments, the G of myosin treated at pH 1.5 was significantly higher than myosin treated at pH 2.5. The authors concluded that the conditions that would result in maximum myosin denaturation and maximum G were unfolding of myosin at pH 1.5 with HCl followed by refolding at pH 7.3 and subsequent addition of 0.6 M NaCl. In a more recent paper, Raghavan and Kristinsson (2008b) studied similar changes in the conformation of catfish myosin as affected by cations, alkaline pH, and salt addition. The bases used to increase the pH to 11.0, 11.5, and 12.0 were NaOH and KOH. After alkaline unfolding of myosin, it was immediately refolded by adjusting the pH back to 7.3. The unfolding/refolding treatment increased the G of thermally treated myosin, especially when a pH of 11 was used. The presence of salt during the treatment stabilized the conformation of myosin against alkali unfolding and denaturation. Furthermore, KOH resulted in greater denaturation and higher gelling ability (G) compared to NaOH. The latter two studies thus indicate that conformational changes in the myosin molecule are not only affected by acid and alkali per se, but also by the type of acid/bases used to achieve the extreme pH. Further, the studies show

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that salt can have a stabilizing effect against denaturation. These findings add another dimension to keep in mind when optimizing the pH-shift processes for a particular raw material.

Patents Related to the Acid and Alkaline Solubilization Process A whole series of patents have been filed to protect the acid and alkaline protein isolation techniques. They differ, e.g., depending on what is actually claimed, the product or the process, and further whether acid or base is used for protein solubilization. Also, the patents differ depending on whether a high-speed centrifugation step to remove cellular membranes is involved or not. The membrane removal is generally believed to reduce the risk for lipid oxidation. A common feature for all the patents is that the process is carried out under cold conditions, which results in the proteins retaining the capacity to form a gel. Below, the different patents are briefly addressed. In the US patent number 6005073 from Dec. 21, 1999 (Hultin and Kelleher 1999), a process is provided for isolating a protein component of animal muscle tissue by mixing a particulate form of the tissue with an aqueous liquid <pH 3.5 to produce a protein-rich solution. The process includes a high-speed centrifugation step which thus allows for membrane removal. The degree of this removal is further discussed under the section Lipid Reduction. What is patented is the actual product, a protein isolate substantially free of membrane lipids. The US patent 6288216 from Sept. 11, 2001 (Hultin and Kelleher 2001) specifically describes the gelling properties of the isolated proteins. The capacity of the proteins to form a gel is, according to the aforementioned study, one of the great advantages with all of the patents listed in this section. In the US patent number 6136959 from Oct. 24, 2000 (Hultin and Kelleher 2000b), a process for isolating edible protein from animal muscle by solubilizing the protein in an alkaline aqueous solution is disclosed. This invention is based on the discovery that if animal muscle is significantly diluted with water and treated with a base to achieve sufficiently low viscosity, and then centrifuged at a sufficient gravitational force, a high yield of membrane lipid-free protein is obtained. This patent thus mainly differs from patent 6005073 in that it uses alkali instead of acid. The US patent number 6451975B (Hultin and Kelleher 2002) from Sept. 17, 2002 covers the acid solubilization process and the derived product that is obtained when utilizing the process. In this patent, the high-speed centrifugation step is not included. This patent thus allows for isolating muscle proteins from complex raw materials

by acid solubilization followed by separation of bones, skin, etc. However, it does not allow for membrane lipid removal which requirs a high g-force centrifugation. In the US patent 2004067551 and the international patent HK1070790 (Hultin et al. 2004, 2007), a process for isolating edible protein from animal muscle by solubilizing the protein in an alkaline aqueous solution is covered. In agreement with patent 6136959, the invention is based on the discovery that alkali-treated muscle proteins can be isolated in a non-oxidized form after centrifugation, compared to the acid isolates which can be highly oxidized. The patents cover the alkaline process both with and without the high-speed centrifugation step. Thus, isolates without and with membrane lipids, respectively, can be obtained. Advantages and disadvantages with the different patents, i.e., the effects of using acid or alkaline protein solubilization and inclusion or exclusion of a high-speed centrifugation step, are further discussed after the section about prior art for acid and alkaline processes. In general, the suitability of the different patents largely depends on the raw material that is under consideration, and what is the intended use of the proteins. As a rule of thumb when selecting among the processes, with raw materials that are susceptible to lipid oxidation, the alkaline process with high-speed centrifugation might be a good starting point to avoid rancidity. However, to be on the safe side, an intitial comparison among the processes, using the particular raw material, will optimize the chances of obtaining the desired result.

Prior Art for the Acid and Alkaline Solubilization Processes for Protein Isolation The principle utilized in the acid and alkaline solubilization process, i.e., pH-driven solubilization and precipitation of muscle proteins has been used in previous patent applications filed to utilize protein-containing by-products. Below, some of these patents are highlighted in order to show how they differ from the patents by Hultin, Kelleher, and co-workers. Already in January 1948, the pH-driven solubilization processing principle was used in patent GB672972 (Anon 1952) by the Swedish company Aktiebolaget Separator. This invention was adapted to protein-containing material such as peanuts and soya beans but was not limited to these. The patent utilizes the technique of dissolving the proteins in either an acid or an alkaline solution, separation of the undissolved substances from the dissolved ones by filtration or centrifugation, and subsequently precipitating the proteins by adjustment to the isoelectric point. This principle is thus almost identical to the one used in the more recent patents, but differs in that nothing is mentioned

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about keeping the temperature low during the processing. The latter factor could thus cause a problem if the proteins are intended for a gelled product. The same principle is also seen in patent US2875061 from Feb. 1959. The inventors Raimund Vogel and Klement Mohler (1959) patented a process of preparing edible protein substances from raw fish material having a fat content of less than 4%. The process utilizes an alkaline solution and later a precipitation at the isoelectric point to recover proteins. It differs from the more recent acid and alkaline solubilization processes in that it also involves heat and and alcohol. Both of these factors can contribute to protein denaturation and thus to reduced or destroyed gelation capacity. In GB patent no. 1108188, applied for in August 1966 and published in April 1968, Cesar Melton Libenson and Ignacio Pirosky (1968) described a process where fish proteins are dissolved by hydrolysis between 40 and 100 C in an alkaline aqueous medium. Then non-protein, insoluble, solid residues, free lipids, and lipoprotein constitutents may be separated by decantation, pressing, filtration, or centrifugation. The proteins are then precipitated by adjusting the pH of the solution to the isoelectric point. Considering the fact that the proteins are hydrolyzed at an elevated temperature, gelation should theoretically not be possible. On behalf of a Swedish Company Astra Nutrition AB, Carpenter et al. (1975) applied for patent in Nov. 1971 (GB1409876). The invention relates to a process for the separation of a protein isolate from fish, particularly from fish waste. This patent utilizes the technique of protein solubilization at alkaline conditions with subsequent acidification and precipitation. However, high temperatures (3070 C) and hydrogen peroxide for decolorization of the processed protein isolates are used. According to the aforementioned study, the elevated temperature prevents gelation. The use of hydrogen peroxide would theoretically constitute a raised risk for lipid oxidation, especially if the material is rich in heme proteins. In the presence of methemoglobin/met-myoglobin, hydrogen peroxide can lead to very reactive ferryl species (Kanner and Harel 1985). As can be seen from the mentioned patents, the process of dissolving and precipitating proteins based on the pH, both from fish and other organic sources, was known before the introduction of the new acidic/alkaline solubilization processes. However, as stated initially, none of these processes specifies that the temperature should be kept low (<15 C) during the whole processing line in order to retain important functional properties like gel strength. The last feature largely raises the applicability of the proteins in food processing and thus makes the newer patents quite unique. The newer patents also implies removal of at least 50% of the membrane lipids, something which is not

specified in the older patents. The last main difference between the newer and older patents is that the newer ones also protect certain products produced utilizing the processes.

Different Parameters Influencing Yield and Quality of Proteins Separated by Acid and Alkaline Solubilization In the literature, many comparisons are made between the acid and alakine processes, as well as between these two processes and traditional surimi processing. In the section below, comparisons made based on protein yield, impurities, color, gel strength, and frozen storage stability, including lipid oxidation stability, are reviewed. It has to be kept in mind that the processes used in the different studies vary in their exact settings. Therefore, cross comparisons of exact numbers from the various studies have to be made with certain precautions. Protein Yield The protein yield obtained during acid and alkaline processing is primarily detemined by three major factors, the solubility of the proteins at extreme acid or alkaline conditions, the size of the sediments formed during the centrifugations, and the solubility of the proteins at the pH selected for precipitation. Ideally, the acid- or alkali-driven solubilization should be high, while the other two factos should be low. During coventinal surimi preparation, the exact yields depend mainly on the number of washes, the pH of the washing solution, and the ionic strength of the washing solution. Below, ten studies are reviewed with respect to their findings on protein yield. The results from these studies are also summarized in Table 1. Using the acid and alkaline processes, Undeland et al. (2002) found protein yields of 744.8% and 684.4%, respectively, from white muscle of herring (Clupea harengus). The lower yield on the alkaline side was linked to a larger sediment formation in the first centrifugation. In a similar comparison between acid- and alkali-aided processing, Kristinsson and Ingadottir (2006) investigated protein yields from tilapia (Orechromis niloticus). From repeated trials, they found yields from 56% to 61% with the acid process, and from 61% to 68% with the alakaline process. Thus, in contrast to the study above, the alkaline method gave better results. In another study, ground catfish muscle was subjected to acid and alkaline processing either immediately or after holding at 4 C or at 25 C for 7 days (Davenport et al. 2005). It was found that the protein recovery from unfrozen materials was slighly higher during the acid process than during the alkaline process. Furthermore, the frozen raw

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Table 1 Overview and comparison of protein yields obtained during acid and alkaline protein isolation as well as traditional surimi processing Study Species Acid process 74.0 64.2/76a 67.4 71.5 73.6 81.2 78.7 68.0 73 53.6 85.5 Acid process (no centrifugation) Alkaline process 68.0 38.2 Alkaline process (no centrifugation) Surimi (3+ wash)

Undeland et al. (2002) Cortes-Ruis et al. (2001) Cortes-Ruis et al. (2001) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kim et al. (2003) Batista et al. (2003)b Batista et al. (2003)b Batista (1999)b Batista (1999)b Kristinsson et al. (2005) Kristinsson and Liang (2006) Kristinsson and Ingadottir (2006) Nolse et al. (2007) Average STD

Herring Sardine Sardine ice-stored 5 days Catfish Spanish mackerel Croaker Mullet Pacific whiting Sardine Blue whiting Hake saw dust Monkfish saw dust Channel catfish Atlantic croaker Tilapia Cod

71.5 78.7 58.5 70.38.0

85.8

85.70.2

70.3 69.3 58.9 65.0 70.0 77 49.1 80.6 62.9 70.3 65.0 64.5 71.0 67.37.6

80.1

62.3 54.1 57.7 59.3

82.1

62.3 57.7

90.0 84.15.2

55.98.3

Results are given as percent of proteins in the isolate based on total proteins in the initial mince. The first column shows source study for the figures. 3+ wash = traditional surimi process with three or more washing cycles. In a few studies, the acid and alkaline processes were run both with and without the high-speed centrifugation step. a Jelly layer reprocessed b A slightly different process was used with salt addition and higher temperature.

material in general gave reduced recovery. In the work by Undeland et al. (2003), storage of herring always increased the viscosity of acidified and alkalized homogenates, something that increased the sediment size. This thickening was ascribed, e.g., crosslinking of proteins and could also explain the present findings on catfish. Kim et al. evaluated how the protein yield during acid and alkaline processing of Pacific whiting was dependent on the soubilization pH selected. In their study, they compared pH 2, 3, 10.5, 11, and 12 and used a 1:10 fish to water ratio. The highest protein yield (70%) was found at pH 12, and the lowest protein yield (60%) was found at pH 10.5. Protein solubility was measured at corresponding pH values, but in much more diluted fish homogenates (1:50). The authors discussed the possibility that the slightly lower solubility found at pH 10.5 than at pH 12 was not enough to explain their recovery difference. Instead they hypothesized that higher ratios of water might be needed at pH 10.5 for optimal solubilization and recovery. However, it must be stressed that there are practical limits regarding the amount of water that can be used in the acid and alakine processes. After isoelectric precipitation of alkali-processed hake (Merluccius merluccius) and monkfish (Lophius piscatorius) saw dust, Batista (1999) found protein yields of 80.6% and 62.9%, respectively. The process used differed slightly from the processes of Hultin and Kelleher since some salt was

added, and since longer extraction times and higher temperatures were used. Batista (1999) also investigated the recovery of proteins in the supernant after the isoelectric precipitation. Here he recovered about 9096% of the proteins using different concentrations of Na6(P6O18) for the pH adjustment. Batista et al. (2003) later investigated protein recovery from sardine (Sardina pilchardus) and blue whiting (Micromesistius poutassou) mince. They used similar acid and and alkaline processes as those described above and obtained 73% and 77% protein recovery, respectively, from sardine mince and 53.6% and 49.1%, respectively, from blue whiting mince. First in a series of five comparisons between the pH-shift techniques and conventional surimi making is the work by Cortes-Ruis et al. (2001). These authors found that the protein yield from fresh sardine mince was 38.25.8% after conventional surimi processing (four washing cycles) and 64.23.2% after acid processing. Performing the acid protein isolation process on mince from sardine fillets stored for 5 days, the protein recovery was 67.41.2%. The authors also investigated the opportunity to re-process the proteins in the gelatinous soft gel discarded from the first centrifugation during processing of the fresh sardines. Together with the precipitate, this gel was mixed with water, adjusted to pH 3.2 and reprocessed according to Fig. 1. In this way, they could increase the total protein yield to 76%.

