3.2. Macronutrients of the Recovered Protein-Enriched Products
Both mechanical separation and pH-shift processing concentrated protein from backbones (Table 1
). Average protein content on dry weight (DW) basis in the original backbones of the three species (34–60%) increased up to 58–81% in the MSM and to 72–85% in pH-shift protein isolates. However, the protein concentration efficiency of both processes was highly species dependent. Previously, also Borgogno et al. [28
] showed that fish species can affect the protein concentrating factor during mechanical separation. The authors found that MSM of beheaded seabass had a significantly (p
< 0.05) lower content of protein than its fillet while beheaded sea bass and rainbow trout resulted in MSM with protein content equal with their fillets. The pH-shift process was significantly (p
< 0.05) more effective compared with mechanical separation in up-concentrating protein when applied on herring and salmon backbone. This is mainly related to the higher efficiency of the pH-shift process in removing fat which becomes evident with high-fat input raw material. On the other hand, when cod, which is leaner, was used as input material, both processes resulted in a similar increase in protein content. Protein concentrating factor for meat-bone separation and pH-shift processing was highest for cod and salmon, respectively. Similarly, the pH-shift process gave rise to highly concentrated protein isolates (up to 80–85%, DW basis) when applied on different bony fish processing coproducts e.g., fish frame, head [29
] and their mixture [14
Salmon MSM showed significantly (p
< 0.05) lower fat content (43% of dw) compared with its original backbones (52% of dw) while MSM from herring and cod backbones did not differ significantly from their corresponding input material (Table 1
). Obviously, the distribution of fat in bone marrow vs. muscle will dictate how much fat can be removed together with the bone residue in mechanic separation. The results from salmon imply that this species, where ≥50% bones were removed, has a relatively large amount of high fat bone marrow or other bone-derived lipid deposits compared to cod and herring. However, for herring backbones, where the relative amount of bone was very small (Figure 1
), the role of bone marrow is expected to play a minor role for the total MSM fat content, explaining why the comparison between salmon and cod backbones is more relevant. Borgogno et al. [28
] found significantly lower fat content in MSM from beheaded sea bream compared with its fillet while there was no such differences in the fat content of MSM vs. fillets for sea bass and rainbow trout.
Salmon and herring pH-shift protein isolates showed substantially lower fat content compared with their original backbones and their corresponding MSM. This is mainly related to the partitioning of neutral storage lipids into the floating emulsion layer emerging after the first centrifugation when using high-fat raw material as salmon and herring backbones. We recently showed that as much as 50–60% of the total fat was removed after the first centrifugation step as floating emulsion layer during pH-shift processing of herring and salmon heads plus backbones [9
]. However, if the centrifugation force is high enough, a large portion of the phospholipids can also be removed into the first sediment, as membranes have a relatively high density. In addition, a certain part of the phospholipids may also be removed into the second supernatant formed during dewatering of the precipitated proteins [30
]. Contrary, there was slightly, but yet significantly (p
< 0.05), higher content of fat in cod protein isolate compared with its backbones explained by the more pronounced removal of collagenous residues as bones, skin and connective tissue, compared to the sedimentation or solubilization of membranes, resulting in an up-concentration of both fat and protein in the final isolate. This is in agreement with previous reports comprising pH-shift processing of lean fish raw materials, including cod head plus backbone [14
Both MSM and protein isolates of the three species showed substantially lower content of ash compared with the initial backbones, which shows the high efficiency of both processes in removing bony residues (Table 1
). However, in all the three cases, protein isolates had significantly (p
< 0.05) lower amount of ash (2.18–2.51%) compared with their corresponding MSM (3.70–5.49%) revealing higher efficiency of the pH-shift process in removing bones and minerals compared with the mechanical separation. The lower protein purity in the MSM samples supports the hypothesis that the higher total protein yield obtained using the mechanical separation goes hand in hand with a less protein-enriched product.
Overall, application of the pH-shift process for the valorization of herring and salmon backbone resulted in much more efficient purification and concentration of proteins via more efficient removal of fat and ash compared with the mechanical separation. In the case of cod, both processes acted similarly in terms of up-concentration of protein and fat, but the pH-shift process more effectively removed ash.
