pH-shift Process Research Articles

Nutritional and Digestive Properties of Protein Isolates Extracted from the Muscle of the Common Carp Using pH-Shift Processing.

This study details the nutritional and digestive properties of protein isolates that are extracted from carp (Cyprinus Carpio L.) muscle using pH shifting methods. Alkaline (ALPI) and acid (ACPI) protein isolates exhibit higher protein yields (87.6%, 76.3%, respectively). In addition to the high recovery of myofibrillar protein, a portion of the water‐soluble proteins is also recovered. The moisture contents of ACPI and ALPI are 85.5% and 88.5%, respectively, and the crude protein contents of these two fractions are 83.20% and 83.0%, respectively, both contents of which are higher than those for fresh muscle. Most part of the ash and fat are removed in the separation process. The protein isolation is also found to be lighter and whiter than the fresh muscle and there is no difference between amino acid content of protein isolation and that of fresh muscle. The maximum solubility of water washed surimi is 73.21%, while solubility of ACPI‐2 and ALPI‐2 (pH 7.0) are 66.67% and 62.08%, respectively. The digestibility of ALPI and ACPI is improved after being treated with chymotrypsin, which is about 7–8 times as that of fresh muscle. The results indicate that the protein isolates have better nutritional and digestive properties than the fresh muscle does in food processing.Practical ApplicationsCommon carp is a lower additional value fish that exists in large amount in China. This study investigates nutritional and digestive properties of protein from carp extracted by pH shifting methods. According to the obtained data in this study, pH shifting method is a good protein recovery method that can effectively remove bone spurs, skin, fat and other impurities. In addition, sarcoplasmic proteins can also be recovered. The nutritional properties of protein isolates of carp were suitable for supplementing as an ingredient for human consumption. The pH‐shift process greatly improves the protein digestibility. Therefore, there are broad application prospects of the protein isolation as protein ingredients in food industry.

Boarfish protein recovery using the pH-shift process and generation of protein hydrolysates with ACE-I and antihypertensive bioactivities in spontaneously hypertensive rats

Abstract Boarfish ( Capros aper Linnaeus) are frequently in European waters. In this work, the generation and identification of angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) inhibitory and antihypertensive peptides from Boarfish proteins are reported for the first time. Boarfish proteins were recovered using the pH shift process and hydrolysates were generated using the commercially available proteolytic enzyme alcalase CLEA, papain and protease AP. Molecular weight cut off (MWCO) filtration methods were used to enrich for peptides less than 10- and 3-kDa in size. The angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) inhibitory activity of the generated hydrolysates and MWCO fractions was assessed in vitro . The 3-kDa Boarfish protein hydrolysate (BPH) generated using protease AP inhibited ACE-I by 85.8% (± 1.81) when assayed at a concentration of 1 mg/mL compared to the positive control captopril. Mass spectrometry was used to characterize the peptides within the BPH generated using protease AP. Moreover, the antihypertensive activity of this BPH was assessed in vivo using spontaneously hypertensive rats (SHRs) over a 24 hour period. Results obtained suggest that BPH generated using protease AP is a source of ACE-I-inhibitory peptides with antihypertensive effects in vivo . This hydrolysate has the potential for use as a functional food ingredient for the maintenance of normal blood pressure in hypertensive individuals. Industrial relevance • This paper provides a potential alternative for marine processors for utilisation of their total catch in the generation of functional foods. • Discarded fish represents an obstacle to the sustainability of global fisheries. Due to the fact that the reformed European Common Fisheries Policy promotes landing of all species under the Total Allowable Catch (TACs) policy in Europe, it will be necessary for processors to utilise all species caught to prevent disposal costs. • Boarfish are caught presently as a co-product with other fish. In light of the new regulation and costs for disposal at landfill it will therefore be necessary to convert Boarfish into value added products. • Industry in Ireland and Denmark are particularly interested in this topic as have the largest quota for Boarfish within the EU. This paper describes a methodology for generation of protein hydrolysates from Boarfish with added health benefits. • This area is of increasing, commercial interest to marine processors and food product developers as fish are a known source of bioactive peptides. Boarfish ( Capros aper Linn) are frequently discarded in European waters. In this work, the generation and identification of angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) inhibitory and antihypertensive peptides from Boarfish proteins is reported for the first time. Boarfish proteins were recovered using the pH shift process and hydrolysates were generated using the commercially available proteolytic enzymes alcalase CLEA, papain and protease AP. Molecular weight cut off (MWCO) filtration methods were used to enrich for peptides less than 10- and 3-kDa in size.