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When comparing the protein yields for four different warm water species, catfish, Spanish mackerel, croaker, and mullet, Kristinsson and Demir (2003) found yields for conventional surimi processing to be 62.3%, 54.1%, 59.3%, and 57.7%, respectively. Using acid and alkaline processing, the protein yields from the four species were 71.5%, 73.6%, 81.2%, and 78.7%, respectively, as well as 70.3%, 69.3%, 58.9%, and 65.0%, respectively. When omitting the first centrifugation during acid and alkaline processing of catfish, the protein yields became 85.8% and 82.1%, respectively (Kristinsson et al. 2005). In somewhat later study on Atlantic croaker from the same laboratory, protein yields of 78.7%, 65.0%, and 57.7%, respectively, were found using acidic, alkaline, and traditional surimi processing methods (Kristinsson and Liang 2006). Park et al. (2003a) investigated protein yields and gel quality during production of traditional surimi from jack mackerel and white croaker muscle and compared it to the use of acid and alkaline processing. In the work with the latter processes, the best fish to water ratio and solubilization/precipitation conditions were detemined. It was found that homogenization of a 1:6 fish/water ratio at <9,500 rpm (30 s), followed by solubilization at pH 2.5 vs. 10.5 and precipitation pH 5 gave the best results. Also, the effect of salt was evaluated in this study, and it was found that an increased level of salt reduced the protein solubilty. With jack mackerel, both acid and alkaline processing gave higher total protein yields than conventional surimi processing: 28% and 31% vs. 25% (which is including filleting loss). With croaker, the corresponding yields were: 27% and 32.5% vs. 31%. When investigating total solid yields on a wet weight basis during conventional surimi processing and acidaided protein isolation from Pacific whiting (Merluccius productus), Choi and Park (2005) found one washing cycle to give 56.7%, three washing cycles to give 44% yield, and acid processing to give 58.3% yield. To summarize these studies, in ten out of 13 cases where the acid and alkaline processes were compared on different species, the acid process gave higher protein yield. Among likely reasons for this could be higher protein solubility under acidic conditions. As an example, Undeland et al. (2002) reported that 92% and 88% of the total proteins of herring light muscle became soluble at pH 2.7 and 10.8, respectively. Another possibility is that the proteinentrapping gel sediment and floating gel layer that sometimes form in the first centrifugation can become larger during alkaline processing than acid processing. In the same study as above, these two layers entrapped in total 16% and 19%, respectively, of acid- and alkali-solubilized herring light muscle proteins. Thereby, they reduced the theoretical protein yields (thus, yields only based on the solubilization) from 88% to 74% and from 83.5% to 68% on the acid and

alkaline side, respectively. It has also been found that these gel layers get larger during processing of stored fish, which can explain the lower protein recoveries obtained with stored raw materials (Undeland et al. 2003; Davenport et al. 2005). As mentioned previously, attempts to re-cycle these gel layers to recover the entrapped proteins have been successfully carried out and could be a way around this problem (Cortes-Ruis et al. 2001). In cases where the first centrifugation has been omitted, protein yields have for natural reasons increased. The studies reviewed above also show that except from one study of mullet (Kristinsson and Demir 2003), the acid and/or alkaline method also gave higher protein yields than traditional surimi processing. The latter result is most likely explained by the recovery of sarcoplasmic proteins using the former processes. Gel Quality Among the most important factors affecting the gel quality of protein isolates and surimi are the pre-process history of the raw material and the temperature used during processing. Fish proteins can denature at any temperuture, but the denaturation rate is slowed at lower temperatures (Lanier et al. 2005). For this reason, rapid chilling of the raw material after catch is of great importance. The fish muscle temperature has a tendency to increase after death by 5 to 10 C because of continuing muscle metabolism (Lanier et al. 2005). When it comes to gel preparation, comparable data can only be achieved if factors like moisture content, salt content, and pH of the cryoprotectant-fortified protein isolate are kept constant. Also, the techniques for measuring the textural properties of the gel, e.g., with a rheometer or the torsion technique, are crucial for whether data can be directly compared or not. The former method gives information on break force and deformation, the latter method on strain and stress. In addition, the folding test, which gives information about elasticity on a scale from 1 to 5, is very commonly performed. Below, 16 studies are reviewed with respect to results on gel quality. The results are also summarized in Table 2 in order to show the ranking order obtained between acid-/alkali-produced gels and gels from conventional surimi. Cortes-Ruis et al. (2001) tested different texture parameters to compare gels from conventional surimi and acidproduced protein isolate from fresh sardines and sardines stored for 5 days on ice. Results from measuring the cohesiveness, elasticity, and folding showed the highest values for gels from conventional surimi followed by freshsardine-acid-produced protein isolate and then the acidproduced isolate from stored fish. The results for the folding tests showed little difference between gels from surimi (4.80.3) and the protein isolate from fresh fish (4.5 0.4), indicating that the quality of the gels is on the same

Food Bioprocess Technol (2009) 2:127 Table 2 Results from the reviewed investigations of gel quality Study Species Acid process 2 3 2 3 3 3 3 2 1 3 3 3 2 3 3 3 Alkaline process Surimi (1 wash)

Surimi (3+ wash) 1 1 1 2 2 2 2

Cortes-Ruis et al. (2001) Cortes-Ruis et al. (2001) Choi and Park (2002) Kristinsson and Demir (2003)a Kristinsson and Demir (2003)a Kristinsson and Demir (2003)a Kristinsson and Demir (2003)a Undeland et al. (2002) Undeland et al. (2002) Park et al. (2003a) Park et al. (2003a) Park et al. (2003a) Kim et al. (2003) Choi et al. (2003) Yongsawatdigul and Park (2004) Perez-Mateos et al. (2004) Chaijan et al. (2006) Chaijan et al. (2006) Kristinsson and Liang (2006) Perez-Mateos and Lanier (2006) Thawornchinsombut and Park (2007)

Sardine Sardine old Pacific whiting Catfish Mackerel Mullet Croaker Herring Herring ice-stored 6 days Horse mackerel Japanese mackerel Redlip croaker Pacific whiting Rockfish Rockfish Atlantic croaker Sardine Mackerel Atlantic croaker Menhaden Pacific whiting

3 1 1 1 1 1 2 2 2 2 1 1 1 1 2 2 1 2 2

1 1 1 2 2 2 1 1 3 1 1

2 3 3

1 means the strongest gel in the comparison, and 3 the weakest gel. 1 wash = traditional surimi process with 1 washing cycle, and 3+ wash = traditional surimi process with three or more washing cycles. a Only storage modulus was measured. In all the other studies, gel strength and deformation values were measured using a 5-mm spheric plunger with a speed of 60 mm/s.

level. A hardness test showed the highest values for the acidproduced protein isolates, especially the one made from sardines stored for 5 days. It was found that the acid-made protein isolate gels contained 15% more proteins, indicating that an excess in proteinprotein interactions may have resulted in a hard and inelastic gel, while an excess of proteinwater interactions may have resulted in a softer and more fragile gel. When investigating Pacific whiting (Choi and Park 2002), it was found that gel force and deformation values were lower for gels from acid-produced protein isolates than for conventional surimi made with three washing cycles. However, the acid-produced gels had higher values than those from surimi made with a single washing cycle. An explanation given was incomplete removal of cathepsin B and L with the acid process compared to surimi making with three washing cycles. Gel force and deformation values for acid-produced protein isolate gels, conventional surimi gels (three washes), and conventional surimi gels (one wash) were 92.39.2 g and 7.50.5 mm, 110.89.6 g and 10.5 0.6 mm, and 83.45.6 g and 7.40.3 mm, respectively. In a study of four different warm and temperate water fish, the gel quality of conventional surimi and acid-/alkaliproduced protein isolates was determined by oscillatory

torsion testing (Kristinsson and Demir 2003). The authors found the best gel quality with the alkali-produced protein isolate, followed by conventionally produced surimi. The acid-produced protein isolate had the poorest gel quality. Low protease activity during/after alkaline processing was one of the explanations given. Investigating alkali and acidic protein isolates from fresh herring (C. harengus) light muscle, Undeland et al. (2002) found stronger gels for the former isolate. It was hypothesized that there was a higher retention of more myosin heavy chains on the alkaline side as a certain degree of hydrolysis took place during acid processing. When herring stored 6 days on ice was used, the gel qualities were generally poorer, which was ascribed to protein denaturation initiated by lipid-free radicals. The TBARS value was much higher in the stored herring. With this raw material, the acid process gave the best gels. For fresh herring, the break force and deformation length was 87162 g and 9.2 0.7 mm as well as 56636 g and 9.20.9 mm for alkali- and acid-produced isolates, respectively. The values for aged herring were 46411 g and 6.20.3 mm as well as 49858 g and 7.30.5 mm, respectively. The folding test gave a score of 5 to both isolates made from fresh herring while both made from the aged material scored 3.