3.3. Amino Acid Composition
Both MSM and protein isolates of the three species had lower content of nonessential amino acids than the backbones, especially proline and glycine (Table 2
) which are enriched in the collagenous residues [31
] being removed in mechanical separation and pH-shift processing. These findings agreed with our previous studies where we subjected mixed fish processing coproducts to pH-shift processing [11
]. For herring, MSM and protein isolates also had reduced content of some other nonessential amino acids including arginine, tyrosine, aspartic acid and glutamic acid compared with its backbones. For the protein isolates, this resulted in a significant (p
< 0.05) enrichment of some essential amino acids (EAA) including leucine and isoleucine, compared with the herring and salmon MSM and backbone raw material. For cod, the content of these EAA ranked the samples as protein isolates > MSM > backbone (p
> 0.05). Both the total content of EAA, and the EAA to total AA ratio, was higher in protein isolates of the three species compared with their MSM and backbones (p
< 0.05) reflecting the efficient up-concentration of the EAA-rich myofibrillar and sarcoplasmic proteins during pH-shift processing. This is in line with the results of Chen et al. [10
] and Taskaya et al. [33
] who found higher content of EAA in protein isolates from trout and carp by-products compared with their original by-products. Overall, the EAA-content of protein isolates and MSM was well above recommendations by FAO/WHO for adults, except for the content of leucine in the MSM which was slightly lower than the recommendations by FAO/WHO for adults [34
]. However, the content of valine, leucine and phenylalanine in both MSM and protein isolate of herring and cod did not meet the recommendations by FAO/WHO for infants. For samples from salmon, it was only the content of phenylalanine which was lower than the recommendation by FAO/WHO for infants [34
3.4. Fatty Acid Composition
Our results reveled that there were no significant differences (p
> 0.05) in the content of total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-3 PUFA and LC n-3 PUFA between herring MSM and its original backbone (Table 3
). For salmon, MSM however had significantly less total SFA and PUFA, while other groups were the same. Mechanical separation also had no significant impact on the relative distribution of the different fatty acid groups when expressed as % of total fatty acids in MSM from herring and salmon compared with their input materials. On the other hand, herring and salmon protein isolates contained significantly (p
< 0.05) lower absolute amounts of all the five fatty acid groups compared with both herring MSM and the backbone raw material, in line with its lower total fat content (see Table 1
). The reduction in total n-3 PUFA after pH-shift processing was from 47 mg/g DW in herring backbones to 15 mg/g DW in its protein isolate, and for salmon from 17 to 11 mg n-3 PUFA/g DW. However, pH-shift processing at the same time caused a dramatic change in the relative distribution of fatty acids in the protein isolated compared with the backbone and MSM of the two species. For example, pH-shift processing increased the % PUFA in the total fatty acid pool from 24% in the herring backbones to 43% in protein isolates, and LC n-3 PUFA from 18 to 39%. In MSM, relative PUFA and LC n-3 PUFA levels were 28% and 16%, respectively. Contrary, the percentages of n-6 PUFA and MUFA were reduced from 7 and 41% in backbones to 3 and 18%, respectively, during pH-shift processing. In herring MSM, relative levels on n-6 PUFA and MUFA were 9 and 37%, respectively. The % of DHA of total fatty acids in protein isolate of herring and salmon doubled and tripled, respectively, compared with their backbone and MSM. Altogether, the relative changes in fatty acid distributions increased the n-3/n-6 ratio 4- and 2-fold for herring and salmon pH-shift produced protein isolates, respectively. This is most probably related to a relatively larger removal of storage lipids than membranes in the pH-shift process; i.e., there was a certain enrichment of phospholipids in the protein isolates, which are known to contain more of the highly unsaturated n-3 PUFA than the lipid droplets [35
]. We have previously shown that the lipids recovered as an emulsion layer in the first centrifugation during pH-shift processing of herring and salmon by-products contained less phospholipids compared to oils extracted from these materials using a classic heat-based method [9
]. This is probably due to the amphiphilic nature and high density of phospholipids which makes them distribute into mainly the solubilized protein fraction and/or into the insoluble sediment during the first centrifugation, while in the second centrifugation, some phospholipids remain in the aqueous supernatant.