Potential Production of Healthier Protein Isolate from Broiler Meat using Modified Acid-Aided pH Shift Process

Protein isolates from broiler meat were produced using acid-aided process (AP) with some modifications including prewashing prior to pH shift process (PW), homogenisation with 5 mM citric acid plus 8 mM CaCl2 (CA) and homogenisation with 5 % ethanol (EA). Significant reduction of total cholesterol, nucleic acids and haemoproteins with improved protein yield and whiteness of the isolate was obtained from all modified process (p < 0.05). CA was the most effective method in elimination of cholesterol (p < 0.05). However, PW was the best way to remove nucleic acids and haem proteins (p < 0.05). Among modified processes, CA gel tended to show the highest breaking force, deformation and whiteness but PW gel had the lowest expressible drip (p < 0.05). EA possessed a negative effect on gel-forming ability. Therefore, CA or PW was a promising means to produce functional protein isolate with reduced nucleic acids, haem pigments and cholesterol.

PH-shift processing of Nannochloropsis oculata microalgal biomass to obtain a protein-enriched food or feed ingredient

Fractionation of plant and animal raw materials by pH-shift processing has been widely applied to purify proteins. The principle is to solubilize proteins at high or low pH, removing debris and precipitating the proteins near their isoelectric point. We here describe the pH-shift process on commercially available Nannochloropsis oculata. The partitioning of major nutrients into the various fractions of the process was studied. Proteins were found to exhibit maximal solubility between pH7 and 10, with a minimal solubility below pH4. Two process versions were investigated in this study, with solubilization at either pH7 (native pH) or 10; both versions were precipitated at pH3. Up to 85% of both the protein and total fatty acids were recovered in the final product, compared to the initial algal slurry. Protein, total fatty acids and carbohydrates were concentrated in the final product, while the ash content was lower compared to the starting material. From a processing point of view, solubilization of Nannochloropsis at native pH was found to be preferable, since less chemicals were consumed compared to high-pH solubilization. Owing to its content of protein and (total) fatty acids (23 and 12% of dry weight, respectively), the latter enriched in eicosapentaenoic acid (EPA), the product of the pH-shift process has potential as a functional food ingredient.

Recovery of Tilapia (Oreochromis niloticus) Protein Isolate by High-Intensity Ultrasound-Aided Alkaline Isoelectric Solubilization/Precipitation Process

High-intensity ultrasound (HIU), which is regarded as “green” technology, was combined with an alkaline pH-shift process to extract tilapia (Oreochromis niloticus) protein isolate (TPI) at various pH conditions. The results showed that HIU significantly decreased the consistency of alkaline muscle homogenate, especially at less extreme pH (pH 10.5), increased the protein solubility, and reduced the sediment ratio. Alkaline volume needed for pH adjustment to the same pH level was slightly increased by HIU, indicating that HIU accelerated the charging of myofibril protein particles and strengthened the electrostatic repulsion forces. Aided by two HIUs, the protein recovery at pH 10.5 was increased from 47.0 to 62.6 %, which was equivalent to that (62.4 %) at pH 11.5 without HIU, suggesting that ~40 % alkaline and corresponding acid in the process could be saved. SDS-PAGE images reveal that HIU induced partial degradation of titin and disassociation of nebulin with thin filament in sarcomere, which possibly facilitated the dispersion and solubilization of myofibril proteins. Those results of ATPase activity loss and surface hydrophobicity increase indicated that HIU also affected the conformation of the dissolved myofibril protein in muscle homogenate. TPI gel strength was elevated when the alkaline pH-shift process was aided by HIU.