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The effect of cold storage and freezing of ground catfish muscle prior to acid- or alkali-aided isolation of muscle proteins has also been investigated by Davenport et al. (2005). Ground catfish muscle was processed immediately, or kept at 4 C or at 25 C for 7 days before processing. What they found was that alkaline processing gave better gel forming ability than the acid process, and just like Undeland et al. (2002), the fresh raw material gave the strongest gels followed by the cold stored and the frozen raw material. Park et al. (2003b) compared protein yield and gel quality of protein isolates originating from acid and alkaline processing with proteins isolated by conventional processing. The studies were performed with different fish species, including horse mackerel, Japanese mackerel, and redlip croaker. The effects from adding sarcoplasmic proteins and NaCl to the gels were also studied. Breaking force, deformation value, and whiteness of the gels were lower with alkaline processing than with conventional processing. Addition of sarcoplasmic protein increased the breaking force and the deformation value. Addition of NaCl had no significant influence on the deformation, but decreased the breaking force of the gel. As shown in the paper by Undeland et al. (2002), acid processing had a strong degrading effect on the myosin heavy chain and actin. The results indicated that alkaline processing could have benefits for production of surimi and kamaboko-type products. The effect from solubilization pH on the gel forming abilities for acid- and alkaline-processed Pacific whiting protein isolates was studied by Kim et al. (2003). The solubilization pHs selected were 2, 3, 10.5, 11, and 12. The authors found that the best textual properties of the gels were obtained solubilizing the fish muscle at pH 11 followed by solubilizing at pH 2. Solubilizing at pH 12 gave the worst textual properties of the gel. The gels looked like coagulants rather than real gels. Solubilizing at pH 3 and 10.5 gave textual properties at almost the same level as pH 11 and 2. This study showed the importance of finding the optimum pH level for solubilization to get the best possible textual properties of the gel. Kim et al. (2005) also studied how gelation was affected by interactions of rockfish sarcoplasmic proteins (SP) or sucrose with myofibrillar proteins from Alaska pollock surimi. The pollock surimi was mixed with SP (0% or 2%) and 2% salt. In another treatment, 2% sucrose replaced the SP. When 2% SP was added, the breaking force significantly increased while the deformation value slightly decreased compared to the control gel without SP. When 2% sucrose was added, both the deformation value and the breaking force decreased compared to the control. These results thus indicate a significant role for SP in the gelation process, especially in relation to breaking force. The

retention of SP following pH-shift processing can therefor be considered an advantage. The influence of insoluble muscle components on the gelation properties of catfish protein isolates made with acid and alkali processing has also been studied (Davenport et al. 2004). Three versions of the acid and alkaline processes were compared: the regular process, a process where filtration replaced centrifugation, and a process where insolubles were kept in the protein isolate. The acid-produced protein isolate gave lower gel strength than the alkali-produced one. Interestingly, the gel with the insoluble components showed the highest gel strength followed by the gel made from the filtered isolate. Gels from isolates made with the regular process were the poorest. Breaking force and deformation values of gels from acid- or alkali-produced rockfish muscle proteins, rockfish mince, and rockfish mince washed three times were investigated in a study by Yongsawatdigul and Park (2004). The breaking force and the deformation values showed that the alkali-processed gel was the strongest, followed by the washed mince gel and the mince gel. The gel from the acid-produced proteins was the weakest. In their study, they also investigated the SH content of the uncooked cryoprotected protein isolates and the heattreated gels from the different treatments. The rockfish mince sample had the highest SH content before and after cooking. A decrease in the SH content was observed after washing of the mince. Oxidation of SH groups further appeared to occur to the greater extent during acid than alkaline processing, resulting in a lower total SH content in the acid-made cryoprotected protein isolates. Therefore, the SH groups available for disulfide formation and/or other interchanges were more limited during gelation of the acidtreated sample than of the alkali-treated sample. Thawornchinsombut and Park (2007) investigated the interactive effect of pH and ionic strength during protein solubilization on gelation properties of acid- and alkaliprocessed protein isolates from Pacific whiting. Results were compared to conventional surimi processing. The strongest gels were those obtained from protein isolate made by solubilization at pH 11 with an ionic strength of 150 mM NaCl and those made from conventionally washed surimi. The protein solubilities per se did not affect the gelation capacities of the different isolates. It was however found that the pH-shift methods, especially the alkaline one, induced more denaturation and lowered the SH content compared to conventional surimi. The results also demonstrated that fish proteins solubilized at pH 3 or 11 with NaCl were only partly refolded at pH 7. Some myosin fragments and actin never refolded. In general, the presence of NaCl affected the rheological properties of the proteins more than different pH values.

Food Bioprocess Technol (2009) 2:127

11

The effect of different pH values and keeping times at these pHs on molecular and gelation properties of proteins from catfish was studied by Davenport and Kristinsson (2004). The pHs used were 2.0, 2.5, 3.0, 10.5, 11.0, and 11.5 and the proteins were kept there for 1 and 30 min before readjusting to pH 5.5. The proteins were then kept at pH 5.5 for 30 min before centrifugation. The results showed that low and high pH had significantly different influences on the gel forming properties of the protein isolates. Only a small influence from the pH and keeping time was seen on the alkali-treated gels, which were very similar in strength. Only a small variation was also found in protein conformation and stability of the alkali-treated proteins. However, a significant variation in gel strength and protein conformation was seen for proteins subjected to low pH. While the pH 3 treatment gave good gels, molecular changes taking place <pH 3 had a detrimental effect on gel forming properties of the proteins. The physiochemical properties, gel forming ability, and myoglobin content of sardine and mackerel surimi produced by conventional washing and the alkaline solubilization process were investigated by Chaijan et al. (2006). Surimi conventionally prepared by water or NaCl washing gave gels with the highest breaking forces and deformation values. Unwashed mince, mince washed three times with water, mince washed three times with 0.2% NaCl, alkaliprocessed pre-washed mince, and alkali-processed unwashed minces were tested. The conventionally NaCl-washed surimi showed the highest gel strength and deformation values, followed by the water-washed and the unwashed mince. Both the alkali-processed samples had the lowest values. The sequence was the same for both species. Chaijan et al. (2006) also investigated Ca2+-ATPase activity and surface hydrophobicity of natural actomyosin (NAM), finding the highest Ca2+-ATPase activity in conventionally NaClwashed surimi. With the NaCl washing, sarcoplasmic proteins, lipids, and other dissolvable material are removed resulting in a higher content of myosin heavy chain with Ca2+-ATPase activity. The results indicated that denaturation of myosin was induced by the alkaline solubilization process resulting in poorer gel quality for the alkaline-processed samples than the regular washed samples. In a study comparing conventional surimi to acid- and alkali-produced protein isolates from Atlantic croaker, the gel quality was investigated by a torsion test for fresh and frozen protein isolates (Kristinsson and Liang 2006). For fresh samples without cryoprotectants, the highest strain values were found for conventional surimi followed by acid-produced protein isolate and alkali-produced protein isolate. The stress values were highest for acid-produced protein isolate followed by conventional surimi and alkali protein isolate. The same tests made with frozen material showed strain values at the same level for all three methods.

The stress values for acid and alkali protein isolates were on a much higher level than for the conventional surimi. This shows that the cryoprotectants and the freezing step substantially improve the gel quality of the protein isolates. The influence of the cryoprotectans and the freezing step on the conventional surimi was on a much lower level with an increase of the strain value and a decrease of the stress value. Testing the same samples in a folding test showed that all samples could stand a double folding test. Textural properties of gels from Atlantic croaker were investigated by Perez-Mateos et al. (2004). They found different gel properties for acidic, alkaline, and conventionally processed surimi. In the investigations, they used three different gel cooking methods: (1) cooking directly in 90 C water bath for 20 min, (2) setting for 2 h in 30 C water bath followed by 90 C water bath for 20 min, and (3) setting for 2 h in 40 C water bath followed by 90 C water bath for 20 min. Also, they tested gels using different additives: 2% NaCl, 2% NaCl plus 0.2% MTGase (commercially developed microbial transglutaminase used for upgrading the gelling quality of surimi), as well as 0.2% MTGase. The tests show that alkali-produced protein isolates gave gels with the highest breaking force, especially when using MTGase alone, independent of the cooking method. Thereafter the acid-produced protein isolate gave the highest gel force for gels with NaCl and MTGase using 30 min setting time. The gel force for the conventional surimi was at the lowest level, giving the highest gel force for 40 min setting time using both NaCl and MTGase. Comparing the deformation for the three methods, it can be seen that the deformation of the alkaliproduced samples with MTGase alone had the highest deformation values, independent of cooking method. The other alkali-processed gels were not higher than the acidprocessed gels and the conventional surimi gels. In the cases with 2% NaCl and MTGase, even lower deformation values were obtained. Interestingly, gels from all three methods failed to show any increase in the gel strength because of the setting periods. To compare gels from conventionally processed menhaden surimi and gels from acid and alkali-produced menhaden protein isolates, Perez-Mateos and Lanier (2006) studied gels from the evaluation described above (Perez-Mateos et al. 2004). Penetration force and the deformation value was measured. The four different gels were /+ 2% NaCl, + 0.2% MTGase and + 2% NaCl and + 0.2% MTGase. For each processing method three different cooking methods were used; cooking directly at 90 C for 20 min, and using 30 min incubation time at 30 C and 40 C before cooking for 20 min at 90 C. For conventional surimi, this test showed break force at the same level for all directly cooked gels. An improvement was seen with the salt containing gels, especially for the gel including MTGase

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which had a fourfold gel strength compared to the 30 C incubation. The salt containing gel without MTGase more than doubled the gel strength. For the 40 C incubation, all the gels showed improved gel strength with a 5-fold increase for the salt containing MTGase gel. The MTGAse gel without salt, gave almost a three-fold greater gel strength compared to direct cooking. The gels without MTGase almost doubled their gel strength with 40 C incubation. Comparing the deformation values, they showed a slightly higher value for the directly cooked gels without additives than for the other two, while the salt containing gels with and without MTGase showed the greatest improvements for both incubation temperatures with the highest deformation values for the gels with both additives. The gels made from acid aided processing were generally of lower value, both for penetration force and deformation compared to conventional surimi. Two exceptions were the 30 C incubated gel with MTGase alone, which had a higher penetration force value than conventional surimi, and the 40 C incubated gel with MTGase alone which was at same level as in the conventional surimi. The alkali-processed gels showed generally higher values for both penetration force and deformation value than the acid-processed gels. The directly cooked alkali-processed gels were at the same level or had slightly higher penetration value than the directly cooked conventional surimi gels while the deformation values for the conventional surimi gels were higher. For the alkali-processed gels incubated at 30 and 40 C, both the MTGase containing gels showed improved penetration forces, especially the latter gels with MTGase alone showed a high penetration force. Both incubation times showed improved deformation values except for incubation at 30 C without additives. In general, the alkali-processed gels were at a lower level than the conventionally processed surimi gels, but at a higher level than the acid-processed gels. Comparing this gel test with the test performed by Perez-Mateos et al. (2004) showed that the gel quality with Atlantic croaker decreased in the following order: alkali-processed protein isolate > acid-processed protein isolate > conventional surimi, and it is obvious that different fish species can give different results in relation to gel quality and processing method. These results also differed compared to what others have found regarding the relation between gel quality and processing method. The more common result has been a higher gel quality for alkali and acid protein isolates compared to conventionally processed surimi (Kim et al. 2003; Kristinsson and Hultin 2003b; Perez-Mateos et al. 2004; Yongsawatdigul and Park 2004). Currently, the salt intake is recommended to be low while the ability to form good gels without addition of NaCl is of interest. It has been described that this feature is species dependent (Perez-Mateos and Lanier 2006) and that

it is enhanced by the pH-shifting method. It has been suggested (Chang et al. 2001; Wright and Lanier 2005) that the more thorough disruption of muscle structure by acid or alkaline processing may play a key role in better distributing proteins for heat-induced gel formation which reduces the need for salt to accomplish the same effect. Kristinsson and Hultin (2003a) found that acid- and alkali-treated cod myofibrillar proteins gelled at lower temperatures than native proteins. The former protein also had a better solubility, which was ascribed to removal of certain solubility restricting proteins during acid and alkaline processing. Also, the functional properties of acid- and alkali-treated proteins have been shown to change after refolding (Kristinsson and Hultin 2003a, b). Summing up the above studies, it is clear that both the fish species and processing method has a large impact on gel strength. In comparisons of the acid- and alkalineproduced protein isolates, the alkali-processed ones in all cases except one (herring stored 6 days on ice) gave the strongest gels. When comparing the acid- and alkaliproduced protein isolates to conventional surimi, it appears that conventional surimi and alkali-produced protein isolates perform fairly equal, and both then performing better than the acid-produced proteins. Based on the 16 studies, half of them showed better gels with alkali-made isolates, and half with conventional surimi. From one study, no significant differences were seen between these two isolates. It has been speculated that the harder gels obtained with alkali-produced proteins compared to acid-produced isolates originate in less hydrolysis during alkaline processing compared to the acid one. Also a larger degree of SS bond formation has been discussed (Yongsawatdigul and Park 2004). The better performance of traditional surimi over acid-made gels could be linked to the increased concentration of myofibrillar proteins in the surimi after washing. Myofibrillar proteins contribute to elasticity of a muscle protein gel (Yongsawatdigul and Park 2004). Color One important parameter when comparing different processing methods is the color of the protein isolate or the gel made thereof. The market is in general most interested in isolates that are as white as possible (Tabilo-Munizaga and Barbosa-Canovas 2004). However, the exact application of course determines how important the color is. The color can be affected, e.g., by the amount of dark muscle, the presence of blood, and the presence of pigments such as melanin. The melanin pigment can come for example from the eyes, skin, and the black lining around the belly. Based on this, the color of course becomes an issue when processing small dark muscle pelagic species and byproducts, both of which are generally right in all of these