For cod, both processes raised the content of PUFA, n-3 PUFA and LC n-3 PUFA in the recovered protein enriched ingredients ≥ 2-fold compared with original cod backbones, with protein isolates showing significantly (p
< 0.05) higher content of PUFA and n-3 PUFA compared with the MSM. However, pH-shift processing reduced the relative amount of n-3 PUFA and LC n-3 PUFA in the fatty acid pool of the cod protein isolate to less than half of its % in cod backbones and MSM. Contrary, the % of n-6 PUFA increased up to 4-fold compared with cod backbones and MSM. The % of EPA and DHA in the fatty acids also dramatically decreased in the cod protein isolates reaching 1.3 and 0.8%, respectively, compared with their percentage in cod backbones (6.5 and 15%, respectively) and MSM (7 and 16%, respectively), which in turn reduced the n-3/n-6 ratio in cod protein isolate down to a tenth of the ratio in cod backbone. As explained before, a visible emulsion layer is not formed during the first step when pH-shift processing cod backbones. This means that the removal of lipids from the cod input raw material takes place via precipitation into the insoluble first sediment or by remaining in the second supernatant. In agreement with this, it has earlier been shown that up to 68–75% of the input lipids could be removed when applying the pH-shift process to menhaden or krill with relatively low lipid content (15 and 24%, dw, respectively) [30
], despite that no emulsion layer was formed. Both these species were ascribed a high content of phospholipids [36
]. Thus, more pronounced removal of phospholipids containing a higher amount of n-3 PUFA in parallel with oxidation of the most unsaturated PUFA as EPA and DHA during the pH-shift process [9
] and Wu et al., in manuscript, may have induced the changes measured in the fatty acid composition in cod protein isolate compared with its input material and MSM.
Overall, the absolute content of the three LC n-3 PUFAs; i.e., EPA, DPA and DHA was highest in products from herring, followed by cod, and then salmon. These findings reflect the large amount of plant-based lipids in the feed for salmon [37
]. The recommended daily intake of DHA and EPA is 250 mg for maintenance of cardiovascular health for children and healthy adults [38
], which could be achieved by eating 32, 123 and 105 g of herring, salmon and cod MSM, respectively, equalized to 80% moisture (Supplementary Table S1
). Corresponding numbers for herring, salmon and cod protein isolated on a 80% moisture basis would be 81, 140 and 96 g, respectively. Based on the general function health claims approved by EFSA (Commission Regulation (EU) No 432/2012) [39
], all products would qualify for the claims related to normal function of the heart (≥40 mg EPA + DHA/100 g) as well as to normal brain function and normal vision (both with a threshold of ≥40 mg DHA/100 g product) when normalized to 80% moisture content.
Fish is a very important dietary source of the lipid soluble vitamin D, which is critical e.g., for bone health since it plays several important roles in our body as a hormone in the regulation of calcium and phosphorus metabolism [40
]. Initial vitamin D content of herring backbones (0.23 µg/g DW) and salmon backbones (0.19 µg/g DW) was significantly (p
< 0.05) higher than its content in cod backbone (0.08 µg/g DW) (Figure 2
a). This shows that all three studied fish backbones can be good sources of vitamin D-rich products but that the backbones of fatty fish indeed are richer sources of this vitamin than lean fish. A large variation in vitamin D content of raw fish muscle and fishery products, ranging from 0 to 47 µg/100 g of fresh fish or product, has been reported [40
]. Mechanical separation did not significantly (p
> 0.05) change the content of vitamin D of salmon backbones while it significantly (p
< 0.05) increased its content in herring MSM (0.30 µg/g DW) compared with its original backbones. pH-shift processing on the other hand caused a product with significantly (p
< 0.05) less vitamin D for both herring (0.05 µg/g DW) and salmon (0.08 µg/g DW) compared with corresponding MSM and backbones, following the results of total fat (Table 1
). Opposite, mechanical separation of cod backbones concentrated vitamin D in the MSM (0.02 µg/g DW), while pH-shift processing gave a cod protein isolate with similar levels (0.05 µg/g DW) compared with its backbone. The daily recommended intake of vitamin D in the Nordic region is 10 μg for persons < 75 years, and 20 μg > 75 years (Nordic Council of Ministers, 2014); based on EFSA, it is 5 µg in all age groups. To achieve the daily recommended intake of vitamin D of 10 μg, the daily intake for herring, salmon and cod MSM with 80% moisture (Supplementary Table S1
) must be approximately 166, 286 and 2042 g, respectively, or for protein isolate it must be 882, 571, and 855 g, respectively. However, all products except the cod MSM qualified for EFSAs general function health claim related to vitamin D’s contribution to normal absorption/utilization of calcium/phosphorus, normal blood calcium levels and maintenance of normal bones, muscle function, teeth, cell division and immune system [39
]; i.e., 15% of the EU RDI/100 g, i.e., 0.75 µg/100 g.