Development of a new method for determination of total haem protein in fish muscle

Using classic haem protein quantification methods, the extraction step in buffer or acid acetone often becomes limiting if muscle is oxidised and/or stored; haem-proteins then tend to bind to muscle components like myofibrils and/or biomembranes. The objective of this study was to develop a new haem protein determination method for fish muscle overcoming such extractability problems. The principle was to homogenise and heat samples in an SDS-containing phosphate buffer to dissolve major muscle components and convert ferrous/ferric haem proteins to hemichromes with a unique absorption peak at 535nm.Hb-recovery tests with the new and classic methods showed that the new method and Hornsey’s method performed significantly better on fresh Hb-enriched cod mince than Brown’s and Drabkin’s methods; recovery was ⩾98%. However, in highly oxidised samples and in cod protein isolates made with acid pH-shift processing, the new method performed better than Hornsey’s method (63% and 87% vs. 50% and 68% recovery). Further, the new method performed well in fish muscle with ⩽30% lipid, <5% NaCl and pH 5.5–7.0; it was also unaffected by freezing/frozen storage.

Effects of pH-Shift Processing and Microbial Transglutaminase on the Gel and Emulsion Characteristics of Porcine Myofibrillar System.

This study investigated the effects of microbial transglutaminase (MTGase) and pH-shift processing on the functional properties of porcine myofibrillar proteins (MP). The pH-shift processing was carried out by decreasing the pH of MP suspension to 3.0, followed by re-adjustment to pH 6.2. The native (CM) and pH-shifted MP (PM) was reacted with and without MTGase, and the gelling and emulsion characteristics were compared. To compare the pH-shifted MTGase-treated MP (PT), deamidation (DM) was conducted by reacting MTGase with MP at pH 3.0. Rigid thermal gel was produced by MTGase-treated native MP (CT) and PT. PM and DM showed the lowest storage modulus (G') at the end of thermal scanning. Both MTGase and pH-shifting produced harder MP gel, and the highest gel strength was obtained in PT. All treatments yielded lower than CM, and CT showed significantly higher yield than PM and DM treatments. For emulsion characteristics, pH-shifting improved the emulsifying ability of MP-stabilized emulsion, while the treatments had lower emulsion stability. PM-stabilized emulsion exhibited the lowest creaming stability among all treatments. The emulsion stability could be improved by the usage of MTGase. The results indicated that pH-shifting combined with MTGase had a potential application to modify or improve functional properties of MP in manufacturing of meat products.

Gelation Characteristics of Surimi Gel from Silver Carp (Hypophthalmichthys molitrix) by Application of Acid-Alkali Processes

Textural characteristics of silver carp surimi gel prepared from acid- and alkali-produced protein isolates were investigated and compared to the surimi gels derived by conventional methods. The acid-aided method showed the most efficiency to recover proteins (86.2%) in comparison to the alkaline-aided (79.8%) and conventional (76.7%) methods. In the washing of fish mince, every step resulted in reduction of sarcoplasmic protein, and consequently the myofibrillar fraction became more concentrated, as shown by the protein patterns in wash water. Some myofibrillar proteins (mainly actin and myosin) were removed along with sarcoplasmic proteins during washing, reducing yield efficiency during the conventional method of making surimi. During the pH shifting process, myosin heavy chain (MHC) was degraded during low (2.5) pH solubilization, whereas there was only a minimal loss of MHC by either hydrolysis or proteolysis during alkaline solubilization. The breaking force and breaking distance of surimi gel prepared in the pH-shifting processes were significantly (p < 0.05) greater than those of surimi gel made by conventional methods, as were expressible moisture, cohesiveness, and springiness. In conclusion, the pH-shifting technique can be considered as an efficient alternative process in recovery of greater quantities of functional protein from silver carp fillet.