Food Bioprocess Technol (2009) 2:127

13

components. It is however important to stress that color, measured for example according to the CIE Lab color scale (a*, b*, and L*), is strongly affected by physical parameters like moisture of the samples or structure. This should thus be kept in mind when considering color data from different isolates and gels. Below, 12 studies are reviewed with respect to findings on color, mainly whiteness and lightness. The internal ranking order of whiteness results from each study is also summarized in Table 3. The color of sardine mince, sardine surimi, sardine surimi gel, acid-produced protein isolate (with cryoprotectants), and finally the acid-produced protein gel was investigated by Cortes-Ruis et al. (2001). The unwashed sardine mince showed a dark red/yellow color because of the presence of myoglobin in the muscle and possible contamination of hemoglobin and skin pigments. The whiteness value was only 37.21.6. They found the highest whiteness values for conventional surimi and surimi gel (51.81.1 and 60.31.2, respectively) followed by acid protein isolate and the gel thereof (45.21.5 and 48.32.2, respectively). Undeland et al. (2002) investigated the colors of acid and alkaline protein isolates from the light muscle of
Table 3 Overview of the whiteness obtained in the reviewed studies Study Species Product

herring. They reported highest L* values (lightness), b* values (yellowness), and whiteness for the alkali-produced protein isolate. a* values (redness) were the same for the acid- and alkali-made isolates. Acid-processed protein isolate and conventional surimi made with one and three washing cycles were investigated for color by Choi and Park (2002). They found the highest L and whiteness values for surimi washed three times followed by surimi washed once and then the acidproduced protein isolate. The acid-produced isolate had a particularly high b* value which reduced the whiteness. The yellowness was due to high levels of hemoglobin and myoglobin. With this background, the same study showed higher L* values for acid-made protein isolates when fillets were washed in ice water before mincing. Kristinsson and Demir (2003) tested the colors of uncooked and cooked protein isolates/conventional surimi from four fish species: Channel catfish, croaker, mullet, and Spanish mackerel. The catfish protein isolates were produced also without the first high-speed centrifugation of the pH-shift method (Fig. 1). They found different results for different species. The lightness order for cooked Channel catfish gels was: alkali-produced protein isolates produced

Acid Acid process Alkaline Alkaline process Surimi Surimi process (no centrifugation) process (no centrifugation) (1 wash) (3+ wash) 2 2 3 3 4 4 1 3 3 2 3 3 3 4 3 1 3 2 2 1 1 1 1 3 5 3 2 1 1 1 1 1 5 1 3 1 3 1 2 1

Cortes-Ruis et al. (2001) Cortes-Ruis et al. (2001) Undeland et al. (2002) Choi and Park (2002) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Kristinsson and Demir (2003) Yongsawatdigul and Park (2004) Kristinsson et al. (2005) Perez-Mateos and Lanier (2006) Kristinsson and Liang (2006) Kristinsson and Liang (2006) Kristinsson and Liang (2006) Kristinsson and Liang (2006) Chaijan et al. (2006) Chaijan et al. (2006)

Sardine Sardine Herring Pacific whiting Catfish Catfish Croaker Croaker Mullet Mullet Spanish mackerel Spanish mackerel Rockfish Channel catfish Menhaden Atlantic croaker Atlantic croaker Atlantic croaker Atlantic croaker Sardine Mackerel

Gel Isolate Gel Gel Isolate Gel Isolate Gel Isolate Gel Isolate Gel Gel Isolate Gel Isolate fresh Gel fresh Isolate Gel Gel Gel

2 2 5 3 2 2 2 1 2 3 2 2 2 2 2 2 2 1 3 1 2 1 1

1 = the whitest gel, 2 = the second whitest gel obtained in the comparison, etc. Some results are reported for cryoprotectant-fortified protein isolates, others for gels and in certain cases for both protein isolates and gel. In a few studies, the acid and alkaline processes were run both with and without the high-speed centrifugation step. All the gels not marked fresh have been made from protein isolate that was frozen before gel making and testing.

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without the first centrifugation > regular alkali-produced protein isolate > conventional surimi > acid-produced protein isolate > acid-produced protein isolate without the first centrifugation. For the raw Channel catfish isolates, the lightness order was: alkali-produced protein isolates produced without the first centrifugation > regular alkaliproduced protein isolate > acid-produced protein isolate without the first centrifugation > acid-produced protein isolate > conventional surimi. For both isolates and cooked gels, the the lightness order for Spanish mackerel was: conventional surimi > alkali protein isolate > acid protein isolate. For mullet, the corresponding lightness order was: conventional surimi > alkali-produced protein isolate > acid-produced protein isolate. For cooked gels from croaker, the lightness order was: alkali-produced protein isolate > conventional surimi > acid protein isolate. For the raw croaker isolates, the order was: acid-produced protein isolate > alkali-produced protein isolate > conventional surimi. From the results using Spanish mackerel and mullet, it thus can be seen that conventional surimi yielded the highest lightness scores for both raw and cooked gels, followed by alkali protein isolate and then the acidproduced isolate. Generally, it can be said that the alkaliproduced protein isolates had higher lightness values than the acid protein isolates. According to the authors, this was probably because more heme proteins are removed in the alkaline process. In a later study (Kristinsson et al. 2005), the above color results for conventional Channel catfish surimi and acid- and alkali-processed protein isolates were repeated, and an attempt was made to explain why the alkaline process gave whiter isolates than the acid process. The authors investigated the UVvisible spectrum of the supernatant collected after the second centrifugation. Here they found that substantial amounts of native heme proteins remained soluble at pH 5.5 after the alkaline process while the opposite was true for the acid process. This shows that a larger part of the heme proteins of the acid process precipitate together with the other proteins at pH 5.5. An interesting finding was that the modified alkaline process without the first centrifugation gave the highest whiteness. Although no explanations were given for this, we hypothesize it could be a physical phenomenon linked to different moisture contents. In contrast to the above findings, Undeland et al. (2005) found that both a* and b* values were lower in acidproduced herring fillet protein isolates when the high-speed centrifugation step was included compared to excluded. L* values were not reported in this study. The same study also showed that the inclusion of a 1:3 pre-wash of the herring mince in water prior to acid processing reduced the b* values of the isolates, but did not affect the a* values. In the pre-washes, both fat and Hb were removed. It was speculated that the lack of effect from the pre-wash on a*

values of isolates was due to the fact that the Hb molecule was completely oxidized after the acid processing that is why its quantity did not affect a* any more. It was a general trend in this study that all acid-produced isolates where no antioxidants had been included were greyish-brown. This is a clear sign of met-Hb/Mb-formation. Another reason could be that the heme is detached after met-Hb formation, leaving behind a yellowish globin residue. In trials with decoloration of slaughterhouse blood by oxidation with H2O2 (37), a yellowish proteinaceous material was obtained (van den Ord and Wesdorp 1979). Using rockfish muscle, Yongsawatdigul and Park (2004) tested the color of acid- and alkali-produced protein isolates and conventionally processed surimi. They obtained the highest whiteness for conventionally processed surimi followed by acid-produced protein isolate, alkali-produced protein isolate, and crude mince. Conventional surimi showed the whitest appearance because of the removal of myoglobin during washing. Kim et al. (2005) found that the addition of 2% freezedried sarcoplasmic protein from rockfish to Alaska pollock surimi negatively affected the color of the cooked gel. The decrease in whiteness value was primarily because of an increased b* value, which in turn was due to the fact that one component related to the sarcoplasmic protein is heme protein. The color of the gels made of conventional surimi as well as acid- and alkali-produced protein isolates from menhaden was investigated by Perez-Mateos and Lanier (2006). They found the highest whiteness for conventional surimi, followed by the alkali- and then acid-produced protein isolates. These results were thus in agreement with what most other studies have shown and were explained by the removal of more myoglobin during the conventional surimi washing. Kristinsson and Liang (2006) tested the color (L*, a*, and b* values) of conventional surimi as well as acid- and alkali-produced protein isolates from Atlantic croaker. They compared the color of isolates and cooked gels. Isolates were used either unfrozen without cryoprotectants or frozen with cryoprotectants added. For unfrozen isolates they found the highest L* value for acid-produced protein isolate, followed by alkali-produced protein isolate, and then conventional surimi. For gels from unfrozen isolates, they found the highest L* value for conventional surimi followed by alkali-produced protein isolate and then acidproduced protein isolate. For frozen paste, the highest L* value was found for alkali-produced protein isolate followed by acid-produced protein isolate and then conventional surimi. However, conventional surimi gels had the highest L* value when frozen isolates were used, followed by the acid- and then alkali-produced protein isolate gels. The L* value for conventional surimi increased substantially

Food Bioprocess Technol (2009) 2:127

15

by cooking the gel. There was a small increase in the L* value for the acid-produced isolate and the fresh alkaliproduced isolate by cooking into gels. However, the frozen alkali isolate decreased in L* value by cooking. Redness values (a* values) were low for all samples. The acidproduced protein isolates had a substantially higher yellowness (b* values) than the other samples. This was probably due to the higher level of retained heme proteins after precipitation. Conventionally processed sardine and mackerel surimi where the washes were made with water or 0.5% NaCl were subjected to color analyses by Chaijan et al. (2006). Comparisons were made with isolates produced with alkaline processing with/without a pre-wash step. With sardine, the order of whiteness was: pre-washed alkaliproduced protein isolate > regular alkali-produced protein isolate > conventional surimi washed in water > conventional surimi washed in 0.5% NaCl. The mackerel samples were ranked as: conventional water washed surimi > prewashed alkali-produced protein isolate > conventional surimi washed in 0.5% NaCl > regular alkali-produced protein isolate. The authors hypothesized that the lower whiteness found for conventional sardine and mackerel surimi washed in NaCl, as well as for the alkali-made mackerel protein isolate might be from oxidized retained myoglobin. They also investigated the expressible moisture and found the highest values for the alkali-produced protein isolates from both species. Thus, possibly a low water holding capacity could also affect the whiteness. In a study with croaker and mackerel (Choi and Park 2005), the possibillity of using air floatation to improve the whiteness of fish protein isolates produced with the pHshift methods was investigated. When a minced muscle homogenate was air-flotated for 1020 min before adjusting it to pH 11, the best improvements in color were seen in the recovered protein isolate; lightness increased, yellowness decreased, and thus whiteness also increased. To sum up, it is obvious that different fish species require different processes to get protein isolates/gels that are as light as possible. In the majority of the cases where the conventional surimi process was included for comparative purposes, this process gave the whitest isolates/gels, most likely due to the washing-induced heme removal. Furthermore, in 13 out of 17 cases, the alkaline process gave whiter isolates/gels than the acid process. According to the aforementioned study, this has been ascribed to the fact that a larger part of the heme proteins are removed with alkaline than with acid processing. Variations in the acid/ alkaline processes like the use of pre-wash or exclusion of the high-speed centrifugation have clear effects on a* and b* values. Regarding high-speed cenrifugation, results on the acid and alkaline side were however contradictory (Kristinsson et al. 2005; Undeland et al. 2005). Pre-washing

was beneficial with both processes (Undeland et al. 2005; Chaijan et al. 2006). Lipid Reduction The capacity of the acid and alkaline processes to remove a large part of both neutral lipids and membrane lipids has been one of the features patented. The amounts of lipids that can be removed are however linked to factors like the lipid content of the starting material, the viscosity of the pH-adjusted homogenate, and the centrifugation speed used in the first centrifugation. The latter factor mainly affects the removal of membrane lipids, as they at this step may go down into the sediment. Lipid reduction can be of importance to reduce the susceptibility of a protein isolate to lipid oxidation. However, it needs to be stressed that as long as strong prooxidants like Hb are present, it seems like extremely low lipids levels need to be reached to prevent lipid oxidation. Richards and Hultin (2001) found severe blood-mediated rancidity odor in washed cod mince containing 0.1% lipids (wet weight, ww). Many comparisons have been made regarding lipid removal by acid and alkaline processing vs. conventional surimi processing. In an investigation of Bristly sardine with a lipid content of 3.3% (ww), Cortes-Ruis et al. (2001) tested the lipid reduction during surimi production with a modified version of the Ishikawa process (Roussel and Cheftel 1988). The latter process applied four 1:5 washing steps using 0.5% sodium bicarbonate solution in the first wash instead of water. Then manual washing and dewatering with a gauze cloth was performed. The outcome was compared to the acid protein isolation process with the follwing settings: a 1:9 mince to water ratio, the first centrifugation for 15 min at 16,000g, 15 min and the second centrifugation for 10 min at 16,000g. The lipid reduction in the surimi process was 67.46.9% and for the acid protein isolate, lipid reduction was 88.34.8% both when fresh Bristly sardine was used. Using 5-day-old iced fish, the lipid reduction was 92.6 2.6% for the acid protein isolate. On a dry weight (dw) basis, the lipid content in the final surimi was 11.15.4%, compared to 4.13.0% and 2.82.3%, respectively, for the acid-produced isolates made from fresh and iced sardines. Undeland et al. (2002) found that the lipid to protein ratio of minced herring light muscle was reduced by 68% and 60%, respectively, using the acid and alkaline process with a g-force of 18,000 (20 min) in the first centrifugation step. On a ww-basis, the total intitial lipid content of the herring light muscle was of 2.4% (ww), on protein basis it was 0.13 g lipid/g protein. The reduction in the phospholipid to protein ratio was from 0.037 to 0.02 g/g protein, thus 46% for both porcesses. In an investigation of the lipid reduction achieved during acid and alkaline processing of catfish, Spanish mackerel,