Fish products can also be good sources of the membrane-bound vitamin E, comprising four tocopherols and four tocotrienols [42
]; molecules which are also important antioxidants. Cod backbones had the highest content of vitamin E (40 mg/kg DW) followed by salmon (34 mg/kg DW) and herring (19 mg/kg DW) (Figure 2
b). Content of tocopherols in fish depends highly on their diet since they cannot synthesize this vitamin. The enrichment of salmon feed with tocopherols [43
] can therefore explain the higher level in salmon than herring. That cod had the highest levels could be a combination of diet and the fact that this species as a lean fish has a relatively higher membrane lipid content (>80%) per amount of total lipids [35
] compared to herring and salmon. That tocopherols are membrane bound was most likely a contributing reason why they responded differently to processing compared to vitamin D. Another likely reason is that they are important antioxidants. Mechanical separation of herring and cod backbones did not change the content of vitamin E in the resulting MSM. However, pH-shift processing resulted in a significantly (p
< 0.05) lower content of vitamin E in protein isolates of both herring (<our detectable level) and cod (26 mg/kg DW) compared with their backbone and MSM. This could be related to the high amount of low molecular weight (LMW) iron and heme iron [44
] measured in these samples (Table 4
), stimulating free radical production which in turn can consume tocopherols during the pH-shift process. As indicated above, we have earlier documented significant lipid oxidation during the pH-shift process [45
]; more so compared with mechanical separation (Wu et al., in manuscript).
In contrast with cod and herring, applying both processing technologies on salmon backbones resulted in a significantly (p
< 0.05) higher content of vitamin E in its MSM (49 mg/g DW) and protein isolate (47 mg/g DW) compared with its backbone (35 mg/g DW). The increase in MSM would imply that bone marrow is not a significant source of vitamin E, while the increase in protein isolates is ascribed the natural abundance of astaxanthin in salmon muscle, protecting it from lipid oxidation during pH-shift processing [45
], as well a relatively larger removal of lipid deposits than membranes.
Salmon backbones had a substantially higher content of vitamin C, i.e., ascorbic acid (61 mg/kg DW), compared with herring (6.3 mg/kg DW) and cod backbones (2.9 mg/kg DW) (Figure 2
c). Additionally, here the results could reflect the enrichment of fish feed with vitamins; in the case of vitamin C to prevent oxidation as well as to stimulate collagen production and the immune system [46
]. Mechanical separation led to significantly (p
< 0.05) higher content of vitamin C in salmon MSM (74 mg/kg DW) compared with its backbone, while it resulted in a significantly (p
< 0.05) lower content of vitamin C in herring MSM compared with its backbone (2.7 mg/kg DW). For cod, levels were the same in MSM and raw material. Applying pH-shift processing on the backbones of the three species resulted in a substantial reduction of vitamin C content (down to 3 mg/kg DW) in salmon protein isolate and values were below the detectable level in herring and cod protein isolates. This is most probably due to the water-soluble nature of vitamin C, and thus, that it is leached out during the pH-shift process. Additionally, occurrence of lipid oxidation can consume vitamin C, as it works as an antioxidant in synergy with tocopherol [47
3.6. Content of Minerals
Both MSM and protein isolates of the three studied species had significantly (p
< 0.05) lower content of calcium, magnesium and manganese compared with their original backbones (Table 4
). Protein isolates of the three species also showed significantly (p
< 0.05) lower content of all the named minerals compared with their MSM counterpart except calcium which was only significant for cod samples. Protein isolate of salmon had significantly lower content of selenium compared with its MSM but this was not significant for cod and herring. Results are in line with the remarkable reduction of ash content after applying mechanical separation and pH-shift processing on the backbones of the three species, which is due to the efficient removal of bones which contain high levels of these minerals. Great removal of calcium and magnesium due to very efficient removal of bone residues when using alkaline pH-shift processing has previously been seen also for trout by-products [10
], gutted silvers carp (Hypophthalmichthys molitrix
] and gutted herring [31
]. Further, a significantly (p
< 0.05) lower content of calcium and magnesium was also found in MSM of beheaded rainbow trout than its fillet [28
Content of potassium in MSM of salmon and cod was significantly (p
< 0.05) higher than in their starting raw materials. pH-shift processing, on the other hand, led to an almost 5–10-fold lower content of potassium in the protein isolates compared with the original backbones for the three species. This is probably due to the high water solubility of potassium [48
], leading to its leaching into the second supernatant formed during the pH-shift process. Similarly, a 20-fold reduction in potassium was found when the pH-shift process was applied on yellowfin tuna (Thunnus albacares
) roe [49
]. These authors confirmed that the largest removal takes place by leaching, as they found very low levels of potassium in the insoluble fraction removed after first centrifugation of the pH-shift process.