Preparation and characterization of gelatin from collagen biomass obtained through a pH-shifting process of mechanically separated turkey meat

Gelatins were extracted from mechanically separated turkey meat following 2 different approaches. The first method was based on a 2-stage batch extraction at 50 and 60°C, respectively, whereas the second method consisted of recovering gelatin from a collagen biomass obtained during a pH-shifting process. The yield of gelatin produced by the latter method was twice that obtained by the former method (13.51 and 6.36%, respectively). The chemical composition, as well as the rheological and the functional properties, of all extracted gelatins were evaluated. Gelatin recovered from the collagen biomass had higher molecular weight components and significantly greater (P < 0.05) bloom value (353.2 g) compared with thermally extracted gelatins. However, gelatin extracted at 60°C possessed higher (P < 0.05) foaming properties, as well as better emulsifying activity, than gelatin extracted from the 50°C treatment and the collagen biomass. The present study revealed that high-quality gelatins can be prepared from mechanically separated turkey meat through precipitation and thermal solubilization of collagen biomass obtained during a pH-shifting process.

Hydrolysis of insoluble fish protein residue from whitemouth croaker (Micropogonias furnieri) by fungi

A significant amount of insoluble fibrous protein, in the form of feather, hair, scales, skin and others are available as co-products of agro industrial processing. These wastes are rich in keratin and collagen. This study evaluated different fungi for the hydrolysis of insoluble fish protein residues. Proteins resulting from Micropogonias furnieri wastes through pH-shifting process were dried and milled for fermentation for 96 h. This resulted the production of keratinolytic enzymes in the medium. Trichoderma sp. on alkaline substrate (28.99 U mL-1) and Penicillium sp. on acidic substrate (31.20 U mL-1) showed the highest proteolytic activities. Penicillium sp. showed the largest free amino acid solubilization (0.146 mg mL-1) and Fusarium sp. the highest protein solubilization (6.17 mg mL-1).

Protein Recovery of Tilapia Frame By-Products by pH-Shift Method

The conditions of isolating protein from tilapia frame by-products (TFB) by a pH-shift process were investigated along with their physicochemical and gelling properties. The minimum solubility of TFB protein was observed at pH 5.5 and gradually increased at both acid and alkaline sides. The highest solubility was observed at pH 2 and 12. Protein solubility of TFB increased as the ratio of alkaline extraction medium and extraction time increased. The highest protein extraction was found at the ratio of ground sample to extraction medium of 1 to 9 and extraction time of 15 min. The maximum protein recovery was found at pH 12 with 19.19%. The color of all protein isolate samples obtained from either acid or alkaline extraction was dark brown. Whiteness of a TFB protein isolate extracted at pH 2 was the highest with a value of 14.69. An alkaline pH-shift process effectively removed fat as much as 95%. Breaking force and deformation of recovered protein gel from an alkaline pH-shift were higher than those from the acid counterpart.

Chemical, rheological and surface morphologic characterisation of spent hen proteins extracted by pH-shift processing with or without the presence of cryoprotectants

The chemical and rheological properties of spent hen proteins recovered by acid and alkaline extraction have been studied with/without cryoprotectants (CP) after 3weeks of frozen storage. Four pH values (2.0, 2.5, 11.5, and 12.0) were used for extraction. CP addition prevented freeze-induced denaturation and oxidation in all extracted proteins, as revealed by significant increases in reactive sulphydryl groups (p<0.0001), and a decrease in the formation of carbonyl groups (p<0.0001). The alkaline extracted proteins with CP formed more viscoelastic gels compared to the others, while samples without CP failed to form a gel network. FTIR of the protein isolates with CP showed a protective effect on the secondary structure of the isolated proteins. Scanning electron micrographs showed a protective shield of CP around the isolated proteins.

Effect of alkaline pH-shift processing on in vitro gastrointestinal digestion of herring (Clupea harengus) fillets

The effect of alkaline pH-shift processing on herring (Clupea harengus) protein oxidation, salt solubility and digestibility, has been evaluated. For the latter, herring mince and pH-shift produced herring protein isolate, both raw and heat-treated, were digested using a static gastrointestinal in vitro model. The pH-shift process resulted in drastically lowered protein salt solubility and increased lipid oxidation while protein carbonyl formation was unaffected. Yet, no significant differences in the degree of hydrolysis (DH) were observed between mince and isolates after completed gastrointestinal digestion, something which was confirmed by a similar release of proteinaceous material <3kDa and similar free amino acid profiles. The polypeptide profiles of digested samples however revealed that two peptides (33 and 36kDa) were present in larger amounts in the digested protein isolate compared to the digested herring mince. The results indicate that alkaline pH-shift processing had limited quantitative influence on the gastrointestinal digestibility of herring proteins despite its negative effects on protein salt solubility and lipid oxidation.