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mullet, and croaker (Kristinsson and Demir 2003), it was found that the highest lipid reduction was given in alkaliproduced protein isolates followed by acid-produced protein isolates and then conventional surimi. On a dry weight basis, the lipid reductions for the alkali-processed protein isolates were 68.4% for croaker, 81.4% for mullet, 79.1% for mackerel, and 88.6% for catfish. For acidprocessed protein isolates, the values for the mentioned species were 38.1%, 58.0%, 76.9%, and 85.4%, respectively. For surimi, the values were 16.7%, 10.4%, 72.1%, and 58.3%, respectively. The surimi was dewatered by manual squeezing through a cheesecloth. The acid and alkaline protein isolates were processed with two centrifugation steps at 10,000g for 20 min. Applying the alkaline protein isolation method on coarsely and finely ground meat from mechanically deboned turkey, Liang and Hultin (2003) found reductions in the total fat content from 10.80.2% to 0.90.0% in coarsely ground meat, and in finely ground meat from 19.3 0.2% to 1.00.0%. The centrifugation was performed at 25,000g for 25 min. In a comparative study between acid- and alkali-aided protein isolation and conventional surimi processing, Kristinsson et al. (2005) investigated lipid reduction in channel catfish muscle. The raw material was skinless fillets containing from 4.7% to 9.8% fat (ww). The two centrifugation steps were done at 10,000g for 20 min. The dewatering of the surimi was performed manually by squeezing it through a cheesecloth. The highest lipid reduction, 88.62.8%, was obtained by alkaline processing followed by acid processing, with 85.42% lipid reduction. Conventional surimi processing gave 58.37.8% lipid reduction. The authors also tested the lipid reduction when omitting the first centrifugation step in production of protein isolates. In this case, the lipid reductions with the alkaline and acid processes were 61.26.3% and 45.4 4.4%, respectively. Using Atlantic croaker fillets with 3.1% fat content, Kristinsson and Liang (2006) studied the lipid reduction obtained with acid and alkaline processing (both centrifugations at 10,000g, 20 min) as well as with conventional surimi processing with a manual dewatering through a cheesecloth. The reductions obtained were 68.4%, 38.1%, and 16.7%, respectively. As described by Kristinsson and Demir (2003), the big difference between the conventional surimi process and the acid and alkaline methods was ascribed the first centrifugation. In a study investigating lipid oxidation during acid processing of herring fillet mince, Undeland et al. (2005) undertook lipid reduction with/without a 1:3 pre-wash, and with/without the high-speed centrifugation step (10,000g, 20 min) was tested. In the pre-wash, the initial lipid content (6.1% on a ww basis, 22.5% on a dw basis) was reduced by

39%. When unwashed and pre-washed minces were processed with high-speed centrifugation, further lipid reductions were 51% and 56%, respectively. The protein isolates thus contained 11% and 10% of lipid on a dry weight basis (1.3% and 0.9%, respectively on ww basis). In cases when unwashed and pre-washed minces were processed without centrifugation, the lipid content became 33.2% and 32.2%, respectively, on dry weight basis (4.3% and 3.9%, respectively, on wet weight basis). The relative increase in lipid content on a dw basis in the latter case was due to the fact that more proteins than lipids were removed. In a series of studies, Liang and Hultin (2005a, b) studied the sedimentation behavior of membranes during the pH-shift methods and looked for practical methods to remove cellular membranes from solubilized muscle proteins at low g-force. The results showed that membrane phospholipids could be selectively removed from solubilized muscle proteins at low g-force. Treatment with CaCl2 or MgCl2 and citric acid and centrifugation at 4,000g could remove up to 90% of the membrane phospholipids from the muscle samples. Citric acid might play a role as a binding agent to the basic amino acid residues of cytoskeletal proteins competing with the acidic phospholipids of membranes. The membranes released from the cytoskeletal proteins could then aggregate or fuse due to the final low pH achieved by the addition of citric acid (pH 4.9 5.3) in the homogenate. Vareltzis and Undeland (2008) compared the acid and alkaline processes on a blue mussel mince that contained 13.5% lipids on dw basis, and had a lipid to protein ratio of 0.3. The g-force in the first centrifugation was 8,000. They found that the acid process resulted in isolates with lower lipid content than the alkaline process: 11% vs. 18.8% on a dw basis, and 0.15 vs. 0.3 on the basis of the lipid to protein ratio. Adding 5 mM citric acid and 10 mM calcium chloride significantly improved lipid reduction in the mussel protein isolates from both the acid or alkaline process. As a result, the lipid to protein ratio was reduced by 73% for both processes compared to the untreated mussel mince. The authors also examined the effect of gforce reduction in the first centrifugation to 800g. None of the two processes then reduced the lipid to protein ratio, and instead, the lipid content increased on a dw basis (to 24% and 18% for the acid and alkaline processes) due to a fairly large protein loss in the process. Liang et al. (2007) applied chitosan and chitin to aid membrane separation during acid- and alkali-aided protein isolation from cod mince. When adding chitosan with a molecular weight of 310375 kDa prior to either acidification or alkalization of the homogenate, about 55% and 80% of the membranes were removed during centrifugation at 4,000g (15 min). In the control, about 31% were removed. The corresponding protein removal was 12%, 28%, and

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7%. Smaller chitosans removed less phospholipids and chitin was not useful. The efficiency of chitosan >310 kDa was ascribed to its high total positive charge/molecule, the positive charges then interacting with the negatively charged membranes. A bridging mechansim between the membranes and chitosan was also suggested. From these studies it can be seen that acid and the alkaline processing clearly removes more lipid that conventional surimi making. From the three studies testing the acid and allakine methods with/without the high-speed centrifugation, it was obvious that this step is crucial for the improved lipid removal. In three out of the four studies comparing the acid and alkaline method, more lipids were removed with alkaline processing. Kristinsson and Demir (2003) described how the lipid emulsifying ability of proteins is improved during the alkaline process compared to during the acid process. This increases the size of the floating lipid layer formed in the first centrifugation, but instead of consisting of pure oil, the layer consists of a proteinlipid emulsion. This implies a greater lipid reduction but also a lower protein yield. In one study combining conventional surimi making (a 1:3 pre-wash of the mince used) with acid processing, it was seen that the final lipid content of the isolates was then slightly reduced. This was most likely since the first removal of some of the neutral fat avoided the risk of proteins emulsifying this fat, which could lead to some lipid entrapment in the isolates. Other ways of improving the lipid reduction, especially membranes include additions of citric acid/calcium chloride and chitosan. Lipid Oxidation An important quality parameter related to seafood products is lipid oxidation which can lead to rancidity if no precautions are taken. This is especially important when producing minced products, and especially when dealing with materials high in prooxidants. After mincing, the lipid surface exposed to oxygen and prooxidants increases. The acid protein isolation process has theoretically been ascribed to possess a higher risk for lipid oxidation than the alkaline process. This is, e.g., as heme proteins can become activated as prooxidants at low pH (Kristinsson 2001). It has also been found that subjection of washed cod mince to low pH (20 min, pH 2.5) made the muscle more susceptible to Hbmediated oxidation than native washed mince (Kristinsson 2001). Although the acid process of course gave more extreme exposure to low pH, it should be remembered that also in the alkaline process, there is a subjection to relatively low pH (around 5.5) during the precipitation step. Lipid oxidation was investigated during production of acid- and alkali-processed protein isolates as well as during conventional surimi making with cold water (4 C) using

catfish, croaker, mullet, and Spanish mackerel (Kristinsson and Demir 2003). Samples from surimi and isolates were placed in plastic weighing cups, put into ziplock freezer bags, and then placed in cold storage set at 4 C. Samples were collected every third day and frozen at 70 C to stop oxidation. Samples were then analyzed for TBARS, i.e., seconday oxidation products. The results showed that the conventional surimi had lower levels of lipid oxidation products than acid-/alkaline-processed isolates, except for in the case of mackerel isolate stored for 6 days. The acid protein isolate was most prone to oxidation while the alkalimade isolates fell in between the other two methods. Investigating channel catfish muscle, Kristinsson et al. (2005) analyzed lipid oxidation products of the ground starting material, after conventional surimi processing as well as after acid- and alkali-aided processing with/without the first centrifugation. The results showed that none of the processes gave significantly higher TBARS values than the starting material. Alkaline processing without the first centrifugation was an exception in that it yielded an isolate with a very low TBARS value. The results indicated that channel catfish is in general very stable towards lipid oxidation. Kristinsson and Liang (2006) investigated lipid oxidation in unprocessed ground Atlantic croaker (Micropogonias undulates) and in conventional surimi as well as acid- and alkali-processed isolates made thereof. Samples were stored on ice for 14 days and analyzed periodically for TBARS. The highest initial TBARS value was found in the acidprocessed protein isolate followed by the ground muscle and then finally the alkali-processed protein isolate. The conventional surimi sample had decreased in TBARS during processing compared to the initial ground raw material. Over the storage period studied, the alkaliprocessed protein isolate and the conventional surimi maintained a low TBARS value while the other samples, especially the acid-produced isolate, had a substantial increase in TBARS. They also analyzed lipid oxidation of cryoprotected protein isolate and gel directly after production and after frozen storage for 1 week. The highest increase in TBARS was also here seen for the acidprocessed protein isolate followed by the conventional surimi and the the alkaline protein isolate. The faster increase in TBARS for the acid-processed material is most likely because of the increased prooxidative potential of the heme proteins when subjected to low pH values (Kristinsson and Hultin 2004). Undeland et al. (2005) studied how lipid oxidation progressed under acid processing of herring fillet mince. They tested how changes in the process and the use of different antioxidants influenced lipid oxidation both under the processing itself and under a subsequential ice storage of protein isolates. Lipid oxidation was followed with TBARS, color analyses, and sensory analyses. The tested