MSM recovered from the backbone of salmon showed significantly (p
< 0.05) lower content of zinc and copper compared with their original backbones. On the other hand, protein isolate of salmon had significantly higher content of zinc compared with salmon MSM and salmon backbone. However, pH-shift processing did not significantly (p
> 0.05) change the content of zinc in the protein isolates of herring and cod compared with their corresponding backbones and MSM. Copper content of salmon, cod and herring protein isolates was also significantly higher than their corresponding backbones and MSM. This could be due to the high binding capacity of these two metals to proteins related to their role as cofactors in enzymes, a phenomenon which even concentrated them in salmon protein isolate. Previously, Marmon and Undeland [31
] found significantly higher content of zinc and copper in protein isolates from gutted herring using the pH-shift process compared with its starting raw material. Lee et al. [49
] showed that precipitation at pH 5.5 led to slightly but significantly (p
< 0.05) higher content of zinc in protein isolates of yellowfin tuna roe compared with its starting raw material, but a significant reduction in the content of zinc was found when precipitating the proteins at pH 4.5.
Mechanical separation and pH-shift processing of salmon and cod backbones did not change the content of iron in MSM and protein isolates of compared with the original backbones, showing an equal partitioning between bones, proteins, lipids and soluble phase. However, herring protein isolates had significantly (p
< 0.05) higher content of iron (71 mg/kg DW) compared with its MSM (52 mg/kg DW) and its original backbone (45 mg/kg DW). Previous studies have also shown an equal or higher iron content of protein isolates produced with pH-shift processing and input material. For example, the iron content of proteins isolated from gutted carp [33
] and trout by-products [10
] did not differ from their original input materials while it was significantly (p
< 0.05) higher in proteins isolated from gutted herring [31
] and tuna roe [49
] compared with starting raw materials.
Herring backbones had two- and four-fold higher content of heme iron (31 mg/kg DW) compared with cod (16 mg/kg DW) and salmon backbone (7 mg/kg DW). Heme iron was concentrated two-fold in the MSM and protein isolate of cod compared with its backbone and it was slightly, yet significantly (p
< 0.05), concentrated in salmon MSM. Other products were not significantly different from their corresponding starting material. Significantly higher content of heme pigments in proteins isolated from cod by-products (head + backbone) compared with the raw material has also been reported earlier [14
]. We have earlier seen that heme-iron can be removed during the pH-shift processing both by precipitation into the first sediment or by solubilization in the second supernatant, the latter accounting for the largest removal [50
]. In the same study, Hb removal with the supernatant increased when recovering proteins at a Ph > or < the pI as this reduced co-precipitation of Hb with the myofibrillar proteins. In the present study we recovered proteins at the pI, which could explain the heme-iron concentrating effect for cod backbones. Another explanation could be a partitioning of heme into cellular membranes upon Hb/Mb oxidation and heme-loss [51
] taking place along with lipid oxidation. More lipid hydroperoxides formed upon pH-shift processing of cod backbones than herring and salmon backbones (Wu et al., in manuscript).
From the perspective of lipid oxidation during subsequent storage of isolates or MSM, a high heme removal is indeed ideal. However, from a nutritional point of view, presence of heme-iron is important due to its higher bioavailability compared to LMW-iron [52
For salmon, sodium was more than twice as high in the pH-shift produced protein isolate than in corresponding backbones. There were also slight, but still significant (p < 0.05), sodium increases in MSM and protein isolates of cod backbones.
Overall, the effects from the mechanical separation and pH-shift process on the mineral content of protein recovered from the fish backbones appeared to be dependent on whether the minerals are located in the bone or muscle fraction of backbones as well as their binding affinity to proteins vs. their water solubility. Minerals enriched in the bone, such as calcium and magnesium, were removed to a higher degree by the pH-shift process than the mechanical separation, while a reverse trend was seen for minerals with high binding affinity to protein e.g., zinc. Highly water-soluble minerals, e.g., potassium, were only slightly affected by mechanical separation while they were severely leached out by the pH-shift process. Fish species and composition of input materials are indeed also factors that define the content of minerals in the final protein-enriched products.
From a health perspective, all three protein isolates contained >15% of RDI for copper when expressed on a 80% moisture basis (Supplementary Table S1
), i.e., levels denoted as significant according to EFSA [39
], allowing for functional health claims related to maintenance of normal connective tissues, energy-yielding metabolism, skin pigmentation and function of the immune system as well as to the protection of cells from oxidative stress. However, the EU Directive 90/496/EEC suggests 10% of RDI to be enough to denote a significant amount of a specific trace element, which applied to selenium (herring MSM), potassium (cod MSM), copper (herring MSM) and iron (herring protein isolate). The same directive also suggests that 5% of RDI can be enough to say there is a significant level, which applied to selenium in all six protein enriched products, to zinc in all three protein isolates and cod MSM, to iron for both of the cod and herring-derived products, to magnesium for cod and herring MSM, and to manganese for both types of cod products and herring protein isolate.