Evaluation of a Modified pH‐Shift Process to Reduce 2‐Methylisoborneol and Geosmin in Spiked Catfish and Produce a Consumer Acceptable Fried Catfish Nugget‐Like Product

Muddy and/or musty off-flavors in farmed-raised catfish occur as a result of the absorption of geosmin (GEO) and 2-methylisoborneol (MIB), compounds produced by algae. Previous research suggests the acid pH-shift method may be able to reduce off-flavors to produce a consumer acceptable product. The objective of this research was to evaluate application of the acid pH-shift method using a shaker sieve for protein recovery and to evaluate consumer acceptability of a resultant batter-coated fried nugget-like catfish product. Farm-raised catfish were either allowed to depurate (control) or treated with 1 ppb GEO or MIB. Fillets from each replicate were collected and ground and treated by the acid pH-shift process. Samples from all treatments and replicates were evaluated for residual GEO and MIB. In addition, batter-coated fried catfish samples were prepared for a consumer sensory evaluation. Results demonstrated that the pH-shift process decreased moisture, ash, and collagen content of catfish fillet tissue (P < 0.05). Flavor of control samples was preferred (P < 0.05). Texture of catfish samples treated by the pH-shift process was preferred (P < 0.05). Results demonstrate the pH-shift process can be utilized to reduce off-flavors and increase the acceptability of a processed catfish product. Use of a sieve as an economic alternative for the pH-shift process was evaluated for removing off-flavors from catfish. Difficulties were encountered with regard to protein recovery using the sieve and suggestions are made to, perhaps, make the process more applicable for a sieve-based recovery step. The process as described reduced off-flavors, but only 2-fold suggesting the process would work best on catfish near or just over off-flavor thresholds. Results also indicated the pH-shift process could be used to improve texture of a fried catfish product designed to be similar to chicken nuggets.

Changes in Salt Solubility and Microstructure of Proteins from Herring (Clupea harengus) after pH-Shift Processing

Salt solubility of pH-shift isolated herring (Clupea harengus) muscle proteins was studied in relation to pH exposure and microstructure using transmission electron microscopy (TEM). Using protein solubilization at pH 11.2 with subsequent precipitation at pH 5.5, salt solubility of the proteins decreased from 78 to 17%. By precipitating the alkali-solubilized proteins at the pH of native herring muscle, 6.5, salt solubility only decreased to 59%, proving that pH values between 6.5 and 5.5 affected protein salt solubility more than the pH cycle 6.5 → 11.2 → 6.5. Precipitation at pH 5.5 resulted in hydrogen bonds, hydrophobic interactions, and S-S bridges, whereas precipitation at pH 6.5 resulted only in the formation of hydrophobic interactions. The alkaline pH-shift isolation process severely rearranged the protein microstructure, with precipitation at pH 6.5 forming a finer, more homogeneous network than precipitation at pH 5.5. The former protein isolate also contained less lipid oxidation products and formed more deformable gels, without affecting protein yield.

Using pH-Shifting Process to Recover Proteins for Low Salt Gel Products from Silver Carp

The pH-shifting processes (include acid and alkali process) and conventional water-wash process was applied to recover protein from bony silver carp muscle. And the new process was evaluated to obtain proteins for low salt (0% and 1% NaCl, w/w) fish products. The alkali isolated protein (AK) exhibited the best solubility at all salt concentrations. Furthermore, the AK had the highest gel strength and water binding capacity at low salt concentrations (0% and 1%). Low salt products of AK containing 1% salt showed similar mechanical properties to the water wash surimi (WM) obtained with 2% salt. The results obtained showed that the alkali process is a good method to recover functional proteins from boney silver carp muscle for low salt fish products.