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processing parameters were +/ a 1:3 pre-wash of the mince, exposure time to pH 2.7 (4, 30, and 75 min), +/ high-speed centrifugation (10,000g, 20 min), and addition of antioxidants. The tested antioxidants were one reducing agent (0.2%, ww, erythorbate), two metal chelators [0.2% sodium tripolyphosphate (STPP), or 0.044% ethylenediaminetetraacetic acid (EDTA)], and one protein concentrate which might act as a radical sink (4% milk proteins). All antioxidants except the milk proteins were added in the prewash or homogenization step. Milk proteins were added into the final isolates. Without high-speed centrifugation and without antioxidants, extensive oxidation took place during the process. Inclusion of a 1:3 pre-wash or varied exposure time to pH 2.7 did not improve the quality. Highspeed centrifugation significantly reduced TBARS values, a* values, and b* values of the isolates. If added in the prewash or homogenization step, erythorbate alone, or combined with STPP or EDTA, significantly reduced lipid oxidation both during processing and subsequent ice storage. The best stabilities were given when both steps were fortified, and when EDTA was used instead of STPP. Petty and Kristinsson (2004) studied how the addition of antioxidants to Spanish mackerel homogenates affected lipid oxidation during alkaline and acid processing. Ascorbic acid and tocopherol were tested separately and combined at a 0.1% and 0.2% (ww basis) level in the homogenate. Homogenates were adjusted to low and high pH values and analyses of primary and secondary oxidation products were made after 0, 2, 4, 8, and 12 h. Both ascorbic acid and tocopherol were more effective in the higher concentrations and at lower pH. At pH 6.5, both antioxidants, but ascorbic acid particularly, served as prooxidants. There was no need for antioxidants during alkaline processing as no oxidation developed here in the absence of antioxidants. Using the acid process, oxidation developed rapidly without antioxidants. Inhibition of lipid oxidation during acid and alkaline isolation of cod muscle proteins was also studied by Vareltzis and Hultin (2007). In their research, the centrifugation of the solubilized homogenate was omitted, and it was shown that both acid and alkaline processing enhanced the oxidative stability of protein isolates. It was suggested that acid processing caused in situ aggregation of the cod muscle membranes rendering them less susceptible to lipid oxidation. Vareltzis and Hultin (2007) showed that acid-treated isolated cod membranes were resistant to Hbmediated and/or NADH-dependent lipid oxidation, exhibiting longer lag phase and lower oxidation rate. This was an indication that bound Hb might have restricted access to the substrate of lipid oxidation, that is, the phospholipids of the acid-treated membranes. Vareltzis et al. (2008) also evaluated the effects of citric acid and calcium chloride to prevent lipid oxidation in ground cod muscle. Their results

suggested that citric acid and calcium chloride added to minced cod can significantly improve the oxidative stability of the protein isolates. It was suggested that citric acid and calcium chloride can act as reducing agents as well as causing higher in situ aggregation of membranes compared to acid only treated muscle. The antioxidative properties of citric acid were partly explained by its ability to break down pre-formed lipid hydro-peroxides of the cod muscle. In a recent study by Raghavan and Hultin (2008), the oxidative stability of acid- and alkali-produced cod proteins in the presence of exogenously added Hb (6 M) was studied during ice storage. Attempts to inhibit such oxidation with 300 ppm -tocopherol, 200 ppm BHA, and 200 ppm propyl gallate were also done. Antioxidants were added using ethanol (1% of the muscle weight) as a carrier. Acid-produced isolates produced rancid odor and TBARS much faster than alkali-produced isolates. Regardless of the process used, the efficacy of the antioxidants was ranked as: propyl gallate > BHA > -tocopherol. Summing up the results, the comparisons between different processes made, e.g., by Kristinsson and coworkers clearly point to the acid-processed protein isolates as more susceptible to lipid oxidation than the alkaliprocessed ones. It is however possible to prevent processinduced lipid oxidation by addition of compounds like erythorbate, EDTA, citric acid, and calcium chloride. Oxidation during storage of isolates can also be prevented with antioxidants. Conventional surimi on the other hand in several studies had the lowest level of oxidation products, which indicates that lipid content and lipid oxidation do not go hand in hand. Better stability of the conventional surimi could be due, e.g., to that the pH of the process never goes far below the physiolgical pH, and thus, that Hb/Mbactivation is avoided. Microbial Stability Microbial stability is an extremely important factor during production and storage of food products. Living and newly harvested fish often has a high number of bacteria on its surface. The nature of these bacteria depends both on the bacteria that are found in the harvesting environment and in the procesing environment. Generally, most of the bacteria growing on fish belong to the psychrotrophic gramnegative genera Huss (1983). Only one study has compared the microbial stability of traditionally processed surimi with acid- and alkali-processed protein isolates (Kristinsson and Demir 2003). The question has however been raised at many occasions since it is expected that the extreme pH values in acid and alkaline processing have a killing effect on microbes associated with the raw material (Jay 1986). Kristinsson and Demir (2003) followed the microbial growth on ground catfish mince, on conventional surimi as

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well as on acid- and alkali-produced protein isolates made thereof. There was a reduction in aerobic plate count numbers directly after all three processes, but the largest reduction was seen for the alkaline-processed isolates followed by the acid-processed isolates. This was probably due to the strong centrifugations that the protein isolates are subjected to causing the microbial cells to sediment together with the undissolved sediment material. The larger reduction during alkaline than acid processing could be due to a larger killing effect of alkali than acid. After 10 days on ice, the aerobic plate count numbers for ground fish had increased from 6.32103 to 7.71105 per gram of tissue, for conventional surimi from 2.38103 to 1.55106, and for acid-produced protein isolate from 1.45103 to 2.45 104. The alkali-produced protein isolate had actually fallen from 1.00103 to 3102 counts per gram of tissue, which was explained by the same hypothesis as above, a more detrimental effect on the bacteria by the alkaline process. Based on the latter result, the question could be raised why the bacteria do not grow on the alkali-processed protein isolate, which is an intriguing observation for further investigation. It needs to be straightened out whether this is a good or bad result. Frozen Storage Stability One of the main advantages with surimi is its stability during frozen storage, which is mainly due to the addition of cryoprotectans. Not much research has been carried out on investigating the frozen storage stability of protein isolates processed either by acidic or alkaline processing. However, it is of course an interesting question how the the stability of these frozen protein isolates compares to that of conventional surimi. Frozen storage stability of alkali-produced rockfish protein isolate under various storage conditions was investigated by Thawornchinsombut et al. (2006) and Thawornchinsombut and Park (2006). In their tests, they adjusted the pH of protein isolates to 5.5 and 7, and they then split each portion into a cryoprotected and an unprotected part. After 4 weeks storage at 80 C, the cryoprotected and the unprotected samples were subjected to three freeze and thaw cycles at 18 and 4 C. Additionally, one cryoprotected sample at each pH level was stored only at 80 C for 4 weeks. Before testing, all samples were adjusted so that they had an equal content of cryoprotectants, an equal moisture content and the same pH (pH 7). The tests comprised salt extractable proteins, surface hydrophobicity, total sulfhydryl content, thermodynamic properties by differential scanning calorimetry, and a gel analysis. They found that regardless of the freeze thaw cycles and storage pH (5.5 or 7.0) the amount of proteins soluble in 0.6 M NaCl was the same, 4345 mg/g

(dw basis). A preliminary study showed that the solubility of conventional surimi from rockfish in 0.6 M NaCl was approximately seven times higher. Altering the pH of samples kept frozen at pH 5.5 to 7 just before analysis considerably increased the amount of salt-extractable proteins. The pH used during the storage did not seem to significantly affect the surface hydrophobicity and the SH content, regardless of exposure to freezethaw cycles used and cryoprotectant addition. Regarding the thermodynamic properties, all samples showed different thermograms as influenced by the different treatments. All samples with cryoprotectants showed three endothermic temperature peaks. The maximum heat input required to denature the protein isolates stored at pH 5.5 were slightly lower than that needed for those kept at pH 7. Storage pH did not seem to affect the heat input required for protein denaturation of cryoprotected isolates. Generally, a decrease in the maximum heat input temperature is interpreted as a decrease in protein stability. The texture properties of the alkaliprocessed protein isolate gels showed that the cryoprotected gels not exposed to freezethaw cycles had the best quality, independent of the pH value at storage. They were followed by the cryoprotected freezethawed gels and with unprotected freezethaw gels at the lowest level. The investigation thus showed the importance of using cryoprotectants for the alkali-processed protein isolates and of storing nonprotected isolates at a higher pH. The study did however not say anything about long-term frozen storage stability.

New Processing Attempts One of the main advantages in the acid and alkaline process is the opportunity to separate the dissolved proteins from other undissolved ingredients. As stated initially, this opportunity should especially be an advantage in the utilization of by-products and small-bone-rich fish, providing a great chance of separating classic feed grade products into a food grade and a feed grade part. However, to date, most of the studies on acid and alkaline processing have involved work with fillets. Only in the recent years, more complex materials have been addressed. Some of those studies are summarized below. In the recent years, some attempts to make the process more applicable for industrial use have also been carried out. These attempts include the recovery of proteins in phases that during lab processing normally are discarded. Also, attempts to replace the costly high-speed centrifugation steps have been carried out. Processing of Whole Fish and Fish By-products Bechtel et al. (2005) investigated the possibilities of using the alkaline process in the production of soluble and

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insoluble protein isolates from pink salmon viscera and red salmon heads. After freeze drying of the isolates, the protein content was analyzed to be 50.7%, 69.4%, 80.0%, and 87.2%, respectively, of the insoluble fraction obtained from red head, the insoluble fraction from pink salmon viscera, the soluble fraction from red head, and the soluble fraction from pink salmon viscera. In another study, Bechtel et al. (2006) investigated the opportunities for utilizing the alkaline process for isolation of proteins from heads of Pollock and wild salmon. They found that soluble protein powders extracted from these raw materials have potential as functional protein ingredients. The insoluble protein isolates on the other hand were ascribed to potential applications as both feed and food ingredients. Using a mix of saw dust and cut-off by-products from Cape hake (Merluccius capensis), Batista et al. (2006) investigated how much proteins could be recovered using the acid and alkaline solubilization processes. After removing skin and bones by hand, the ground by-product mix, which had been previously frozen, was either processed unwashed or pre-washed. They obtained protein yields of of 58.7% and 63.0%, respectively, from unwashed acid- and alkali-processed raw materials. The corresponding protein yields from pre-washed raw material were 43.6% and 50.1%, respectively, when calculated based on the initial ground muscle. Chen and Jaczynski (2007) applied the acid and alkaline methods on trout processing by-products (fish meat left over on bones, head, skin, etc.) using two-phase continuous centrifuges (20,000g) with 600 mL/min in the first step and 50 mL/min in the second step. Protein yields were between 77.7% and 89.0%, depending of the pH used for solubilization and precipitation. Acid processing generally gave higher yields than the alkaline one, and also isolates with more lipids (about 19% on dw basis as compared to 9 11% after alkaline processing and 15.2% in the raw material). The fairly poor lipid removal was ascribed the continuous centrifuges used. Ash analyses showed that the processes effectively removed impurities such as bones, scales, skin, etc., from the trout processing by-products. Due to high proteolytic activity in the raw material, the recovered proteins failed to gel unless beef plasma protein (BPP) was added. Even with BPP, the recovered protein showed some proteolysis between 40 and 55 C. Addition of potato starch, transglutaminase, and phosphate to the recovered proteins resulted in good texture of trout gels. Higher shear stress and strain were measured for gels developed from basic pH treatments than the acidic counterparts. The gels from acidic treatments were whiter than those from the basic ones, probably due to the higher lipid content. The same research team, Chen et al. (2007), also performed a study to determine the amino acid, fatty acid,

and mineral profiles of the trout (Oncorhynchus mykiss) processing by-products as well as the protein, lipids, and insolubles recovered thereof by the acid and alkaline methods. Solubilization was performed at pH 2, 2.5, 3, 12, 12.5, and 13 and precipitation at pH 5.5. Both centrifugations were at 10,000g for 10 min. The results showed that the use of basic pH yielded proteins with higher concentrations of essential amino acids than using acid pH. Based on the essential amino acid profile, the quality of the recovered proteins seemed to be better than that of soy protein, comparable to that of milk protein, but slightly below that of egg protein. The fatty acid analyses showed that the acid and alkaline methods did not change the composition of polyunsaturated fatty acids. In another study, Chen and Jaczynski also investigated the opportunity of using the solubilizing process to recover whole krill (Euphausia superba) proteins in a continuous mode. Centrifugations were made at 20,000g. Ash analyses showed that shells, appendages, etc. were efficiently removed in the process. Lipid removal was better after alkaline than acid solubilization. The krill initially contained 12.1% lipids (dw basis) and the acid-processed as well as alkali-processed isolates contained 11.619% and 7.48.1% lipids (dw basis), respectively. The protein isolates produced both at acid and alkaline pH had about 65% of the protein content found in the initial raw material (dw basis). Because of high proteolytic activity, the proteins failed to gel unless 1% of the protease inhibitor bovine plasma protein (BPP) was added. Proteins solubilized at alkaline pH, especially pH 12, gelled better than those produced with acidic pH. This was explained by higher proteolytic activity and denaturation at the acidic pH. Acidproduced gels were however whiter and brighter, which was ascribed the higher lipid content. As the first study of its kind, it should be highlighted that freeze-dried alkali-isolated krill protein concentrates were tested for their nutritional value, health benefits, and safety (Gigliotti et al. 2008). The isolates used contained 78% protein and 8% fat on a dw basis. About 27% of the fat was n-3 PUFA. A study of female SpragueDawley rats that were given isocaloric diets containing either 10% krill proteins or 10% casein showed that the krill protein digestibility ratio, corrected for amino acid score and protein efficiency, was equal to that of casein. Safety of the krill proteins was indicated by the fact that there were no differences in clinical measures of kidney function compared to casein. An interesting observation in the study was that the rats consuming krill proteins obtained greater total retroperitoneal and gonadal fat pads than the rats fed casein. This was despite equal body weights and weight gains between the two groups. Greater nitrogen excretion in krill protein-fed rats than in casein-fed rats suggested more amino acid deamination in the former group. Possibly, the