Protein isolation from blue mussels (Mytilus edulis) using an acid and alkaline solubilisation technique—process characteristics and functionality of the isolates

The pH shift method was developed to isolate proteins from low-value raw materials by solubilisation at high or low pH followed by precipitation. In this paper the application of the pH shift method on isolated mussel (Mytilus edulis) meat and whole mussels is reported. Highest protein solubilisation was achieved at pH values of 2.6 and 12. The optimum precipitation pH values were established as around 5.8 following acid solubilisation and 5.2 following alkaline solubilisation. Protein recoveries were 430 and 580 g kg(-1) with the acid and alkaline processes respectively. Using whole crushed mussels, the corresponding recoveries were 310 and 480 g kg(-1). Process modifications to further improve protein recovery resulted in only a marginal increase. Lipid oxidation was not induced during pH shift processing, but heavy proteolysis occurred during the acid process version. Proteolysis could not be prevented by porcine plasma protein. Alkali-produced proteins performed better in all functionality tests compared with acid-produced proteins. The acid process removed slightly more lipids, recovered relatively more cysteine, methionine and lysine and resulted in whiter isolates. The pH shift method can be successfully used to extract functional proteins from mussels and add value to blue mussels unsuitable for human consumption (with or without shells).

Properties of hydrolysed saithe protein isolates prepared via pH shift process with and without dewatering

Fish protein hydrolysates (FPH) possess various bioactivities making them a desirable ingredient in health foods. Saithe is an underutilized fish species and an excellent candidate for FPH. To produce high quality FPH removing undesirable components prior to the hydrolysis process is preferred and possible via the pH-shift process. The objective was to produce and investigate the properties of hydrolyzed protein isolates produced from minced saithe trimmings. Saithe trimmings were minced, homogenized in water and subjected to two variations of a pH shift process with (A) and without (B) dewatering. The latter process has the advantage of being less complicated and less expensive which is beneficial to the industry. Both systems were adjusted to 3% protein, pH 8 and 45 °C and subjected to enzyme hydrolysis. Both hydrolysis processes led to almost identical SDS-page peptide makeup. Both FPHs had similar antioxidative activities evaluated using oxygen radical absorbance capacity (ORAC), iron chelation ability and (DPPH) radical scavenging ability assays. FPH from process A had significantly higher ACE inhibition ability compared to FPH from process B.These results demonstrate that saithe FPH has good antioxidative activities and a strong ability to inhibit ACE, and may thus have a potential as a bioactive food ingredient.

Channel Catfish (Ictalurus punctatus) Muscle Protein Isolate Performance Processed under Different Acid and Alkali pH Values

Channel catfish (Ictalurus punctatus) muscle was subjected to 6 protein extraction and precipitation techniques using acid solubilization (pH 2.0, 2.5, and 3.0) or alkaline solubilization (pH 10.5, 11.0, 11.5) followed by precipitation at pH 5.5. The catfish protein isolate was compared with ground defatted white muscle. Alkali-processed catfish showed increased gel rigidity, gel strength, and gel flexibility compared to acid-processed catfish, which exhibited inconsistent functional performance, increasing and decreasing gel rigidity, gel strength, and gel flexibility. The gel rigidity (G') at pH 3.0 in the absence of salt had the highest G' of the acid treatments and was not significantly different from the alkaline-treated catfish muscle (P>0.05). However in the presence of added salt pH treatment it had the lowest G' and was different from alkaline treatments (P<0.05) during break force testing. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a potentially broad range of applications. This range of applications is possible due to the modification of the textural properties of catfish muscle protein produced using different acidic or alkaline pH solubility treatments.

가열 젤 형성능을 가진 오징어 Surimi와 Surimi-based 제품을 위한 첨가물의 최적화

We investigated the optimum formulation to improve the gelling ability of squid, Dosidicus gigas, surimi. The solubility of minced squid muscle was highest at pH 10.7, and lowest at pH 5.0. The yields of conventional surimi and protein recovery after alkaline pH-shift processing were 68.1±2.4% and 65.3±2.6%, respectively, whereas the protein recovery with acidic pH-shift processing was only 21.2±1.6%. The addition of 5% starch decreased the breaking force regardless of the kind of starch, while the mixture of corn, potato, and wheat starch (total 15%) increased the breaking force by up to 1.9 fold. The addition of 5% egg white, 5% porcine plasma protein, 0.3% CaCl