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excess carbon skeleton was converted into fat from energy storage. The latter result needs further investigations to be confirmed. Strong parallels to the above protein isolation studies were seen in a very recent investigation of protein isolation from blue mussels (Mytilus edulis). Mussels are also a whole raw material in the sense that the intestines are present (Vareltzis and Undeland 2008). Here the process was primarily applied to remove diarrheic shellfish poisoning (DSP) toxins, but information on lipid removal and protein yields were also obtained. A 1:6 homogenate of minced mussel meat was solubilized at low pH (2.8) or high pH (11.1), followed by centrifugation at 8,000g and re-precipitation of the solubilized mussel proteins at pH 5.8 for the acid-solubilized proteins and 5.3 for the alkaline solubilized. These processes resulted in 81% (acid solubilization) and 72% (alkaline solubilization) reduction in the DTX-1 toxin content of the mussel meat on an equal moisture content basis. This result can thus provide an alternative way of processing blue mussels in periods when they are toxic. Acid processing of mussel meat resulted in 50% reduction in the total lipid content expressed in terms of lipid to protein ratio, while alkaline treatment did not significantly affect the lipid content. The latter result was thus different from the findings of Kristinsson and coworkers using fillets from various tropical species. Interestingly enough, a poor correlation factor was obtained between the removal of DTX-1 toxins and removal of lipids. Mass balance calculations also showed that a large part of the DSP toxin ended up in the precipitation supernatant in which only traces of lipids are present. Lipid oxidation as followed by TBARS was not induced during acid or alkali solubilization process. The protein yields were fairly low; the alkaline process gave 53% protein yield and the acid one 35%. This was primarily ascribed to a high solubility of the proteins at all pHs tested for precipitation. In turn, this could be a result of proteolytic peptide formation during the process. Peptides are known for their high solubility in water. SDS-PAGE electrophoresis revealed proteolysis during the storage of blended mussel meat even at 80 C. In a preliminary study of acid- and alkali-aided protein isolation from whole and gutted herring, the use of a 1:9 homogenate solubilized at either pH 2.7 or 10.8 and precipitated at pH 5.5 gave promising data (Rkaeus and Undeland 2007) The total protein yields were 6065% from whole herring mince and somewhat lower for gutted herring mince. For the protein isolates, the acid process gave whiteness values of 59 and 54 when using gutted and whole herring, respectively. For the alkaline process, the corresponding values were 58 and 55, respectively. Considering the heavy pigmentation of the eyes, belly, and dark muscle, these results indicated that a substantial part, e.g., of melanin pigment are lost during the process.

From the results above, it is obvious that the acid and alkaline processes provide very promising ways of raising the value of fish by-products and certain whole fish/ shellfish species. However, some new problems may arise when the acid and alkaline processes are applied on raw materials with high proteolytic activity. The presence of proteolytic enzymes can via their action reduce protein yield and gelation capacity. Addition of BPP or other protease inhibitors could however be one way to combat these problems. Recovery of Additional Fractions Using pH-shift Methods The possibility of utilizing the precipitation supernatant from alkaline protein isolation processing of Cape hake saw dust was investigated (Pires et al. 2006). The collected supernatant at pH 5.5 was concentrated by ultra-filtration and then spray-dried. The protein content and ash content of dried proteins from the supernatant were 74.0% and 9.1%, respectively. The color parameters of the protein powder were L* 86.26, a* 0.06 and b* 5.47. As described in the section addressing protein yield, Cortes-Ruis et al. (2001) investigated the opportunity to reprocess the proteins in the gelatinous soft gel discarded from the first centrifugation during processing of sardines. This gel layer and the protein precipitate was mixed with water and adjusted to pH 3.2 after which it was reprocessed according to Fig. 1. In this way, they could increase the total protein yield from 64.2% to 76%. Okada and Morrissey (2007) developed a new oil extraction method involving pH adjustment and compared it with the traditional heat extraction. This method is related to the acid and alkaline processing methods, but is not at all identical. Minced sardines were mixed with deionized water (9:1 v/v), and the pH was adjusted to 5.5 with several acids, including HCl or the food-grade organic acids citric acid and tartaric acid (+/ Ca) followed by centrifugation. Oil extracted with citric acid + Ca showed the best quality, along with good recovery, the lowest haze value, and the highest level n-3 polyunsaturated fatty acids. Oil recoveries with citric acid + Ca were 40.6% followed by oil extracted by citric acid alone (37.6%) and classic heat extraction (35.1%). Compared to the heat process, all oils extracted by pH adjustment showed high oxidative stability and had fewer impurities. There was a significant beneficial effect of Ca addition prior to pH adjustment in terms of lipid oxidation stability and color of the final products. According to the authors, the pH adjustment method is a novel process for oil extraction and a promising method that can be utilized for high oil pelagic species such as Pacific sardines. These studies show that there is great room for increasing the protein yields from acid and alkaline

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processing by utilizing the fractions normally discarded. Using an alternative pH-shift method, high quality oil can also be obtained. Processing Simplifications and Attempts for Upscaling During the years that have passed since the acid and alkaline processes were patented, a series of attempts have been made to improve, e.g., lipid removal, lipid stability, color, protein yields, gelation capacity, and the simplicity/ economy of the methods. Some of the successful lipidrelated improvements have already been highlighted, e.g., antioxidant additions, membrane-removing agents (citric acid/calcium chloride, chitosan), and pre-washing/air floatation of the minced raw material. In the section below, some strategies tested, e.g., to reduce the costs of the process by avoiding the requirements for the high-speed centrifuges are given. Some attempts for upscaling are also highlighted. Kristinsson and Demir (2003) evaluated the pH-shift methods without the first centrifugation step. With this modified process they obtained a high protein yield from channel catfish mince. Compared to ordinary processed surimi and regular alkali-processed surimi where the first centrifugation was included, they also obtained high gel strength. Using impure materials, it is however important to stress that skipping the first centrifugation calls for some other way of removing, e.g., bones. The simplest strategy would be just time, i.e., gravity or filtration. Nolse et al. (2007) found protein yields between 58% and 75% from cod and haddock mince using the alkaline process with centrifugation in the first separation step. When replacing the first centrifugation with sieving, the protein yields obtained were between 82% and 89% of the ground fish muscle. However, the gel quality obtained from the sieved material was inferior to the quality of the centrifuged material. Vareltzis and Undeland (2008) made an attempt to reduce the g-force used in the first centrifugation from 8,000g to 800g in order to simplify acid- and alkaliaided protein isolation from blue mussels. The findings showed that the lower centrifugation force did not affect the lipid reduction of the alkaline-produced samples, while with the acid process the lipid removal was less efficient. However, when citric acid and calcium chloride (Cc) was used in the alkaline solubilization combined with the low centrifugation g-force, the lipid reduction was still significant compared to the untreated blended mussels. In summary, the lipid/protein ratio in the various samples analyzed decreased in the following order: blended mussels alkaline process, 800g alkaline process, 8,000g acid process, 800g > acid process, 8,000g > Cc alkaline process, 800g Cc acid process, and 8,000g Cc alkaline process 8,000g.

Based on the trials with conventional surimi, it was found that protein losses could be reduced by 50% by using decanter technology instead of using rotary screen and presses (Lanier et al. 1992). This might be applicable also to acid and alkaline processing. Regarding upscaling and attempts to make the process continuous, the work by Chen and Jaczynski (2007) has already been addressed. Here, the process steps, including the centrifugation steps, were made in a continuous manner with a flow rate of 120 L/h. Although this flow rate is not large scale, it shows that it is possible to get the process working at a larger scale than normally performed in the laboratory. In a recent paper by Mireles DeWitt et al. (2007), catfish fillets were subjected to pilot scale acid processing after which gels were made from the isolates. Centrifugation was carried out at 3,000g and with a flow rate of 1 m3/h. The outcome was compared with non-solubilized catfish proteins. The acid solubilization process removed 74% of the initial fat (2.8% on ww basis). Gels from treated protein were less red and yellow than non-solubilized muscle. There was no difference in whiteness or in cooking yield. The water holding capacity of gels made from control protein was 0.73 g/g protein higher than treated. The texture analysis indicated that acid-processed proteins had significantly higher hardness, springiness, chewiness, and cohesiveness compared with non-solubilized muscle gels. The resilience was equivalent for both treated and control gels. The results indicated that the protein solubilization process in the pilot scale produced a low-fat protein product with good gel strength properties. To summarize, protein yields can be improved by using a sieve instead of centrifugation in the first separation step. This is most likely since the formation of large proteinentrapping gel sediments is prevented. The gelation ability was however lower after sieving, probably due to a lower purity of the proteins. Reduced g-force in the first centrifugation step reduced lipid removal, especially with acid processing. Successful attempts with continuous processing at 120 and 1,000 L/h have been carried out indicating good potentials for upscaling of the processes.

Tentative Uses of the Protein Isolates and the pH-shift Process The most obvious and widespread application of the acidand alkali-processed protein isolates have so far been as an alternative to traditional surimi. Recently, there have however been some interesting applications reported, e.g., as coatings for fried foods, as injection/tumbling marinades to improve water holding of muscle products, and as substrates for proteolytic enzymes in the production of very

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clean protein isolates. The latter product could have important functions, e.g., as antioxidants and bioactive ingredients in functional foods. Finally, the use of acidproduced cod proteins as emulsifiers has also been reported (Petursson et al. 2004). Proteus Industries (www.proteusindustries.com) has succeeded in using the acid solubilizing process in the production of dried water-soluble muscle protein isolates that are applied as coatings for different bathered and breaded products. The patented fat-blocking protein technology when produced is called Nutrilean. According to Proteus industries, Proteus proteins work by forming a barrier around the coated product, partially preventing fat from entering the product during cooking. The barrier also assists in the binding of moisture in the final product, thus improving the juiciness of the product. Commercial results to date have seen up to 50% reduction in fat content and 13% increase in final moisture in the cooked chicken. Proteus Industries further states that the process can be replicated across species and can be applied to fish, chicken, beef, pork, and other animal muscle, and the resulting isolates can be used in liquid and powder forms in order to achieve the desired benefits in food processing and cooking. GRAS statements have been submitted for seafood and poultry (FDA GRAS Notices No. 000147 and GRN 000168). In a recent study, select grade strip loins were enhanced with either a phosphate- or an acid-solubilized protein solution (Vann and Mireles DeWitt 2007). The authors then followed a color score, aerobic plate count, lipid oxidation, purge loss, cook yield, shear force, and sensory analysis during storage. The phosphate injection consisted of 4.5% phosphate, 3.6% sodium chloride, 90.9% water, and 1% Herbalox seasoning. Two solutions were prepared for the protein injection: one consisting of 1:9 protein to water and the other an aqueous solution of 1% Herbalox seasoning and 3.6% sodium chloride. Enhancement solutions were injected twice at 5% to create a 10% total injection. It was found that the protein-enhanced steaks were comparable to phosphate-enhanced steaks for percent discoloration and overall acceptability. The phosphate-enhanced steaks performed better than the protein-enhanced steaks for lean color, fat color, aerobic plate count, lipid oxidation, percent purge, cook yield, and WarnerBratzler shear force. It should be noted that for protein-enhanced steaks, lean color and cook yield, although statistically different, did perform similar to phosphate-enhanced steaks. It should be stressed that the protein-containing injection solutions can be made in two ways. One is by resolubilizing proteins isolated by the acid/alkaline methods, and the other is to directly use solubilized ground fish muscle, in the acid or alkaline state, for injection into fillets without using any precipitation step.

The ability of a high-salt-extracted fraction and a pH 3acid-extracted protein fraction from cod to form and stabilize 5 wt.% corn oil-in-water emulsion was examined by Petursson et al. (2004). Emulsions could be formed that had relatively small mean droplet diameters and good creaming stability at protein concentrations 0.2% ww for both fractions. The emulsions were stable to droplet flocculation and creaming at pH 4 and NaCl concentrations 150 mM when stored at room temperature. In the absence of salt, the emulsions were also stable to thermal treatment, but in the presence of 100 mM NaCl droplet flocculation and creaming were observed in some of the emulsions, particularly those stabilized by the AE fraction. The results suggest that protein fractions extracted from cod can be used as emulsifiers to form and stabilize food emulsions. Raghavan and Kristinsson (2008a) prepared alkaliproduced tilapia protein isolates that were then subjected to proteolysis using five different enzymes. Hydrolysis was carried out to 7.5%, 15%, and 25%. All hydrolysates significantly inhibited Hb-mediated TBARS and PV development in a washed cod muscle system when 0.35 g hydrolyzed protein was added per 100 g model. Higher inhibition was seen with higher degree of hydrolysis. The proteolytic enzyme used also had an impact on the antioxidant capacity with the Cryotin F being most effective and Flavourzyme as well as Neutrase being least effective. The capacity of the hydrolysates to scavence DPPH radicals was different from the capacity to prevent Hb-mediated lipid oxidation. In a later study (Theodore et al. 2008), alkali-isolated catfish proteins hydrolyzed to 5%, 15%, and 30% degree with Protamex protease were separated into insoluble and soluble fractions and tested for their antioxidative capacity using five different measures. These were metal chelation, DPPH-radical scavenging, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and ability to prevent Hb-mediated TBARS in washed tilapia muscle. Results showed that low molecular weight peptides gave the highest chelating ability, ORAC values and TBARS inhibition. High molecular weight peptides gave higher FRAP values and scavenged more DPPH radicals. To summarize, several new applications have been tested in recent years that broadens up the use of proteins isolated with the acid and alkaline technology. They all provide promising ways of raising the value of underutilized muscle sources.

Conclusions A fairly large body of literature has emerged on the acid and alkaline protein isolation methods since the first patent

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came in 1999. A wide variety of species have been used, in most cases fillets, but also separated light muscle. Studies on more complex raw materials have however emerged in the later years, which shows that great opportunities open up for pH-shift isolation of proteins from fish by-products and underutilized species. In fact, isolation and value adding of underutilized muscle proteins appear to be the area where the pH-shift processes have a really large potential. Among utilizations of the protein isolates that have been reported so far are an alternative surimi, a coating for low-fat fried products, a basis for a marinade/ injection solution for improved water holding, e.g., of fillets, an emulsifier, and a substrate for hydrolysis making. The latter product can then, e.g., be used as an antioxidant. A first study has also emerged which shows that alkaliproduced krill proteins have equally high nutritional properties as the milk protein casein. This study opens up for many additional applications for protein isolates made with the acid and alkaline methods. It is important to point out though that most of the studies that so far have been published on the pH-shift processes are from laboratory scale trials. The processes are likely to perform quite differently in larger scale processing. From the comparisons that have been made between the acid and alkaline methods, it is obvious that there in most cases is one important advantage of the acid process over the alkaline one. This is the generally higher protein yields that have been obtained. Likely explanations are a higher solubility at acidic pHs and the formation of smaller sediments. The alkaline process, on the other hand, performs better in many other aspects. Almost all published studies gave stronger gels with the alkaline than the acid process, with the former process performing fairly equal to conventional surimi. It has been speculated that the harder gels obtained with alkali-produced proteins compared to acid-produced isolates originate in less hydrolysis during alkaline processing and also a larger degree of SS bond formation. Conformational changes of key myofibrillar proteins occuring during the acid-/alkali-induced unfolding/refolding are also likely to be involved. Further, the alkaline process in most cases has given whiter isolates/gels than the acid process as a larger part of the heme proteins is removed with alkaline than acid processing. Pre-washing the raw material is therefore beneficial with respect to color. In most studies covering lipid reduction with the two processes, more lipids have been removed with alkaline processing. This might be since the lipid emulsifying ability of proteins is improved during the alkaline process compared to during the acid process which increases the size of the floating lipid layer formed in the first centrifugation. In studies of lipid oxidation, it has been pointed out that the alkali-processed protein isolates are

much more stable towards lipid oxidation than the acidprocessed ones as heme proteins get more catalytic after subjection to low pH. Additions like erythorbate, EDTA, citric acid, and calcium chloride can however prevent process-induced lipid oxidation. Lastly, one study showed that the alkaline process proved to give isolates with a lower total bacterial count than the acid process and another study showed that the alkaline process yielded proteins with higher concentrations of essential amino acids than the acid one. From the advantages and disadvantages listed, it is obvious that the choice of method depends on the aim of the processing and the intended uses of the protein isolates. In several studies, attempts have been made to improve or modify the pH-shift methods. Some of these attempts have been focused on recovering proteins in the phases that are normally discarded, such as the gelatinous soft layer from the first centrifugation and the precipitation supernatant. Attempts have also been made to run the process in a continous mode, to introduce a pre-washing step of the raw material, and to utilize alternatives to centrifugation in the two separations. According to the aforementioned studies, additions of various kinds to aid lipid removal, to reduce lipid oxidation, and to reduce proteolysis have also been tested with good results. When comparing the pH-shift methods to classic surimi making, one major advantage of the former method is the possibility of directly processing whole fish and complex raw materials without first mechanically separating the meat from bones/skin. This makes the pH-shift methods very useful in utilization of by-products such as heads, collarbones, and backbones and also for whole underutilized species. Some studies are already published along these lines that show promising results both on protein yields and removal of lipid-soluble toxins. A drawback in the use of whole fish and shellfish can however be high proteolysis caused by the abundance of proteolytic enzymes. Strategies to prevent this have therefore been addressed. Other advantages of the pH-shift methods are that they use less water than a classic surimi process and that they generally give higher protein yields than traditional surimi processing. The latter advantage is most likely explained by the recovery of sarcoplasmic proteins using the pH-shift processes. Further, the acid and alkaline processing clearly removes more lipids that conventional surimi making, something that can limit lipid oxidation and aid in removal of lipid-soluble contaminants. However, in favor of conventional surmi making, it often gives whiter isolates/ gels than the pH-shift methods, most likely due to the washing-induced heme removal. Another advantage of classic surimi making is that there is no necessity of using chemicals in the production. The use of acid and base in the pH-shift methods will require stronger safety requirements. Using acid in connection with metals can cause corrosion.

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25 Carpenter, R. P., Main, G., & Sutton, A. H. (1975). Process for the isolation of protein from fish. Patent GB1409876. Chaijan, M., Benjakul, S., Visessanguan, W., & Faustman, C. (2006). Physiochemical properties, gel-forming ability and myoglobin content of sardine (Sardinella gibosa) and mackerel (Rastrelliger kanagurta) surimi produced by conventional method and alkaline solubilization process. European Food Research and Technology, 222, 5863. Chang, H. S., Feng, Y., & Hultin, H. O. (2001). Role of pH in gel formation of washed chicken muscle at low ionic strength. Journal of Food Biochemistry, 25(5), 439457. Chen, Y. C., & Jaczynski, J. (2007). Protein recovery from rainbow trout (Oncorhynchus mykiss) processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives. Journal of Agricultural and Food Chemistry, 55(22), 90799088. Chen, Y. C., Tou, J. C., & Jaczynski, J. (2007). Amino acid, fatty acid, and mineral profiles of materials recovered from rainbow trout (Oncorhynchus mykiss) processing by-products using isoelectric solubilization/precipitation. Journal of Food Science, 72(9), C528C536. Choi, Y. J., Kim, Y. S., & Park, J. W. (2003). New approaches for the effective recovery of fish proteins and their physiochemical characteristics. Fisheries Science, 69(6), 12311239. Choi, Y. J., & Park, J. W. (2002). Acid-aided protein recovery from enzyme-rich Pacific whiting. Journal of Food Science, 67(8), 29622967. Choi, Y. J., & Park, J. W. (2005). Improvement of whiteness of recovered proteins obtained by alkaline processing. IFT89B-18 (2005 IFT Annual Meeting) New Orleans, Louisiana. IFT Annual Meeting. 7-15-2005. Conference Proceeding Cortes-Ruis, J. A., Pacheco-Aguilar, R., Garcia-Sanchez, G., & LugoSanches, M. E. (2001). Functional characterization of a protein concentrate from bristly sardine made under acidic conditions. Journal of Aquatic Food Product Technology, 10(4), 523. Davenport, M., & Kristinsson, H. G. (2004). Effects of different acid and alkali treatments on the molecular and functional properties of catfish muscle proteins. IFT49G-14(2004 IFT Annual Meeting) New Orleans, Louisiana, IFT. 7-12-2004. Conference Proceeding Davenport, M., Kristinsson, H. G., & Nguyen, H. Q. (2005). Effect of cold storage and freezing of ground catfish muscle prior to acid or alkali-aided isolation of muscle proteins. Annual IFT Meeting 2005 New Orleans, Louisiana, IFT. 7-15-2005. Conference Proceeding Davenport, M., Theodore, A. E., & Kristinsson, H. G. (2004). Influence of insoluble muscle components on the gelation properties of catfish protein isolates made with acid and alkaliaided processing. 2004 IFT Annual Meeting83A-21Las Vegas, FDA GRAS Notice No. GRN 000147, http://www.cfsan.fda.gov/ ~rdb/opag147.html NV, IFT. 7-12-2004. Conference Proceeding FDA (2004). GRAS Notice No. GRN 000147, http://www.cfsan.fda. gov/~rdb/opag147.html. Gigliotti, J. C., Jaczynski, J., & Tou, J. C. (2008). Determination of the nutritional value, protein quality and safety of krill protein concentrate isolated using an isoelectric solubilization/precipitation technique. Food Chemistry. On line 2008. doi:10.1016j. foodchem.2008.03030). Huang, L., Chen, Y., & Morrissey, M. T. (1997). Coagulation of fish proteins from frozen fish mince wash water by ohmic heating. Journal of Food Process Engineering, 20, 285300. Hultin, H. O., & Kelleher, S. D. (1999). Process for isolating a protein composition from a muscle source and protein composition. Patent US6005073. Hultin, H. O., & Kelleher, S. D. (2000a). Surimi processing from dark muscle fish. In J. W. Park (Ed.), Surimi and surimi seafood (pp. 5977). New York: Marcel Dekker.

HCl, for example, is a strong oxidizing agent for metals. This property has also been seen in connection with the heme proteins of the fish during acid production. If lipids are to be efficiently removed, the need of a relatively long high-speed centrifugation step during the pH-shift methods is another feature that makes these processes more complex to work with than classic surimi making. However, if lipid removal is not an issue and if the requirements regarding gel strength are not the same as the requirements usually set for surimi, the production can be facilitated accordingly. Then it is for example possible to remove the undissolved material from the dissolved material using a filter, with or without pressure. In summary, we see the pH-shift methods coming up as an important complement to classic surimi making for complex and unstable materials where it is difficult to mechanically pre-separate the meat. The possiblity to remove lipids and lipid-soluble contaminants also strengthens its role in the processing of low value materials that otherwise mainly go to animal feed or that even are discarded. Since each material appears to respond differently to the acid and alkaline processes, it is recommended to try them both in an initial stage.
Acknowledgment We wish to sincerely thank Prof. Herbert Hultin, one of the two main inventors of the acid and alkaline technology, for so kindly sharing a lot of his knowledge on these processes to us. Professor Hultin sadly passed away on Dec. 12, 2007, but through his fantastic scientific accomplishments, he is still with us.

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